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NIS-Elements C (For MULTIPHOTON CONFOCAL MICROSCOPE A1 MP)

Instructions

(Ver. 4.00)

M487E 11.9.NF.13 (2/4)

i

Preface

Thank you for purchasing the Nikon products.

This instruction manual has been prepared for the users of the Camera Settings function of Nikon NIS-Elements.

To ensure correct usage, read this manual carefully before operating the instrument.

• It is prohibited to reproduce or transmit this manual in part or whole without Nikon’s expressed permission.

• The contents of this manual are subject to change without notice.

• Although every effort has been made to ensure the accuracy of this manual, if you note any points that are unclear or incorrect, contact your nearest Nikon representative.

• Be sure to read the manuals for any other products that you are using with this product.

• Usage in a way not specified by the manufacturer may impair the product safety.

• Reference spectrum data of dyes on NIS-Elements are provided from Invitrogen Corporation / Molecular Probes Clontech Laboratories, Inc.

Invitrogen Corporation http://www.invitrogen.com/

Clontech http://www.clontech.com/

• Spectral data of fluorescence dyes CoralHue ® Kaede and CoralHue ® Kusabira-Orange referenced in the NIS-Elements is provided from Amalgaam Co., Ltd.

Amalgaam Co., Ltd. http://www.amalgaam.co.jp/

ii

Contents Preface .................................................................................................................................................................. i Chapter 1 Before You Use ............................................................................................................................... 1

1.1 A1 Multi Photon ............................................................................................................................. 1 1.2 Differences in Window Names Depending on Models Used.......................................................... 1 1.3 Combinations of Detection Modes and Functions ......................................................................... 2

Chapter 2 Basic Operations ............................................................................................................................ 4 2.1 Acquiring the Live Image and Setting the Scan Area..................................................................... 4

Chapter 3 Detection Mode IR DU4................................................................................................................. 14 3.1 Filter and Dye Window................................................................................................................. 14

3.1.1 Structure of Filter and Dye Window................................................................................ 14 3.1.2 Setting the Optical Path ................................................................................................. 15 3.1.3 Optical Path Window...................................................................................................... 16

3.2 Acquisition Window...................................................................................................................... 18 3.2.1 Structure of Acquisition Window..................................................................................... 18

3.2.1.1 Recommended Value Indication/Automatic Application ........................................ 20 3.2.2 HV Linear Correction...................................................................................................... 22 3.2.3 Auto Gain ....................................................................................................................... 23

Chapter 4 Detection Mode IR SD................................................................................................................... 25 4.1 Filter and Dye Window................................................................................................................. 25

4.1.1 Structure of Filter and Dye Window................................................................................ 25 4.1.2 Setting the Optical Path ................................................................................................. 26 4.1.3 Optical Path Window...................................................................................................... 27 4.1.4 Optical Path Window Switching Tab .............................................................................. 28

4.1.4.1 Detector Tab ......................................................................................................... 28 4.1.4.2 Binning / Skip Tab ................................................................................................. 29


4.2.1.1 Recommended Value Indication/Automatic Application ........................................ 31 4.2.2 Auto Gain ....................................................................................................................... 33

4.3 Various Views (IR SD-use) .......................................................................................................... 35 4.3.1 Spectrum Profile............................................................................................................. 35 4.3.2 Spectral Unmixing Setting .............................................................................................. 35 4.3.3 Live Unmixing................................................................................................................. 35 4.3.4 Blind Unmix .................................................................................................................... 35

Chapter 5 Detection Mode IR VF ................................................................................................................... 36 5.1 Filter and Dye Window................................................................................................................. 36

5.1.1 Structure of Filter and Dye Window................................................................................ 36 5.1.2 Setting the Optical Path ................................................................................................. 37 5.1.3 Optical Path Window...................................................................................................... 38 5.1.4 Optical Path Window Switching Tab .............................................................................. 39

5.1.4.1 Detector Tab ......................................................................................................... 39 5.1.4.2 Grating Settings Tab ............................................................................................. 40

5.2 Acquisition Window...................................................................................................................... 41

Contents

iii

5.2.1 Structure of Acquisition Window..................................................................................... 41 5.2.1.1 Recommended Value Indication/Automatic Application ........................................ 43

5.2.2 Auto Gain ....................................................................................................................... 45 5.3 Various Views (IR VF-use) .......................................................................................................... 47

Chapter 6 Detection Mode IR NDD................................................................................................................ 48 6.1 Filter and Dye Window................................................................................................................. 48

6.1.1 Structure of Filter and Dye Window................................................................................ 48 6.1.2 Setting the Optical Path ................................................................................................. 49 6.1.3 Optical Path Window...................................................................................................... 50


6.2.1.1 Recommended Value Indication/Automatic Application ........................................ 54 6.2.2 HV Linear Correction...................................................................................................... 56 6.2.3 Auto Gain ....................................................................................................................... 57 6.2.4 Filter Cube Setting Dialog Box ....................................................................................... 59

6.3 Using Nikon Microscope ECLIPSE Ni-E ...................................................................................... 61 6.3.1 NDD Live Image Acquisition Settings............................................................................. 61 6.3.2 Optical Path Window in IR NDD Mode when Ni-E is in Use........................................... 71 6.3.3 Structure of Acquisition Window in IR NDD Mode when Ni-E is in Use ......................... 73

6.4 GaAsP NDD Detector Usage Notes (Only FN1 Microscopes)..................................................... 75 Chapter 7 Photo Activation Experiment Using A1 Multi Photon................................................................ 76

7.1 Laser for Photo Activation Experiment......................................................................................... 76 7.2 Setting Optical Path for Photo Activation Experiment Using A1 Multi Photon.............................. 77 7.3 Available Lasers for Simultaneous Photo Activate Observation .................................................. 82

Chapter 8 A1plus MP GUI Dialog Box........................................................................................................... 83 8.1 Structure of A1plus MP GUI Dialog Box ...................................................................................... 83

Chapter 9 A1plus Simple GUI ........................................................................................................................ 86 9.1 Displaying the A1plus Simple GUI............................................................................................... 86 9.2 Functions of A1plus Simple GUI.................................................................................................. 87

Chapter 10 Remote Control Function ............................................................................................................. 94 10.1 Starting and Stopping the Remote Controller .............................................................................. 94 10.2 Structure of NIS-Elements C with the Remote Controller ............................................................ 94

10.2.1 Operation of Remote Controller and Display in the Window .......................................... 95 10.2.1.1 Channel Select...................................................................................................... 95 10.2.1.2 Remote Controller Operation and the Corresponding Window

for Each Detector Mode ........................................................................................ 95 10.2.1.3 Channel Assignment ............................................................................................. 99

Chapter 11 Fast Galvano Mode ..................................................................................................................... 100

1

Before You Use1 1

This instruction manual describes how to make settings when capturing images with the NIS-Elements C using a Multiphoton Confocal Microscope system (referred to as “A1 Multi photon” hereinafter).

For operations common to the Confocal Microscope system A1, see “NIS-Elements C (For Confocal Micro-scope A1) Instructions (Ver. 4.00)”.

1.1 A1 Multi Photon

The A1 Multi photon uses the 2-photon excitation principle using near-infrared light. Therefore, light absorption is limited only to a region close to the focus apex and the other regions are less discolored. Furthermore, long-wavelength near-infrared light (IR pulse laser) is used as the excitation light. Thus the A1 Multi photon is also superior in deeper observation of samples.

* When using the IR pulse laser, first turn on the power to the IR pulse laser and complete its warm-up, and then start NIS-Elements C. (If NIS-Elements C is started in the course of the warm-up, the [A1 MP GUI] dialog box is grayed out and cannot be used.) Note that it takes approximately 30 minutes to warm up the IR pulse laser.

* If the connection to the IR pulse laser fails in the course of the start-up of NIS-Elements C, NIS-Elements C is started without connection to the A1 controller. (An error message appears.) In that case, check the IR pulse laser status and connection and then restart NIS-Elements C.

1.2 Differences in Window Names Depending on Models Used

If a model that supports the Fast Galvano mode is in use, a name “A1plus” is displayed at the upper left of each window. For models that do not support the Fast Galvano mode, a name “A1” is displayed.

Figure 1.2-1 Identification of models

Table 1.2-1 Window Names of Models That Support or Do Not Support Fast Galvano Mode

Window Names of Models That Support Fast Galvano Mode

Window Names of Models That Do Not Sup-port Fast Galvano Mode

A1plus Settings A1 Settings

A1plus MP GUI A1MP GUI

A1plus Simple GUI A1 Simple GUI

A1plus Stimulation A1 Stimulation

A1plus Scan Area A1 Scan Area In this instruction manual, window names of models that support Fast Galvano mode are written as “A1plus” hereinafter. When a model that does not support Fast Galvano mode is in use, replace window name “A1plus” with “A1.”

Models that support Fast Galvano mode Models that do not support Fast Galvano mode

Chapter 1 Before You Use

2

1.3 Combinations of Detection Modes and Functions

The following tables lists settable functions in each detection mode.

Table 1.3-1 Combinations of Detection Modes and Functions (1/2)

Detection Mode Function

DU4 SD VF VAAS IR DU4 IR SD IR VF IR NDD

Unidirectional Y Y Y Y Y Y Y Y Galvano

Bidirectional Y Y Y Y Y Y Y Y

Unidirectional Y N N Y Y N N Y Resonant

Bidirectional Y N N Y Y N N Y

Unidirectional Y N N Y N N N N Galvano Bidirectional Y N N Y N N N N

Unidirectional Y N N Y N N N N

Channel S

eries

Resonant Bidirectional N N N N N N N N

Multi position acquisition (A1 Chapter 3)

Available in all detection modes

(However, only Galvano scan mode is available and only square and band scan areas

are available without rotation. No photo activation experiment is available.)

Fast Galvano (Only Galvano) (A1 Chapter 8)

Y N N Y Y N N Y

CLEM (Only Galvano) (A1 Chapter 15) Y N N N N N N N

HV linear correction Y N N Y Y N N Y

Pinhole setting Y Y Y N N N N N

External detector (A1 Chapter 10)

Y Y Y Y N N N N

Unidirectional Y Y Y Y Y Y Y Y Galvano Bidirectional N N N N N N N N

Unidirectional Y N N Y Y N N Y

Line skipping (A1 C

hapter 8)


Filter cube setting (A1 MP Chapter 7)

N N N N N N N Y

Y: Available N: Unavailable

Chapter 1 Before You Use

3

Table 1.3-1 Combinations of Detection Modes and Functions (2/2)

Detection Mode Function

DU4 SD VF VAAS IR DU4 IR SD IR VF IR NDD

Normal photo activation (A1 Chapter 10)

Y Y Y Y Y Y Y Y

Simultaneous photo activation (A1 Chapter 10)

Y N N Y N N N N

Unidirectional Y N N N N N N N Galvano Bidirectional Y N N N N N N N

Unidirectional Y N N N N N N N

(A1 C

hapter 4) Line S

equential


Selection of stimulation laser for photo activation experi-ment (Visible light/IR)

Available in all detection modes only A1 Multi photon is used.

(However, only Galvano scan mode is available.)


4

Basic Operations2 2

This chapter describes the basic instructions for acquiring image when the A1 Multi photon is used.

2.1 Acquiring the Live Image and Setting the Scan Area

1 Selecting the Scan mode Select [Resonant] or [Galvano] for using the Scan

mode.

* In [SD], [VF], [IR SD] and [IR VF], only the Galvano scan is effective. If [Resonant] is selected and then detection mode is changed to [SD], [VF], [IR SD] or [IR VF], the scan mode changes automatically to Galvano.

Figure 2.1-1 Selecting the scan mode

2 Setting the Optical path 1. Display the Optical path window.

Click the [Setting] button in the Filter and Dye window.

Figure 2.1-2 Filter and Dye window

Figure 2.1-3 Optical path window

Setting button

Chapter 2 Basic Operations

5

2. Select the detection mode (detector).

Acquire images using the normal visible light laser with A1 Multi photon. According to each purpose, select [DU4] to use the Standard Detector; [SD] to use the Spectral Detector; [VF] to use the Virtual Filter; [VAAS] to use the VAAS.

* For details of the Optical path settings for each mode, see the following sections in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

[DU4] ··· see Section 4.1, “Filter and Dye Window.” [SD] ··· see Section 5.1, “Filter and Dye Window.” [VF] ··· see Section 6.1, “Filter and Dye Window.” [VAAS] ··· see Section 7.1, “Filter and Dye Window.”

Acquire images using the 2-photon excitation with A1 Multi photon. Select [IR DU4] to combine the IR pulse laser with the Standard Detector, select [IR SD] to combine it with the Spectral Detector, select [IR VF] to combine it with the Virtual Filter, and select [IR NDD] to combine it with the NDD.

* For details of the Optical path settings for each mode, see the following sections in this instruction manual.

[IRDU4] ··· see Section 3.1.1, “Filter and Dye Window.” [IR SD] ··· see Section 4.1.1, “Filter and Dye Window.” [IR VF] ··· see Section 5.1.1, “Filter and Dye Window.” [IR NDD] ··· see Section 6.1.1, “Filter and Dye Window.” When A1 Multi photon and Nikon microscope ECLIPSE Ni-E are in use, there are a few differences in setting items. For details, see “6.3 Using Nikon Microscope ECLIPSE Ni-E.”

(The following shows an example of the screen where [IR NDD] is selected for Detector mode.)

Figure 2.1-4 Selecting the Detection mode (Detector)

Detection mode button


6

3. Select the channels and fluorescence dye names to be used.

Figure 2.1-5 Selecting channels and fluorescence dye names (IR NDD-use)

4. Select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the combination of the selected laser wavelength and the 1st Dichroic mirror, “!” appears following the 1st Dichroic mirror name.

Figure 2.1-6 Selecting Dichroic mirror (IR NDD-use)

Check the channels to be used.

Select/enter fluorescence dye names used.

Select the 1st Dichroic mirror.


7

5. Furthermore, the scan mode that was set in step 1 can be changed. (When [SD], [VF], [IR SD] or [IR VF] is selected, the scan mode is fixed to Galvano.)

Figure 2.1-7 Changing the scan mode

6. Click the [OK] button to determine the Optical path settings.

At this time, if a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in step 4, a confirmation dialog box “1st DM is Invalid! Will you apply?” appears. When you apply the selected 1st Dichroic mirror, click the [OK] button. The Optical path settings are determined, and the Optical path window closes. Clicking the [Cancel] button returns the display to the Optical path window.

Figure 2.1-8 Determining the Optical path settings

OK button

Change the scan mode


8

3 Making preparations for the IR pulse laser 1. Call the [A1plus MP GUI] dialog box.

Right-click on the gray area (without any dialog box and setting window displayed) to display a menu. Then select [Acquisition Controls] -> [A1plus MP GUI] in the menu to open the [A1plus MP GUI] dialog box.

Figure 2.1-9 Displaying the A1plus MP GUI dialog box

2. Confirm that the IR pulse laser status indicator is lit green, and then click the Emission [ON] button in the [A1plus MP GUI] dialog box.

* While the IR pulse laser status indicator is blinking yellow, the IR laser is being warmed up.

* If a detection mode [DU4], [SD], [VF] or [VAAS] is selected and [Vis Stim] (visible laser light) is specified for the stimulation laser, the Emission [ON] button is disabled.

3. When the IR pulse laser status indication changes to green in color again, the preparation is completed.

* It takes a few minutes until the preparation is completed in some cases.

Figure 2.1-10. A1plus MP GUI dialog box

* To perform photo activation with the IR laser, check the [Use IR laser for Stimulation] check box.

Figure 2.1-11 A1plus MP GUI dialog box

Emission ON button

IR pulse laser status indicator


9

4 Adjusting the Laser Alignment 1. In the [A1plus MP GUI] dialog box called up in

Step 1 , adjust the IR pulse laser. Set an appropriate wavelength and laser power.

2. Perform the Laser Alignment. When the shutter opens, the Laser Alignment field is made available.

Click the [Auto Alignment] button. For fine adjustment, use the [Fine/Coarse] buttons in the Laser Alignment field.


* The Auto Alignment takes time in some cases.

* Each time the IR pulse laser wavelength has been changed, be sure to click the [Auto Alignment] button to perform the Laser Alignment. Or check the [Auto] check box to perform the Auto Alignment automatically when changing the wavelength.

5 Applying Scan settings In the Scan setting window, apply various scan settings to acquire the live image.

For details of Scan settings, see Chapter 8, “Scan Setting Window” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

Figure 2.1-13 Scan setting window (Galvano scan mode-use)

Selects the scan method (Unidirectional / Bidirectional). Selects scan magnification.

IR pulse laser wavelength

IR pulse laser power

Auto Alignment button

Open the shutter

Auto check box

Selects resolution.Selects scan speed.

Selects averaging and integration methods.

Selects line skipping.


10

6 Acquiring the live image Click the [Live] button.

The live image is acquired and the Live window appears.

Figure 2.1-14 Acquiring the live image (IR NDD-use)

Figure 2.1-15 Live window

Live button


11

7 Adjusting the brightness of the live image In the Acquisition window, adjust the brightness of the live image for each channel.

Acquire images using the normal visible light laser with A1 Multi photon.

* For details of the Optical path settings for each mode, see the following sections in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

[DU4] ... See 4.2, “Acquisition window” [SD] ... See 5.2, “Acquisition window” [VF] ... See 6.2, “Acquisition window” [VAAS] ... See 7.2, “Acquisition window”

Acquire images using the 2-photon excitation with A1 Multi photon.

* For details of the Optical path settings for each mode, see the following sections in this instruction manual.

[IR DU4] ... See 3.2, “Acquisition window” [IR SD] ... See 4.2, “Acquisition window” [IR VF] ... See 5.2, “Acquisition window” [IR NDD] ... See 6.2, “Acquisition window” When A1 Multi photon and Nikon microscope ECLIPSE Ni-E are in use, there are a few differences in setting items. For details, see “6.3 Using Nikon Microscope ECLIPSE Ni-E.”

Figure 2.1-16 Acquisition window (IR NDD-use)


12

8 Setting the scan area Set the scan area for the acquired live image.

For details of the scan area, see Chapter 9, “Navigation Mode” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

1. Switch the Live window to the navigation mode. Click the [Show Scan Area] button in the Live window.

Figure 2.1-17 Switching to navigation mode

2. Select the scan area setting tool to be used. The scan area setting tools differ in their available shapes depending on the scan area selected.

Figure 2.1-18 Selecting the scan area setting tool

3. Set the scan area with the tool selected. For instructions on selecting and using scan area setting tools, see “Scan Area Setting Tools” in Section 9.3, “About Scan Areas” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

Figure 2.1-19 Setting the scan area

Scan area

Scan area setting tool.

Show Scan Area button


13

9 Acquiring the image of the set scan area 1. Right click on the drawn scan area.

Figure 2.1-20 Acquiring the live image of scan area

Figure 2.1-21 Live window after changing the scan area

* While working with Frozen image, the live image in the set scan area can also be acquired by clicking the [Live] button.

Figure 2.1-22 Acquiring the live image

Right click on scan area

Live button

14

Detection Mode IR DU43 4

This chapter describes settings for the case when a detection mode in combination of the IR pulse laser and the Standard Detector (DU4) is selected.

3.1 Filter and Dye Window

This window enables to set the Optical path.

3.1.1 Structure of Filter and Dye Window

Figure 3.1-1 Filter and Dye window (IR DU4-use)

Table 3.1-1 Functions of Filter and Dye window (IR DU4-use)

Name Function

(1) Detector Indicates the name of the detection mode in current use.

(2) Setting button Opens the Optical path window. To use, select the detector, the dichroic mirror, the channel, laser wavelength and others.

(3) Status Indicates for the settings for each channel (fluorescence dye name, IR pulse laser wavelength, and wavelength band to be acquired).

(4) Close mechanical shutter during experiment

If unchecked, the shutter remains open during the ND image acquisition. As the shutter is not opened/closed every image acquisition, the time for the image acquisition can be shortened.

* During the interval period, laser power is automatically changed to the minimum but the laser cannot be shut off completely because the shutter is left open.

(5) PreScan button Makes settings of the pre-scan mode for correction of the position shift in a zoom change or of the image acquisition area shift in the photo activation by the prior scanning.

(6) Eye Port button Changes optical path to eye port.

• Optical Configuration

Individual data items set in the IR DU4 mode can be managed collectively with the [Optical Configuration] dialog box. For storing and retrieving the [Optical Configuration] settings, see the sections concerning the optical configuration in the “NIS-Elements Advanced Research User's Guide.”

(2)

(3)

(5)

(6) (1) (4)

Chapter 3 Detection Mode IR DU4

15

3.1.2 Setting the Optical Path

Click the [Setting] button of “Filter and Dye” window to display the Optical path window. Select the IR DU4 mode (IR DU4).

Figure 3.1-2 Filter and Dye window (IR DU4-use)

Figure 3.1-3 Optical path window (IR DU4-use)

Setting button


16

3.1.3 Optical Path Window

Figure 3.1-4 Optical path window (IR DU4-use)

Table 3.1-2 Functions of Optical path window (IR DU4-use) (1/2)

Name Function

(1) Detection mode selection button

Enables to select the IR DU4 mode. Enables to acquire up to 4 channel images.

(2) Excitation laser indicator

Displays the current setting for the laser. The currently set laser icon is displayed in a large size, and the optical path is indicated.

Used to select the setting window for Detector or Stimulation.

Detector Sets the wavelength of image acquisition laser. (3)

Optical path window switching tabs

Stimulation

Sets the wavelength of stimulation laser.

For the Stimulation tab, see Section 7.2, “Setting Optical Path for Photo Activation Experiment Using A1 Multi Photon.”

(4) Sorting fluorescence dye list

Sorts the fluorescence dye list according to the selected type.

ABC: Displays the list in alphabetical order. Emission: Displays the list in excitation wavelength order. Excitation: Displays the list in the order of peak wavelength of fluorescence intensity.

(9)

(1)

(13)

(14)

(15)

(3) (4) (5)

(8)

(7)

(6)

(2)

(10)

(11) (12)


17

Table 3.1-2 Functions of Optical path window (IR DU4-use) (2/2)

Name Function

(5) Rainbow chart

Provides the following information: - Wavelength band for which to acquire images (shown in color and value) - Excitation IR pulse laser - Stimulation laser for photo activation - A color band indicating the wavelengths in the entire band (400 to 750 nm) - Scale of the wavelengths in the entire band (400 to 750 nm)

(6) Channel selection check box

Enables to select the channels to be used.

(7) Channel color setting button

Displays the [Color Selection] dialog box, enables to set the desired color for each channel.

(8) Fluorescence dye name select / enter

Selects the in-use fluorescence dye name for each channel or enters an arbitrary channel name.

(9) IR pulse laser wavelength setting

Enables to set the IR pulse laser wavelength.

(10) 1st Dichroic mirror select

Enables to manually select the 1st Dichroic mirror to be used.

If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the combination of the selected laser wavelength and the 1st Dichroic mirror, “!” appears following the 1st Dichroic mirror name.

(11) Stimulation laser select

Enables to select IR pulse laser or visible laser as stimulation laser.

For setting the stimulation laser wavelength, see Section 7.2, “Setting Optical Path for Photo Activation Experiment Using A1 Multi Photon.”

Selects the scanner unit to be used.

Galvano Galvano scan mode allows high-quality imaging of up to 4096 × 4096 pixels. (12) Scan mode select

Resonant Resonant scan mode allows high-speed imaging of 420 frames per second.

(13) Filter cube display / select Selects the filter cube installed on the detector.

(14) OK button Determines the Optical path settings applied and closes the Optical path window.

(15) Cancel button Discards the Optical path settings applied and closes the Optical path window.


18

3.2 Acquisition Window

The Acquisition window enables to set PMT brightness (detection sensitivity).

3.2.1 Structure of Acquisition Window

Figure 3.2-1 Acquisition window (IR DU4-use)

Table 3.2-1 Functions of Acquisition window (IR DU4-use) (sheet 1/2)

Name Function

(1) Channel selection Selects the channels (Ch1 to Ch4) to acquire the desired images. Do this by adding a check mark.

For each of the channels (Ch1 to Ch4), use the HV and Offset to adjust the brightness of the live image.

HV

Sets the voltage to be applied to the PMT.

Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value.(2)

Brightness adjustment for each channel

Offset

Sets the BL offset value of the PMT.

Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value.

(3) Channel name indication Displays the channel name entered in the Optical path window.

(4) Channel color Displays the channel color specified in the Optical path window.

(5) IR pulse laser wavelength indication

Displays the currently selected IR pulse laser wavelength.

(6) thickness of optical section Indicates the FWHM (full width at half maximum) of z airy disk.

(7) Optical Resolution The actual size of 1 pixel square calculated from the optical information (for objectives and scan parameters) and the size acquired from an image.

(1)

(2)

(4)

(6) (7)

(8)

(11)

(3) (5) (13)

(9) (10) (12)


19

Table 3.2-1 Functions of Acquisition window (IR DU4-use) (sheet 2/2)

Name Function

(8) HV Linear CorrectionEnables or disables HV Linear Correction. For HV Linear Correction, see Section 3.2.2, “HV Linear Correction.”

(9) AG button

Automatically adjusts the HV value (HV gain) of the currently selected channel to the optimum values.

For Auto Gain, see Section 3.2.3, “Auto Gain.”

(10) Auto Gain setting button

Sets the ratio of saturation pixels used for automatic HV gain correction. The range of the Ratio of saturation pixels setting dialog box appears when this button is clicked. For Setting for Ratio of saturation pixels, see “Setting for Ratio of saturation pixels” in the Section 3.2.3, “Auto Gain.”

(11) Remote controller selector icon

Displayed for the channel group which is currently adjustable with the remote controller, see Chapter 11 in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

(12) Optimize button

Opens the [XYZ Size Setup] dialog box. In the [XYZ Size Setup] dialog box, the calculation method of the recommended values of the resolution, zoom magnification, and Z stack step size can be set.

For the [XYZ Size Setup] dialog box, see Section 3.2.1.1, “Recommended Value Indication/Automatic Application” on the next page.

(13) Select All Channels button

Selects all channels for acquiring images.

PMT Overload

If too much gain is applied with reference to the illumination intensity, the gain is automatically shut down to protect PMT. In this case, Live is aborted, the PMT HV value of the excitation laser in which the overload occurs becomes “0”, and the following [PMT Overload] dialog box is displayed. To acquire the live image again, reset the PMT HV value.

Figure 3.2-2 PMT Overload dialog box


20

3.2.1.1 Recommended Value Indication/Automatic Application

By the function of the recommended value indication/automatic application, the recommended values of the appropriate resolution, zoom magnification, and Z stack step size are calculated based on the objective type and the selected excitation wavelength. Using the calculated recommended values enables the image acquisition clearer and with less damage to the sample.

Recommended Value Automatic Application

To automatically apply the recommended values to the parameters, set the [Nyquist XY] button of the Scan Area window to ON.

Figure 3.2-3 Scan Area window

Figure 3.2-4 Location of Recommended Value Indication

* When the laser or objective in use is changed, the recommended values are recalculated, and newly indicated and automatically applied.

Nyquist XY button

Indicates the recommended value of the resolution.

Indicates the recommended value of the scan magnification.


21

Recommended Value Settings

Detailed settings of the recommended values are made in the [XYZ Size Setup] dialog box that is displayed by clicking the [Optimize] button of the Acquisition window. If the [Nyquist XY] button of the Scan Area window is ON, the recommended values are automatically applied to the parameters. Or if the [Nyquist XY] button is OFF, the recommended values of the scan size and zoom are indicated in the Scan setting window.

Figure 3.2-5 XYZ Size Setup dialog box

Table 3.2-2 Functions of XYZ Size Setup dialog box

Name Function

(1) Zoom Preference When the [Nyquist XY] button is ON, keeps the scan size and applied the recommended value of the zoom.

(2) Resolution Preference

When the [Nyquist XY] button is ON, keeps the zoom and applied the recommended value of the scan size.

(3) Use Cropping Fits the scan size in detail by using Crop Scan.

Sets the Z step size calculation method.

Recommend (Z~1/3 FWHM) Approximately one third of the thickness of optical section (FWHM value).

Half Overlap (Z=1/2 FWHM) One half of the thickness of optical section (FWHM value).

Minimum Overlap (Z=FWHM) The thickness of optical section (FWHM value).

(4) Suggested Step (Z)

Perfect Voxel (Z=X=Y) Value same as the pixel size.

(5) OK button Determines the XYZ Size Setup applied and closes the [XYZ Size Setup] dialog box.

(6) Cancel button Discards the XYZ Size Setup applied and closes the [XYZ Size Setup] dialog box.

(6)

(1)

(5)

(4)

(2) (3)


22

3.2.2 HV Linear Correction

When HV changes, Gain changes as shown in the graph captioned “Without HV Linear Correction” of Figure 3.2-6. As HV increases, the gain variation (the variation of image brightness) is gradual initially, and it becomes steep beyond a certain point. The gain variation can be automatically corrected to be linear with HV variation by the function called “HV Linear Correction.” With this correction, gain varies at the same rate as the HV adjustment.

Figure 3.2-6 Gain vs. HV

To enable HV Linear Correction, check the HV Linear Correction check box.

Figure 3.2-7 HV Linear Correction

• When HV Linear Correction is enabled or disabled, HV is reset to 0 V once.

Without HV Linear Correction With HV Linear Correction HV HV

GainGain


23

3.2.3 Auto Gain

Auto Gain is a function to automatically correct the value of HV gain to set the optimum image brightness. Automatic HV gain correction is performed within the predetermined range of the ratio of saturation pixels.

Both Galvano mode and Resonant mode are supported.

Automatic HV gain correction is performed only when channels are selected.

After execution of Auto Gain, in the dialog box indicating the progress of Auto Gain, the correction values actually used (Ratio of saturation pixels) are displayed by channel. For a channel on which Auto Gain failed, “x” is indicated and the HV value returns to its original value.

• Auto Gain cannot be started during Scan.

• In line scan, Auto Gain is not executable.

• During execution of Auto Gain, do not execute manual adjustments in the Acquisition window and adjustments by remote control.

Figure 3.2-8 Execution of Auto Gain (IR DU4-use)

Figure 3.2-9 Auto Gain progress

Auto Gain does not execute on unselected channels.

Auto Gain setting button Auto Gain button

Auto Gain status ... Failed ... Completed

Channel name

Option button

If checked, the window is automatically closed when Auto Gain is completed.


24

Setting for Ratio of saturation pixels

Set the maximum and minimum value for the Ratio of saturation pixels used for automatic HV gain correction. Click the [Auto Gain Setting] button to display the [Auto gain setup] dialog box. Set the maximum and minimum value for the ratio of saturation pixels in [Auto gain setup] dialog box.

Figure 3.2-10 Displaying the Auto gain setup dialog box

Figure 3.2-11 Setting for Ratio of saturation pixels

Table 3.2-3 Setting for Ratio of saturation pixels

Name Function

(1) Target Maximal Intensity Specifies the application ratio of the setting of the ratio of saturation pixels. Sets the percentage (%) of the maximum value to be applied.

(2) Advanced Settings If checked, advanced settings of the ratio of saturation pixels are enabled.

Minimum Sets the minimum value for Ratio of saturation pixels. (3)

Overillumination Tolerance (Area) Maximum Sets the maximum value for Ratio of saturation pixels.

(4) Perform in Find Mode (fast) If checked, execution in the Find mode is enabled.

(5) OK button Determines the settings of Auto gain setup applied and closes the [Auto gain setup] dialog box.

(6) Cancel button Discards the settings of Auto gain setup applied and closes the [Auto gain setup] dialog box.

(7) Default button Resets the set values to the default values.

(1)

(2)

(3)

(6)

(7)

(4)

(5)

25

Detection Mode IR SD4 5

This chapter describes settings for the case when a detection mode in combination of the IR pulse laser and the Spectral Detector (SD) is selected.




Figure 4.1-1 Filter and Dye window (IR SD-use)

Table 4.1-1 Functions of Filter and Dye window (IR SD-use)

Name Function


(2) Setting button Opens the Optical path window. To use, select the detector, the dichroic mirror, the laser wavelength, number of channels and others.

(3) Status Indicates for the settings for each excitation laser (IR pulse laser wavelength, and wavelength band to be acquired).

(4) Spectral Detector setting information

Displays the information set on the Spectral Detector.







Individual data items set in the IR SD mode can be managed collectively with the [Optical Configuration] dialog box. For storing and retrieving the [Optical Configuration] settings, see the sections concerning the optical configuration in the “NIS-Elements Advanced Research User's Guide.”

(7) (1) (3) (5)

(4)

(2)

(6)

Chapter 4 Detection Mode IR SD

26


Click the [Setting] button of “Filter and Dye” window to display the Optical path window. Select the IR SD mode (IR SD).

Figure 4.1-2 Filter and Dye window (IR SD-use)

Figure 4.1-3 Optical path window (IR SD-use)

Setting button


27


Figure 4.1-4 Optical path window (IR SD-use)

Table 4.1-2 Functions of Optical path window (IR SD-use)

Name Function


Enabled to select the IR SD mode. Enables to acquire up to 32-channel spectral images simultaneously.

(2) Excitation laser indicator Displays the current setting for the laser. The currently set laser icon is displayed in a large size, and the optical path is indicated.







Used to select the setting window for Detector, Binning/Skip or Stimulation.

Detector Sets the wavelength of image acquisition laser.

Binning/Skip Sets the binning and skip of channels. (5) Optical path window switching tabs

Stimulation





(1)

(6)

(7)

(5)

(2)

(3)

(4)


28

4.1.4 Optical Path Window Switching Tab

The tab for switching between [Detector] and [Binning/Skip] is displayed on the right top of the Optical path window.

4.1.4.1 Detector Tab

Figure 4.1-5 Optical path window (Detector tab)

Table 4.1-3 Functions of Detector tab (sheet 1/2)

Name Function

(1) Resolution Selects a wavelength resolution. Selectable from 2.5, 6, or 10nm.

(2) Channels Selects the number of channels (number of PMTs). Up to 32 channels can be selected in the wavelength range of 400 nm to 650 nm. (Up to 25 channels can be selected when 10nm is selected in Resolution.)

(3) Binning Displays the number of channel binning currently set.

(4) IR pulse laser wavelength setting Enables to set the IR pulse laser wavelength.

(5) Rainbow chart


(6) Wavelength range setting bar

Sets a wavelength range in a wavelength range from 400 nm to 650 nm. Sets a range by shifting the wavelength range setting bar to the right or left or by enlarging or reducing it. (Linked with the above setting of the number of channels.) * A part of the wavelength range may be displayed in black depending on the

setting conditions. In the wavelength range displayed in black, no wavelength range can be set.

(7) Start Displays the start wavelength of the wavelength range currently selected. Enabled to enlarge or reduce the range in units of wavelength resolution with the right or left button.

(8) All Enabled to shift to the right or left within the currently selected wavelength range without the wavelength interval being changed.

(9) End Displays the end wavelength of the wavelength range currently selected. Enabled to enlarge or reduce the range in units of wavelength resolution with the right or left button.

(10) Enlarge button Enlarges the rainbow chart. The display is switched in three levels.

(1)

(2)

(3)

(4)

(7) (8)

(5)

(6)

(9)

(10)


29

4.1.4.2 Binning / Skip Tab

With the inter-channel binning, the dark image can be brightened. Further, channels within the set wavelength range can be arbitrarily skipped. Since masked channel data is not acquired, the data volume can be reduced. Set this tab after the setting of the [Detector] tab is determined.

Figure 4.1-6 Optical Path window (Binning / Skip tab)

Table 4.1-4 Functions of Binning / Skip tab

Name Function

(1) Binning

Sets the number of channels to be combined into one channel. Two to four channels can be set. When Binning is set, the number of channels set with the [Detector] tab is automatically re-set to the closest number of channels that can be divided by the binning value.

Resets all PMT skips that have been set. (2)

PMT All on / off button

Leaves one channel and skips all of other PMTs.

(3) PMT skip selection check box

Sets skip in each channel. If this box is clicked, ■ (black) is displayed and skip is set. Channel data with skip set is not acquired during scan.

* If the setting of the [Detector] tab is changed, the setting with the [Binning / Skip] tab is cancelled.

(1)

(1)

(2)

Wavelength indication

Enlarge button

(3)


30




Figure 4.2-1 Acquisition window (IR SD-use)

Table 4.2-1 Functions of Acquisition window (IR SD-use)

Name Function

(1) Si HV

Adjusts HV of the Spectral Detector. (Applied to all excitation lasers.)

Slider bar: Slides to the right or left to set the Si HV value. Arrow buttons: Click either arrow button to increase or decrease the Si HV value stepwise. Direct entry in Si HV value display field: Type the desired setting value.

(2) thickness of optical section Indicates the FWHM (full width at half maximum) of z airy disk.


(4) AG button Automatically adjusts the Si HV value (Si HV gain) of the currently selected excitation laser to the optimum values.



Sets the ratio of saturation pixels used for automatic Si HV gain correction.The range of the Ratio of saturation pixels setting dialog box appears when this button is clicked. For Setting for Ratio of saturation pixels, see “Setting for Ratio of saturation pixels” in the Section 4.2.2, “Auto Gain.”

(6) Optimize button



PMT Overload

If too much gain is applied with reference to the illumination intensity, the gain is automatically shut down to protect PMT. In this case, Live is aborted, the PMT Si HV value of the excitation laser in which the overload occurs becomes “0”, and the following [PMT Overload] dialog box is displayed. To acquire the live image again, reset the PMT Si HV value.


(1)

(2) (3)

(4)

(5)

(6)


31









Nyquist XY button



32





Name Function


(2) Resolution Preference When the [Nyquist XY] button is ON, keeps the zoom and applied the recommended value of the scan size.










(1)

(2)

(4)

(5)

(3)

(6)


33

4.2.2 Auto Gain

Auto Gain is a function to automatically correct the value of Si HV gain to set the optimum image brightness. Automatic Si HV gain correction is performed within the predetermined range of the ratio of saturation pixels.

Automatic Si HV gain correction is performed only Si HV.

After execution of Auto Gain, in the dialog box indicating the progress of Auto Gain, the correction values actually used (Ratio of saturation pixels) are displayed. If Auto Gain failed, “x” is indicated and the Si HV value returns to its original value.




Figure 4.2-6 Execution of Auto Gain (IR SD-use)


Auto Gain button

Option button



Auto Gain setting button


34


Set the maximum and minimum value for the Ratio of saturation pixels used for automatic Si HV gain correction.

Click the [Auto Gain Setting] button to display the [Auto gain setup] dialog box. Set the maximum and minimum value for the ratio of saturation pixels in [Auto gain setup] dialog box.




Name Function




Overillumination Tolerance (Area)

Maximum Sets the maximum value for Ratio of saturation pixels.





(1)

(2)

(3)

(6)

(7)

(4)

(5)


35

4.3 Various Views (IR SD-use)

This section describes various spectral image views.

There are various spectral image viewing methods in accordance with the usage, such as Channel View Setting (Channel Mixed View, Split Channel View and Ratio Image View), Color Mode Setting, and Spectrum Separation.

For details of the spectral image various views with the IR SD mode selected, see Section 5.3, “Various Views (Spectral Detector-use)” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

4.3.1 Spectrum Profile

Brightness of the ROI area specified in the spectral image acquired in the IR SD mode can be decomposed and displayed for each 32 channels.

For details of the Spectrum Profile, see Section 5.3.3, “Spectrum Profile” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

4.3.2 Spectral Unmixing Setting

Separate the wavelength information of the spectral image acquired in the IR SD mode and display an Unmixing image. If wavelengths overlap (because multiple reagents are in use) and differences are hard to identify, wavelength information can be separated and displayed.

For details of the Spectral Unmixing Setting, see Section 5.3.4, “Spectral Unmixing Setting” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

4.3.3 Live Unmixing

Live observation is available in the state where the spectral image acquired in the IR SD mode is separated for each wavelength.

For details of the Live Unmixing, see Section 5.3.5, “Live Unmixing” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

4.3.4 Blind Unmix

Automatically search for typical spectra and display an Unmix image separated by the spectral wavelength information form the spectral image acquired in the IR SD mode. If wavelengths overlap (because multiple reagents are in use) and differences are hard to identify, wavelength information can be separated and displayed.

For details of the Blind Unmixing, see Section 5.3.6, “Blind Unmixing” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

36

Detection Mode IR VF5 5

This chapter describes settings for the case when a detection mode in combination of the IR pulse laser and the Virtual Filter (VF) is selected.

The Virtual Filter is a function that provides up to four binning groups for 32-channel spectral data and adjusts brightness of each group.




Figure 5.1-1 Filter and Dye window (IR VF-use)

Table 5.1-1 Functions of Filter and Dye window (IR VF-use)

Name Function










Individual data items set in the IR VF mode can be managed collectively with the [Optical Configuration] dialog box. For storing and retrieving the [Optical Configuration] settings, see the sections concerning the optical configuration in the “NIS-Elements Advanced Research User's Guide.”

(2)

(3)

(5)

(6) (1) (4)

Chapter 5 Detection Mode IR VF

37


Click the [Setting] button of “Filter and Dye” window to display the Optical path window. Select the IR VF mode (IR VF).

Figure 5.1-2 Filter and Dye window (IR VF-use)

Figure 5.1-3 Optical path window (IR VF-use)

Setting button


38


Figure 5.1-4 Optical path window (IR VF-use)

Table 5.1-2 Functions of Optical path window (IR VF-use)

Name Function


Enables to select the IR VF mode. Binning is performed for the spectral data of the concurrent 32 channels to group it into up to four groups, enabling acquisition of an image of light of a specified wavelength range.

(2) Excitation laser indicator

Displays the current setting for the laser. The currently set laser icon is displayed in a large size, and the optical path is indicated.







Used to select the setting window for Detector, Grating settings or Stimulation.


Grating Settings Sets the range for grating. (5)

Optical path window switching tabs

Stimulation





(1)

(6)

(7)

(5)

(2)

(3)

(4)


39

5.1.4 Optical Path Window Switching Tab

The tab for switching between [Detector] and [Grating Settings] is displayed on the Optical path window.

5.1.4.1 Detector Tab

Figure 5.1-5 Optical path window (Detector tab)

Table 5.1-3 Functions of Detector tab

Name Function (1) Resolution Displays the wavelength resolution currently set.

(2) Channels Displays the number of channels (number of PMTs) currently set.

(3) Binning The number of channel binning is fixed to 1.


Sorts the fluorescence dye list according to the selected type. ABC: Displays the list in alphabetical order. Emission: Displays the list in excitation wavelength order. Excitation: Displays the list in the order of peak wavelength of fluorescence intensity.

(5) Channel selection check box

Enables to select the channels to be used. (Up to 4 channel.)

(6) Channel color setting button






(9) Rainbow chart

Provides the following information: - Wavelength band for which to acquire images (shown in color and value) - Excitation IR pulse laser - Stimulation laser for photo activation - A color band indicating the wavelengths in the entire band (400 to 750 nm)- Scale of the wavelengths in the entire band (400 to 750 nm)

(10) Acquisition range for each virtual channel Slider bar

Specifies the laser wavelength range to be acquired for each virtual channel.

(6) (7) (8) (10)

(9)

(1)

(2)

(3)

(4)

(5)


40

5.1.4.2 Grating Settings Tab

Set the range for grating and set the wavelength range for the channels selected within the range.

Figure 5.1-6 Optical Path window (Grating Settings tab)

Table 5.1-4 Functions of Grating Settings tab

Name Function

(1) Resolution Selects a wavelength resolution. Selectable from 2.5, 6, or 10nm.

(2) Channels

Once the Resolution is selected, number of channels (number of PMTs) is automatically selected. To change the number of channels to 32, drag the edge of the Grating range setting bar.

(3) Binning The number of channel binning is fixed to 1.

(4) Grating range setting bar

Sets a wavelength range in a wavelength range from 400nm to 650nm. The range depends on the grating resolution. It is shiftable horizontally but the width of the bar cannot be reduced.

* A part of the wavelength range may be displayed in black depending on the setting conditions. In the wavelength range displayed in black, no wavelength range can be set.

(5) Start

Displays the start wavelength of the Grating range currently selected. Enabled to enlarge or reduce the range in units of Grating resolution with the right or left button. When the start wavelength is changed, it shifts without changing the grating range, thus the value of the end wavelength is changed simultaneously.

(6) All Enabled to shift to the right or left within the currently selected Grating range.

(7) End Displays the end wavelength of the Grating range currently selected. When the end wavelength is changed, it shifts without changing the grating range, thus the value of the start wavelength is changed simultaneously.

(8) Enlarge button Enlarges the rainbow chart. The display is switched in three levels.

Rainbow chart

(5) (6) (8)

(4)

(7)

(1)

(2)

(3)


41




Figure 5.2-1 Acquisition window (IR VF-use)

Table 5.2-1 Functions of Acquisition window (IR VF-use) (sheet 1/2)

Name Function

(1) Channel selection Selects the Virtual channels (Ch1 to Ch4) to acquire the desired images. Do this by adding a check mark.

(2) Gain

For each of the virtual channels, use the Gain to adjust the brightness of the live image.

Sets the PMT Gain.

Slider bar: Slides to the right or left to set the gain value. Arrow buttons: Click either arrow button to increase or decrease the gain value by 0.01. Direct entry in gain value display field: Type the desired setting value.

(3) Si HV

Adjusts HV of the Spectral Detector. (Applied to all channels.)

Slider bar: Slides to the right or left to set the Si HV value. Arrow buttons: Click either arrow button to increase or decrease the Si HV value stepwise. Direct entry in Si HV value display field: Type the desired setting value.

(4) Fluorescence dye name indication

Displays the fluorescence dye name entered in the Optical path window.




(7) thickness of optical section

Indicates the FWHM (full width at half maximum) of z airy disk.


(1)

(2)

(3) (7) (8)

(11)

(4) (5) (13) (6)

(9) (10) (12)


42

Table 5.2-1 Functions of Acquisition window (IR VF-use) (sheet 2/2)

Name Function


Displayed for the channel group which is currently adjustable with the remote controller, see Chapter 11 in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

(10) AG button

Automatically adjusts the Si HV value (Si HV gain) of the currently selected channel to the optimum values.



Sets the ratio of saturation pixels used for automatic Si HV gain correction. The range of the Ratio of saturation pixels setting dialog box appears when this button is clicked. For Setting for Ratio of saturation pixels, see “Setting for Ratio of saturation pixels” in the Section 5.2.2, “Auto Gain.”






PMT Overload

If too much gain is applied with reference to the illumination intensity, the gain is automatically shut down to protect PMT. In this case, Live is aborted, the PMT Si HV value of the excitation laser in which the overload occurs becomes “0”, and the following [PMT Overload] dialog box is displayed. To acquire the live image again, reset the PMT Si HV value.



43








Nyquist XY button




44





Name Function












(1)

(2)

(4)

(5)

(3)

(6)


45

5.2.2 Auto Gain

Auto Gain is a function to automatically correct the value of Si HV gain to set the optimum image brightness. Automatic Si HV gain correction is performed within the predetermined range of the ratio of saturation pixels.

Automatic Si HV gain correction is performed only Si HV.

After execution of Auto Gain, in the dialog box indicating the progress of Auto Gain, the correction values actually used (Ratio of saturation pixels) are displayed. If Auto Gain failed, “x” is indicated and the Si HV value returns to its original value.




Figure 5.2-6 Execution of Auto Gain (IR VF-use)


Auto Gain button

Option button



Auto Gain setting button


46


Set the maximum and minimum value for the Ratio of saturation pixels used for automatic Si HV gain correction. Click the [Auto Gain Setting] button to display the [Auto gain setup] dialog box. Set the maximum and minimum value for the ratio of saturation pixels in [Auto gain setup] dialog box.




Name Function










(1)

(2)

(3)

(6)

(7)

(4)

(5)


47

5.3 Various Views (IR VF-use)

For details of the various views with the IR VF mode selected, see Section 6.3, “Various Views (Virtual Filter mode-use)” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

48

Detection Mode IR NDD6 5

This chapter describes settings for the case when the NDD available only for the A1 Multi photon is selected for Detection Mode.

“NDD” (Non-Descanned Detector) is a detector that is able to detect the fluorescence signal from a sample with less loss close to the sample.

If you use the A1 Multi photon and a Nikon microscope ECLIPSE Ni-E equipped with both of transmission- and reflection-side NDD, the transmission-side NDD can be used in combination with the reflection-side NDD when the IR NDD mode is selected. For details, see Section 6.3, “Using Nikon Microscope ECLIPSE Ni-E.”




Figure 6.1-1 Filter and Dye window (IR NDD-use)

Table 6.1-1 Functions of Filter and Dye window (IR NDD-use)

Name Function




(4) NDD IN/OUT button

Sets/removes NDD in/from the optical path. (IN = Set in the optical path/OUT = Remove from the optical path)

For an observation with the eyepiece, remove NDD from the optical path.






(1) (4)

(5)

(7)

(2)

(3) (6)

Chapter 6 Detection Mode IR NDD

49


Individual data items set in the IR NDD mode can be managed collectively with the [Optical Configuration] dialog box. For storing and retrieving the [Optical Configuration] settings, see the sections concerning the optical configuration in the “NIS-Elements Advanced Research User's Guide.”


Click the [Setting] button of “Filter and Dye” window to display the Optical path window. Select the IR NDD mode (IR NDD).

Figure 6.1-2 Filter and Dye window (IR NDD-use)

Figure 6.1-3 Optical path window (IR NDD-use)

Setting button


50


Figure 6.1-4 Optical path window (IR NDD-use)

Table 6.1-2 Functions of Optical path window (IR NDD-use) (sheet 1/2)

Name Function


Enables to select the IR NDD mode. Enables to acquire up to 4 channel images.



(2) Optical path window switching tabs

Stimulation

Sets the wavelength of stimulation laser



Sorts the fluorescence dye list according to the selected type.

ABC: Displays the list in alphabetical order. Emission: Displays the list in excitation wavelength order. Excitation: Displays the list in the order of peak wavelength of fluorescence intensity.

(4) Rainbow chart


(5) Channel selection check box Enables to select the channels to be used.

(9)

(1)

(13)

(14)

(15)

(2) (3) (4)

(5)

(6) (7)

(8)

(10)

(11) (12)


51

Table 6.1-2 Functions of Optical path window (IR NDD-use) (sheet 2/2)

Name Function

(6) Channel color setting button Displays the [Color Selection] dialog box, enables to set the desired color for each channel.















(13) Filter cube setting button

Used to exchange the filter cube, create new filter cube information, or change the details of the registered filter cube. Clicking this button displays the [Filter Cube Setting] dialog box.

For the editing procedure, see Section 6.2.4 “Filter Cube Setting Dialog Box.”




52





Table 6.2-1 Functions of Acquisition window (IR NDD-use) (sheet 1/2)

Name Function



HV

Sets the voltage to be applied to the PMT. Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value.

(2) Brightness adjustment for each channel

Offset

Sets the BL offset value of the PMT. Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value.








(8) HV Linear Correction Enables or disables HV Linear Correction. For HV Linear Correction, see Section 6.2.2, “HV Linear Correction.”

(1)

(2)

(4)

(6) (7)

(8) (11)

(3) (5) (13)

(9) (10) (12)


53

Table 6.2-1 Functions of Acquisition window (IR NDD-use) (sheet 2/2)

Name Function

(9) AG button






Displayed for the channel group which is currently adjustable with the remote controller, see Chapter 11 in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).






PMT Overload




54








Nyquist XY button




55





Name Function












(1)

(2)

(4)

(5)

(3)

(6)


56

6.2.2 HV Linear Correction

When HV changes, Gain changes as shown in the graph captioned “Without HV Linear Correction” of Figure 6.2-6. As HV increases, the gain variation (the variation of image brightness) is gradual initially, and it becomes steep beyond a certain point. The gain variation can be automatically corrected to be linear with HV variation by the function called “HV Linear Correction.” With this correction, gain varies at the same rate as the HV adjustment.

Figure 6.2-6 Gain vs. HV

To enable HV Linear Correction, check the HV Linear Correction check box.

Figure 6.2-7 HV Linear Correction

• When HV Linear Correction is enabled or disabled, HV is reset to 0 V once.

Without HV Linear Correction With HV Linear Correction HV HV

GainGain


57

6.2.3 Auto Gain

Auto Gain is a function to automatically correct the value of HV gain to set the optimum image brightness. Automatic HV gain correction is performed within the predetermined range of the ratio of saturation pixels.

Both Galvano mode and Resonant mode are supported.

Automatic HV gain correction is performed only when channels are selected.

After execution of Auto Gain, in the dialog box indicating the progress of Auto Gain, the correction values actually used (Ratio of saturation pixels) are displayed by channel. For a channel on which Auto Gain failed, “x” is indicated and the HV value returns to its original value.




Figure 6.2-8 Execution of Auto Gain (IR NDD-use)


Auto Gain does not execute on unselected channels.

Auto Gain setting button Auto Gain button


Option button


Channel name


58


Set the maximum and minimum value for the Ratio of saturation pixels used for automatic HV gain correction. Click the [Auto Gain Setting] button to display the [Auto gain setup] dialog box. Set the maximum and minimum value for the ratio of saturation pixels in [Auto gain setup] dialog box.




Name Function










(1)

(2)

(3)

(4)

(5)

(6)

(7)


59

6.2.4 Filter Cube Setting Dialog Box

This dialog box opens when the [Filter cube setting] button is clicked on the Optical path window. The new filter cube to be created or the content of the registered filter cube can be changed.

Figure 6.2-12 Filter Cube Setting dialog box (for DM/BA1 BA2 Filter-use)

Table 6.2-4 Functions of the Filter Cube Setting dialog box (sheet 1/2)

Name Function

(1) Filter cube tab Selects the [FL Block] tab for registration and editing the Filter cube.

(2) Preset data Selects a set of the Filter cube for NDD recommended by Nikon.

(3) Filter cube name display / Edit

Displays / edits the Filter cube name in the combination of the dichroic mirror and the barrier mirror. (Input words: 1 - 24 (in Alphabetical letters))

(4) Filter cube name creation button

Generates the filter cube name automatically using Wavelength, Type, and BandWidth of the barrier filter.

(5) Use BA2 check box When checked, the second barrier filter (BA2) can be used.

(6) Dichroic mirror wavelength input

Inputs the wavelength of the dichroic mirror. (Input range: 400 - 750 mm)

The following wavelength is transmitted according to the selected Type.

[LP] Long pass: Wavelength equal to or longer than the Wavelength value is transmitted.

[SP] Short Pass: Wavelength equal to or shorter than the Wavelength value is transmitted.

(7) Filter type select Selects the dichroic mirror type.

(8) Barrier filter wavelength input

Inputs the wavelength of the barrier filter (Input range: 400 - 750 mm)

The following wavelength is transmitted according to the selected Type.

[LP] Long pass: Wavelength equal to or longer than the Wavelength value is transmitted.

[SP] Short Pass: Wavelength equal to or shorter than the Wavelength value is transmitted.

[BP] Band Pass: Wavelength of the Wavelength value ± BandWidth value/2 is transmitted.

(1)

(3)

(5)

(6)

(8)

(9) (11)

(2) (4)

(7) (10)

(12)


60

Table 6.2-4 Functions of the Filter Cube Setting dialog box (sheet 2/2)

Name Function

(9) Barrier filter type select

Selects the barrier filter type. ( [LP] Long pass/[SP] Short Pass/[BP] Band Pass)

(10) Barrier filter band input When the barrier filter type is [BP], inputs the range from the center wavelength for the barrier filter. [Input range: 1 - 350 mm normally (but not limited to)]

(11) Apply Determines the Filter Cube Settings.

(12) Exit Closes the [Filter Cube Setting] dialog box. If the setting has not been determined with the Apply button, a confirmation dialog box appears.


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6.3 Using Nikon Microscope ECLIPSE Ni-E

If you use a Nikon microscope ECLIPSE Ni-E equipped with both of transmission- and reflection-side NDD, the transmission-side NDD can be used in combination with the reflection-side NDD when the IR NDD mode is selected.

6.3.1 NDD Live Image Acquisition Settings

This section describes the basic instructions for acquiring live images in combination of the reflection-side NDD and the transmission-side NDD when the IR NDD mode is selected.


mode.


2 Setting the Optical path 1. Click the [Setting] button in the Filter and Dye window.

For details of the Optical path settings, see Section 6.3.2, “Optical Path Window in IR NDD Mode when Ni-E is in Use.”


2. Select [IR NDD] in the Optical path window.

Figure 6.3-3 Selecting the Detection mode (Detector)

[IR NDD]

Setting button


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3. Select or enter channels and fluorescence dyes to be used in the Reflected (reflection-side NDD) field and the Transmitted (transmission-side NDD) field.

Figure 6.3-4 Selecting channels and fluorescence dyes (IR NDD-use)

4. Select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the combination of the selected laser wavelength and the 1st Dichroic mirror, “!” appears following the 1st Dichroic mirror name.

Figure 6.3-5 Selecting 1st Dichroic mirror (IR NDD-use)

Check channel check boxes to be used for transmission-side NDD.

Select or enter fluorescence dyes to be used for transmission-side NDD.

Check channel check boxes to be used for reflection-side NDD.

Select or enter fluorescence dyes to be used for reflection-side NDD.

Reflection-side NDD

Transmission-side NDD

Select the 1st Dichroic mirror


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5. Furthermore, the scan mode that was set in step 1 can be changed.

Figure 6.3-6 Changing the scan mode

6. Click the [OK] button to determine the Optical path settings.

At this time, if a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in step 4, a confirmation dialog box “1st DM is Invalid! Will you apply?” appears. When you apply the selected 1st Dichroic mirror, click the [OK] button. The Optical path settings are determined, and the Optical path window closes. Clicking the [Cancel] button returns the display to the Optical path window.

Figure 6.3-7 Determining the Optical path settings

The mode in current use is displayed in the Detector field of the Filter and Dye window. Display of the detection mode depends on channels selected on the Optical path window.

[IR R-NDD]... When channels of the reflection-side NDD are selected [IR T-NDD]... When channels of the transmission-side NDD are selected [IR RT-NDD]... When channels of both reflection-side NDD and transmission-side NDD are selected

Figure 6.3-8 Displaying the detection mode (IR RT-NDD-use)

Change the scan mode

OK button


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3 Preparing the IR pulse laser 1. Call the [A1plus MP GUI] dialog box.

Right-click on the gray area (without any dialog box and setting window displayed) to display a menu. Then select [Acquisition Controls] -> [A1plus MP GUI] in the menu to open the [A1plus MP GUI] dialog box.

Figure 6.3-9 Displaying the A1plus MP GUI dialog box

2. Confirm that the IR pulse laser status indicator is lit green, and then click the Emission [ON] button in the [A1plus MP GUI] dialog box.

* While the IR pulse laser status indicator is blinking yellow, the IR laser is being warmed up.

* If a detection mode [DU4], [SD], [VF] or [VAAS] is selected and [Vis Stim] (visible laser) is specified for the stimulation laser, the Emission [ON] button is disabled.

3. When the IR pulse laser status indication changes to green in color again the preparation is completed.

* It takes a few minutes until the preparation is completed in some cases.


* To perform photo activation with the IR laser, check the [Use IR laser for Stimulation] check box.


Emission ON button

IR pulse laser status indicator


65

4 Adjusting the Laser Alignment 1. In the [A1plus MP GUI] dialog box called up in

Step 1 , adjust the IR pulse laser. Set an appropriate wavelength and laser power.

2. Perform the Laser Alignment. When the shutter opens, the Laser Alignment field is made available.

Click the [Auto Alignment] button. For fine adjustment, use the [Fine/Coarse] buttons in the Laser Alignment field.


* The Auto Alignment takes time in some cases.

* Each time the IR pulse laser wavelength has been changed, be sure to click the [Auto Alignment] button to perform the Laser Alignment. Or check the [Auto] check box to perform the Auto Alignment automatically when changing the wavelength.

5 Applying Scan settings In the Scan setting window, apply various scan settings to acquire the live image.

For details of Scan settings, see Chapter 8, “Scan Setting Window” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

Figure 6.3-13 Scan setting window (Galvano scan mode-use)

IR pulse laser wavelength

IR pulse laser power

Auto Alignment button

Open the shutter

Selects the scan method (Unidirectional / Bidirectional). Selects scan magnification.

Selects resolution.Selects scan speed.

Selects averaging and integration methods.

Selects line skipping.


66

6 Acquiring the live image Click the [Live] button.

The live image is acquired and the Live window appears.


Figure 6.3-15 Live window

7 Adjusting the brightness of the live image In the Acquisition window, adjust the brightness of the live image for each channel.

For details of the Acquisition window, see Section 6.3.3, “Structure of Acquisition Window in IR NDD Mode when Ni-E is in Use.”

Figure 6.3-16 Acquisition window (IR RT-NDD-use)

Channels of reflection-side NDD

Channels of transmission-side NDD


67

8 Setting the scan area Set the scan area for the acquired live image.

For details of the scan area, see Chapter 9, “Navigation Mode” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).

1. Switch the Live window to the navigation mode. Click the [Show Scan Area] button in the Live window.

Figure 6.3-17 Switching to navigation mode

2. Select the scan area setting tool to be used. The scan area setting tools differ in their available shapes depending on the scan area selected.

Figure 6.3-18 Selecting the scan area setting tool

3. Set the scan area with the tool selected. For instructions on selecting and using scan area setting tools, see “Scan Area Setting Tools” in Section 9.3, “About Scan Areas” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

Figure 6.3-19 Setting the scan area

Scan area

Scan area setting tool.

Show Scan Area button


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9 Acquiring the image of the set scan area 1. Right click on the drawn scan area.

Figure 6.3-20 Acquiring the live image of scan area

Figure 6.3-21 Live window after changing the scan area

* While working with Frozen image, the live image in the set scan area can also be acquired by clicking the [Live] button.


Right click on scan area

Live button


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10 Improving the S/N ratio of the Live image by combining channels on the reflection side and transmission side

1. Click the [NDD Live] button.

The [NDD Live Settings] dialog box appears.

Figure 6.3-23 Acquiring the NDD Live image

2. Select the channel combining button in the [NDD Live Settings] dialog box.

Figure 6.3-24 NDD Live Settings

3. Select the transmission-side channel to be combined with the reflection-side channel.

Figure 6.3-25 NDD Live Settings

4. Select a channel combining method.

Averaging Keep Bit Depth: The Reflection NDD image and the Transmitted NDD image will be averaged (12 bits). The combined image will be darker than images before combination.

Sum Keep Bit Depth: The Reflection NDD image and the Transmitted NDD image will be summed (12 bits). Bright images requiring 13 bits or more are saturated.

Sum Increase Bit Depth: The Reflection NDD image and the Transmitted NDD image will be summed (13 bits).

Figure 6.3-26 Selecting a combining method

NDD Live button

Select the transmission-side channel to be combined

Select the channel combining button


70

5. Turning on the [Apply on Live] button in the [NDD Live Settings] dialog box displays an NDD Live image combining each NDD channel in the Live window.


When channels have been combined, channel tabs are merged to a single tab. Turning off the [Apply on Live] button returns the merged channel tab to the previous tabs.


Apply on Live button

Combined channels


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6.3.2 Optical Path Window in IR NDD Mode when Ni-E is in Use

Figure 6.3-29 Optical path window (IR RT-NDD-use)

Table 6.3-1 Functions of Optical path window (IR RT-NDD-use) (1/2)

Name Function


Enables to select the IR NDD mode. Enables to acquire up to 4 channel images.



(2) Optical path window switching tabs

Stimulation

Sets the wavelength of stimulation laser. For the Stimulation tab, see Section 7.2, “Setting Optical Path for Photo Activation Experiment Using A1 Multi Photon.”


Sorts the fluorescence dye list according to the selected type. ABC: Displays the list in alphabetical order. Emission: Displays the list in excitation wavelength order. Excitation: Displays the list in the order of peak wavelength of

fluorescence intensity.

(4) Reflection-side NDD channel selection check box Enables to select reflection-side NDD channels to be used.

(7) (6)

(10)

(1)

(15) (16)

(17) (18)

(2) (3)

(4) (5) (8) (9)

(11) (12) (13) (14)

Reflection-side NDD

Transmission-side NDD


72

Table 6.3-1 Functions of Optical path window (IR RT-NDD-use) (2/2)

Name Function

(5) Reflection-side NDD channel color setting button


(6) Reflection-side NDD fluorescence dye name select / enter

Selects the fluorescence dye name to be used for each channel or enters an arbitrary channel name.



(8) Transmission-side NDD channel selection check box

Enables to select transmission-side NDD channels to be used.

(9) Transmission-side NDD channel color setting button


(10) Transmission-side NDD fluorescence dye name select / enter

Selects the fluorescence dye name to be used for each channel or enters an arbitrary channel name.











(15) Reflection-side NDD filter cube setting button

Used to exchange the filter cube of the reflection-side NDD, create new filter cube information, or change the details of the registered filter cube. Clicking this button displays the [Filter Cube Setting] dialog box.


(16) Transmission-side NDD filter cube setting button

Used to exchange the filter cube of the transmission-side NDD, create new filter cube information, or change the details of the registered filter cube. Clicking this button displays the [Filter Cube Setting] dialog box.





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6.3.3 Structure of Acquisition Window in IR NDD Mode when Ni-E is in Use


Table 6.3-2 Functions of Acquisition window (IR RT-NDD-use) (1/2)

Name Function







HV

Sets the voltage to be applied to the PMT.

Slider bar: Slides to the right or left to set the HV value. Arrow buttons: Click either arrow button to increase or decrease the HV value stepwise. Direct entry in HV value display field: Type the desired setting value.(5)

Brightness adjustment for each channel

Offset

Sets the BL offset value of the PMT.

Slider bar: Slides to the right or left to set the offset value. Arrow buttons: Click either arrow button to increase or decrease the offset value stepwise. Direct entry in offset value display field: Type the desired setting value.





(1) (2) (3) (4) (5) (13)

(7) (8) (9) (10) (11) (12)

Reflection-side NDD channels

Transmission-side NDD channels

(6)


74

Table 6.3-2 Functions of Acquisition window (IR RT-NDD-use) (2/2)

Name Function


(9) AG button









Displayed for the channel group which is currently adjustable with the remote controller.

When acquiring images using normal visible laser of the A1 Multi photon

See Chapter 11 in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

When acquiring images using 2-photon excitation of the A1 Multi photon

See Chapter 10 in this instruction manual.

(13) HV Linear Correction Enables or disables HV Linear Correction. For HV Linear Correction, see Section 6.2.2, “HV Linear Correction.”

PMT Overload




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6.4 GaAsP NDD Detector Usage Notes (Only FN1 Microscopes)

If the GaAsP device is used in the PMT of the NDD detector in the FN1 microscope in use, note that the gain setting differs from the setting for the normal NDD detector.

- No voltage is applied while the gain is 0 when the GaAsP NDD is used, but a voltage of 300 [V] is applied when the gain is changed to 1.

- When the gain is set to 171 or more, a voltage of 900 [V] is applied. However, the sensitivity remains unchanged even if the gain is further increased.

Figure 6.4-1 Relationship between HV Gain and voltage

Voltage [V]900

300

0

0 1 171 HV Gain value

Sensitivity

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Photo Activation Experiment Using A1 Multi Photon 7

7

This chapter describes operations different from those of the confocal microscope system A1 in the photo acti-vation settings when the A1 Multi photon is used.

For operations common to the Confocal Microscope system A1, see “NIS-Elements C (For Confocal Micro-scope A1) Instructions (Ver. 4.00)”.

7.1 Laser for Photo Activation Experiment

When the A1 Multi photon is used, IR pulse laser or visible laser is selectable as stimulation laser.

When acquiring an image with the IR pulse laser while the resonant scan mode is selected, no photo activation experiment can be made.

Table 7.1-1 Combinations of detection mode and laser (Galvano scan mode-use)

Stimulation Laser Detection Mode Motion

Select Vis Stim Select IR Stim

Image acquisition Visible laser Visible laser DU4

Photo activation Visible laser IR pulse laser

Image acquisition Visible laser Visible laser SD


Image acquisition Visible laser Visible laser VF


Image acquisition Visible laser Visible laser VAAS


Image acquisition IR pulse laser IR pulse laser IR DU4


Image acquisition IR pulse laser IR pulse laser IR SD


Image acquisition IR pulse laser IR pulse laser IR VF


Image acquisition IR pulse laser IR pulse laser IR NDD


Table 7.1-2 Combinations of detection mode and laser (Resonant scan mode-use)

Stimulation Laser Detection Mode Motion

Select Vis Stim Select IR Stim

Image acquisition Visible laser Visible laser DU4

Photo activation Visible laser IR pulse laser (less than 800 nm)

Image acquisition Visible laser Visible laser VAAS

Photo activation Visible laser IR pulse laser (less than 800 nm)

Image acquisition IR pulse laser IR pulse laser IR DU4

Photo activation Photo activation disabled Photo activation disabled

Image acquisition IR pulse laser IR pulse laser IR NDD

Photo activation Photo activation disabled Photo activation disabled

Chapter 7 Photo Activation Experiment Using A1 Multi Photon

77

7.2 Setting Optical Path for Photo Activation Experiment Using A1 Multi Photon


mode.

* To make a photo activation experiment using the IR pulse laser, select the Galvano scan mode.


2 Setting the Optical path 1. Display the Optical path window.

Click the [Setting] button in the Filter and Dye window.


2. Select the detection mode (detector) in the Optical path window. For details of the Optical path settings, see “Filter and Dye” in the chapters concerning detection modes.

Figure 7.2-3 Selecting image acquisition mode (detector)

Setting button

Detection mode button


78

3. Select the laser for photo activation from the [Stimulation Laser] field in the Optical path window.

Vis Stim ... Visible laser is used for photo activation. IR Stim ... IR pulse laser is used for photo activation.

Figure 7.2-4 Selecting stimulation laser

* The following settings vary depending on the selected detection mode.

When [DU4],[SD],[VF] or [VAAS] is selected, go to step 4. When [IR DU4], [IR SD], [IR VF]or [IR NDD] is selected, go to step 5.

Select laser for photo activation


79

4. When [DU4],[SD],[VF] or [VAAS] is selected

4-a. Select the stimulation laser.

When visible laser [Vis Stim] is selected for photo activation, select channels to be used and visible laser wavelengths.

Figure 7.2-5 Setting stimulation laser

When IR pulse laser [IR Stim] is selected for photo activation, enter the IR pulse laser wave-length to be used directly in the IR pulse laser wavelength display area under the [Excitation laser indicator] field with the keyboard.

* When performing photo activation using the IR pulse laser and acquiring an image with the visible laser, set the IR pulse laser wavelength to a value less than 800 nm.


4-b. Make other settings on the Optical path window, and then click the [OK] button. The Optical path settings are determined and the Optical path window closes.

Optical path settings when making a photo activation experiment using the IR pulse la-ser when [DU4], [SD], [VF] or [VAAS] is se-lected have been completed.


When [Vis Stim] is selected, select channels and visible laser wavelengths.

When [IR Stim] is selected, enter the IR pulse laser wavelength

OK button


80

5. When [IR DU4], [IR SD], [IR VF] or [IR NDD] is selected

5-a. Select the [Stimulation] tab.


5-b. Set the stimulation laser.

When visible laser [Vis Stim] is selected, select the Visible laser wavelength to be used.

When IR pulse laser [IR Stim] is selected, enter the IR pulse laser wavelength to be used in the [IR] field.

* When performing photo activation using the IR pulse laser and acquiring an image with the visible laser, set the IR pulse laser wavelength to a value less than 800 nm.


5-c. Select the 1st Dichroic mirror to be used. If a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in the combi-nation of the selected laser wavelength and the 1st Dichroic mirror, “!” appears following the 1st Dichroic mirror name.

Figure 7.2-10 Selecting the 1st Dichroic mirror

Stimulation tab

When [IR Stim] is selected, enter the IR pulse laser wavelength.

When [Vis Stim] is selected, select the visible laser wavelength.

Select the 1st Dichroic mirror


81

5-d. Make other settings in the [Detector] field of the Optical Path window, and then click the [OK] button to determine the Optical path settings.

At this time, if a 1st Dichroic mirror that the NIS-Elements does not recommend is selected in step 5-c, a confirmation dialog box “1st DM is Invalid! Will you apply?” appears. When you apply the selected 1st Dichroic mirror, click the [OK] button. The Optical path set-tings are determined, and the Optical path window closes. Clicking the [Cancel] button returns the display to the Optical path window.


Optical path settings when making a photo activation experiment using the IR pulse laser when [IR DU4], [IR SD], [IR VF] or [IR NDD] is selected have been completed.

Combinations of 1st Dichroic mirror and laser

If a combination of the image acquisition laser and the stimulation laser does not match the selection of the 1st Dichroic mirror, “!” appears next to the 1st Dichroic mirror selection box. In that case, reselect the 1st Dichroic mirror or change the laser combination.

The following table lists NIS-Elements-recommended combinations of 1st Dichroic mirror and laser.

Table 7.2-1 Combinations on 1st Dichroic mirror and laser

Laser Wavelength (nm)

IR Pulse Laser Visible Laser 1st Dichroic Mirror

800 or more

Less than 800

640 561 543 514 488 471 457 440 405

405/488 N N N N N N Y N N N Y

405/488/561 N N N Y N N Y N N N Y

405/488/561/640 N N Y Y N N Y N N N Y

400-457/514/IR N Y Y N N Y N N Y Y Y

405/488/543/640 N N Y N Y N Y N N N Y

BS 20/80 Y* Y Y Y Y Y Y Y Y Y Y

IR-DM Y Y N N N N N N N N N

405/488/561/IR N Y N Y N N Y N N N Y


* When a BS20/80 1st Dichroic mirror is used in the normal photo activate observation, the IR pulse laser wavelength can be set to 800 nm or more, but note that the illumination intensity becomes low.

OK button


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7.3 Available Lasers for Simultaneous Photo Activate Observa-tion

In the A1 Multi photon, simultaneous photo activate observation is enabled only when visible laser is selected as the image acquisition laser. In that case, only 405-nm laser and IR pulse laser (less than 800 nm) are available as the stimulation laser. (If IR pulse laser is selected as the image acquisition laser, simultaneous photo activate observation is disabled.)

When the confocal microscope system A1 is in use, 405-nm and 488-nm laser wavelengths are available as the stimulation laser for simultaneous photo activate observation, but note that 488-nm laser is not available in the A1 Multi photon.

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A1plus MP GUI Dialog Box8 8

This chapter describes IR pulse laser control, photo activation settings, and laser alignment settings.

8.1 Structure of A1plus MP GUI Dialog Box


Table 8.1-1 Functions of A1plus MP GUI dialog box (sheet 1/2)

Name Function

(1) Emission switch ON/OFF button

Selects ON/OFF of the IR pulse laser.

When the IR pulse laser is used for NIS-Elements, first turn ON the Emission switch.

* When Emission is turned ON from NIS-Elements C, it will take approximately 5 minutes at maximum until the Emission actually enters the ON status.

* To turn off Emission, hold down the left mouse button on [Off] button of Emission switch for about 3 seconds.

(2) Wavelength

Enables to set IR pulse laser wavelength.

* Each time the IR pulse laser wavelength has been changed, be sure to click the [Auto Alignment] button to perform the Laser Alignment.

* When the wavelength of the IR pulse laser is changed, it may take approximately 80 seconds at maximum.

(1)

(2)

(5)

(6) (7)

(8)

(9)

(10) (11)

(3)

(4)

(12)

(13)

(14)(15)

Chapter 8 A1plus MP GUI Dialog Box

84

Table 8.1-1 Functions of A1plus MP GUI dialog box (sheet 2/2)

Name Function

(3) IR pulse laser status indication

Indicates the IR pulse laser status.

No color: Preparation not completed Blinking in yellow: In process Green: Process completed

Selects open/close of the IR pulse laser hardware shutter. The shutter is opened after the preparation of the IR pulse laser is completed.

Opens the shutter. (4) Shutter button

Closes the shutter.

(5) Laser Power for observation

Enables to set IR pulse laser power value for observation.

* If the laser power value is modified while the IR pulse laser is assigned to the remote controller, the laser power value in the A1plus MP GUI dialog box is also modified at the same time.

* The IR pulse laser power is linearly corrected.

(6) Use IR laser for Stimulation check box

When checked, IR pulse laser is used for stimulation laser.

(7) Synchronize lasers for all Stimulation Areas check box

When checked, laser settings to be irradiated to entire photo activation area are linked.

(8) Laser Power for Photo Activation

Enables to set IR pulse laser power value for Photo Activation.

* The IR pulse laser power is linearly corrected.

Stimulation Areas 1, 2, and 3 correspond to photo activation areas 1, 2, and 3 respectively specified for the image.

(9) Laser power monitor button

Displays the laser power value (integer obtained after A/D conversion divided by 10) of the current channel by clicking this button.

During the image acquisition, the laser power cannot be measured and this button is grayed out.

(10) Auto check box Automatically executes the laser alignment in synchronization with the change of the laser wavelength.

(11) Auto Alignment

Adjusts the emission laser position automatically and performs the Laser Alignment.

* Each time the IR pulse laser wavelength has been changed, be sure to click the [Auto Alignment] button to perform the Laser Alignment.

(12) Photo activation position shift correction (X and Y directions)

Displays the [Manual Shift Alignment] dialog box that enables the photo activation position to be corrected manually if the position is shifted in the X and Y directions. Specifies the shift amount in pixel units for each photo activation group to correct the position shift.

(13) Photo activation position shift correction (Z direction)

Displays the Beam Expander that can manually correct the photo activation position shift in the Z direction, if any, during visible laser observation and IR photo activation. For details of the Beam Expander, see the next page.

(14) Coarse motion buttonManually make coarse adjustment of the emission laser position. When adjusting Alignment manually, adjust it while watching the brightness of the image.

(15) Fine motion button Manually make fine adjustment of the emission laser position. When adjusting Alignment manually, adjust it while watching the brightness of the image.

Chapter 8 A1plus MP GUI Dialog Box

85

Beam Expander

If the photo activation position is shifted in the Z direction when observed with visible laser and execution of photo activation using the IR pulse laser, the position shift can be adjusted manually using the Beam Expander. (Normally, the position shift is automatically corrected.)

1. Click the [Beam Expander setting] button.


2. Click the buttons to correct the position shift while checking the image acquired during the first photo activation experiment sequence.

Figure 8.1-3 Beam Expander

3. Clicking the [Recommend] button restores to the recommended value of automatic correction. If any condition (such as IR pulse laser wavelength) is changed, the manually corrected value is discarded and the recommended value is restored. (The recommended value is also restored when the NIS-Elements is restarted.)

Figure 8.1-4 Beam Expander

Moves inside the cell when viewed from the cover glass side.

Returns to the recommended value

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A1plus Simple GUI9 4

NIS-Elements allows you to use [A1plus Simple GUI], which is a simplified configuration screen supporting functions equivalent to those of [A1plus Settings], so that you can sufficiently use the window spaces. This section describes how to show the screen and the functions available on it.

9.1 Displaying the A1plus Simple GUI

As shown below, right-click on the gray area (without any dialog box and setting window displayed) to display a menu. Then select [Acquisition Controls] -> [A1plus Simple GUI] in the menu to open [A1plus Simple GUI] dialog box. (If you are using a Galvano scanner that is not compatible with Fast Galvano, select [Acquisition Controls] -> [A1 Simple GUI] from the menu.)

Figure 9.1-1 To display the A1plus Simple GUI

Figure 9.1-2 A1plus Simple GUI (VAAS-use)

A1plus Simple GUI

Chapter 9 A1plus Simple GUI

87

9.2 Functions of A1plus Simple GUI

[A1plus Simple GUI] allows you to configure settings for use of the Confocal Microscope A1 in the same manner as you configure with [A1plus Settings].

The following shows an example of the screen where VAAS is selected for Detector mode. For details of each item by detector, see the appropriate chapter.

(VAAS-use) (IR NDD-use)

Figure 9.2-1 A1plus Simple GUI

(6)

(7)

(9)

(12)

(15)

(16) (18) (20)

(23)

(24) (26)

(29)

(4)

(8)

(10) (11)

(13)

(14)

(17)

(19)

(21)

(22)

(25)

(27)

(28)

(1) (2) (3) (5)


88

Table 9.2-1 Functions of A1plus Simple GUI (sheet 1/6)

Name Function

(1) Scan button Starts/stops live image acquisition.

(2) Capture button Captures the image.

(3) Find... button

Starts/stops live image acquisition in Find mode.

Find mode is the mode where the live image acquisition is executed by temporarily switching to the high-frame-rate setting in order to ease the detection of the observation object such as a cell. For details, see Section 3.1.2, “Find Mode” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”


DU4, VAAS, IR DU4 or IR NDD-use


* Since the following items are common to the confocal microscope system A1, see “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

DU4: Section 4.2.5, “Auto Gain”

VAAS: Section 7.2.4, “Auto Gain”

For the following items, see each item in this instruction manual.

IR DU4: Section 3.2.3, “Auto Gain”

IR NDD: Section 6.2.3, “Auto Gain” (5) Auto Gain

SD, VF, IR SD or IR VF-use

Automatically adjusts the Si HV value (Si HV gain) to the optimum values.

* Since the following items are common to the confocal microscope system A1, see “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

SD: Section 5.2.4, “Auto Gain”

VF: Section 6.2.4, “Auto Gain”


IR SD: Section 4.2.2, “Auto Gain”

IR VF: Section 5.2.2, “Auto Gain”

(6) Scan mode selection

Enables to alternate between the Resonant scan mode and the Galvano scan mode. Scan modes cannot be alternated while the Live window is displayed. When selecting [SD], [VF], [IR SD] or [IR VF] for detection mode, only Galvano scanner is available. If the detection mode is changed to [SD], [VF], [IR SD] or [IR VF] after the Resonant scan mode is selected, the scan mode is automatically changed to the Galvano scan mode.


89


Name Function

(7) Line skipping

Sets the line skipping mode to be applied during scanning. Line skipping increases scan speed. (Pixel dwell does not change.)

Pull-down menu: Selects “None” (no line skipping), 2, 4, 8, or 16.

Example: If “2” is selected, scanning runs on the 1st line, skips the 2nd line, runs on the3rd line, etc.

(8) Laser power monitor button

Displays the laser power value (integer obtained after A/D conversion divided by 10) of the current channel by clicking this button.

(9) Scan Direction

Toggles between Unidirectional and Bidirectional scan. Bidirectional scan is only selectable if the Square scan area or Band scan area is set. By default, Unidirectional scan is selected.

* For details, see the following sections in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

Galvano scan mode: For details, see Section 8.1.3, “Scan Setting Parameters.” Resonant scan mode: For details, see Section 8.2.3, “Scan Setting Parameters.”

(10) Control by: Switches the Scan Speed selection form.

(11) Fast Mode

This setting is available only in the Galvano scan mode.

Switches to the Fast Galvano mode and the Galvano scanner processing speed becomes higher than the normal high-speed mode.

* For details, see Chapter 11 in this instruction manual.

Galvano scan mode-use

Sets scan speed. (Setting unit: Frame/Sec)

Pull-down menu: Selects the desired scan speed from this list. [▲] and [▼] buttons: Click these to select scan speeds one after another.

(12) Scan Speed Resonant scan mode-use

Displays the scan speed automatically selected by the scan area. (Display unit: Frame/sec)

For details, see “Relationship between scan area and Scan setting” in Section 8.2.2, “Relationships among Scan Area Shape, Resolution, and Scan Speed” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”


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Name Function

Galvano scan mode-use

Sets the scan resolution in the X-direction. (Setting unit: Pixel) The resolution in the Y-direction is automatically calculated from the X to Y ratio of the scan area.

Pull-down menu: Selects the desired resolution from this list. [▲] and [▼] buttons: Click these to select resolutions one after another. (13) Scan Size

Resonant scan mode-use

Displays the resolution automatically selected by the scan area. (Display unit: Pixel)For details, see “Relationship between scan area and Scan setting” in Section 8.2.2, “Relationships among Scan Area Shape, Resolution, and Scan Speed” in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).

(14) Average/Integral

Provides options for scanning a given line or area a number of times to display an image of the averaged or integrated values.


Galvano scan mode: For details, see Section 8.1.5, “Averaging and Integration.” Resonant scan mode: For details, see Section 8.2.4, “Averaging and Integration.”

(15) Ch Series button

Settable only in DU4 and VAAS-use. Selects whether to perform scanning by simultaneously firing all lasers for the channels in use or by sequentially firing one laser after another. Lasers can be selected in the order of Ch1 to Ch4 or Ch4 to Ch1.


DU4: Section 4.1.4, “Selecting the Channel Series.” VAAS: Section 7.1.4, “Selecting the Channel Series.”

(16) Ch.Setup check box

Displayed when the [Ch Series] button is ON. When checked, the setting by the channel is facilitated. Automatically enters the state where only one channel is selectable.

(17) Fps: Indicates the current scan settings.

(18) Settings button Displays the menu to open dialog boxes for various settings such as HV Linear Correction.


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Name Function

DU4, SD or VF-use

Adjusts the pinhole size.

Sets a pinhole size in Airy units (units of airy disk size).

Slider bar: Slides to the right or left to set the pinhole size. (Unit: A.U.)

Arrow buttons: Click either arrow button to increase or decrease the pinhole size stepwise.

Direct entry in pinhole size display field: Type the desired setting value.


DU4: Section 4.2.3, “Setting the Pinhole” SD: Section 5.2.3, “Setting the Pinhole” VF: Section 6.2.3, “Setting the Pinhole”

(19) Pinhole

IR DU4, IR SD, IR VF, VAAS or IR NDD-use

Not available

Standard Detector, Spectral Detector or Virtual Filter-use

Changes the pinhole to the predetermined home position. The default is 1.0 A.U.

The value of the home position can be changed in the [A.U. Calculation Settings] dialog box.


DU4: Section 4.2.3.1, “Calculation Settings for Pinhole Size” SD: Section 5.2.3.1, “Calculation Settings for Pinhole Size” VF: Section 6.2.3.1, “Calculation Settings for Pinhole Size”

The [A.U. Calculation Settings] dialog box is displayed by selecting [AU settings...] from the setting menu displayed by the [Settings] button.

(20) AU button

VAAS-use

Not available

(21)

Reference excitation wavelength for the pinhole size calculation

Selects the excitation wavelength as the reference of the automatic calculation of the pinhole size from the laser wavelengths, or enter it manually in the [A.U. Calculation Settings] dialog box.


DU4: Section 4.2.3.1, “Calculation Settings for Pinhole Size” SD: Section 5.2.3.1, “Calculation Settings for Pinhole Size” VF: Section 6.2.3.1, “Calculation Settings for Pinhole Size”

(22) Optical path Setting button

Opens the Optical path window. To use, select the detector and the dichroic mirror, the channels as well as the fluorescence dye, laser, and for each channel.


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Name Function


Selects the Detection mode for use.

* For details of each item for each Detection mode, see the following. Since the following items are common to the confocal microscope system A1, see “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

: Chapter 4, “Detection Mode DU4”

: Chapter 5, “Detection Mode SD”

: Chapter 6, “Detection Mode VF”

: Chapter 7, “Detection Mode VAAS”


: Chapter 6, “Detection Mode IR NDD”

+ Use IR Laser: Chapter 3, “Detection Mode IR DU4”

+ Use IR Laser: Chapter 4, “Detection Mode IR SD”

+ Use IR Laser: Chapter 5, “Detection Mode IR VF”

(24) Use IR Laser

This check box is only effective while DU4, SD, or VF is selected.

When checked, the mode is switched to the detection mode of 2-photon excitation observation using the IR pulse laser.

(25) In/Near Link check box

Displayed only when VAAS is selected. The In/Near link check box is checked, the PMT adjustment for In focus and Near focus are linked. When you adjust either PMT, the other PMT is also adjusted automatically.

Displayed only when VAAS is selected. Used to switch between the In focus and Near focus PMT adjustment windows. To acquire the VAAS image, PMT of an In focus image (image of the focus area) and a Near focus image (image of the non focus area) can be adjusted respectively.

ON status

Displays the PMT adjustment window of Near focus.

The light that passes through the normal pinhole is detected. (Normally-acquired confocal image)

(26) Define Near Focus

OFF status

Displays the PMT adjustment window of In focus.

The light that does not pass through the normal pinhole is detected.


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Name Function

(27) Brightness adjustment for each channel

For each of the channels (Ch1 to Ch4), use the HV, Offset, and Laser controls to adjust the brightness of the live image.

Note that these items vary depending on the selected detector.

* For details of each item in each detection mode, see the following. Since the following items are common to the confocal microscope system A1, see “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

DU4: Section 4.2.1, “Structure of Acquisition Window” SD: Section 5.2.1, “Structure of Acquisition Window” VF: Section 6.2.1, “Structure of Acquisition Window” VAAS: Section 7.2.1, “Structure of Acquisition Window”


IR DU4: Section 3.2.1, “Structure of Acquisition Window” IR SD: Section 4.2.1, “Structure of Acquisition Window” IR VF: Section 5.2.1, “Structure of Acquisition Window” IR NDD: Section 6.2.1, “Structure of Acquisition Window”

(28) Brightness adjustment for transmitted detector

For the transmitted detector, use the HV and Offset controls to adjust the brightness of the live image.

* This item is not displayed when the detection mode of 2-photon excitation observation using the IR pulse laser is selected because the transmitted detector is not available.

This button is displayed only when the IR NDD mode is selected. Switches brightness adjustment windows for each channel of the reflection-side NDD and transmission-side NDD.

ON status

Displays brightness adjustment windows for each channel of the transmission-side NDD.

(29) T NDD button

OFF status

Displays brightness adjustment windows for each channel of the reflection-side NDD.

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Remote Control Function10 4

This chapter describes the structure of “NIS-Elements C” window and cautions for the use of the remote controller.

With “NIS-Elements C”, you can use the remote controller supplied with A1 to perform image adjustment such as laser power output and PMT brightness (detector sensitivity) or start/stop of scanning.

For remote controller functions for acquiring images with normal visible laser, see Chapter 11 in “NIS-Elements C (For Confocal Microscope A1) Instructions (Ver. 4.00).”

For details about how to use the remote controller, refer to the section concerning the remote controller operation in “Confocal Microscope A1 <Hardware>.”

10.1 Starting and Stopping the Remote Controller

To synchronize “NIS-Elements C” with the remote controller, turn “ON” the power switch of the remote controller before launching “NIS-Elements C.”

When you turn “OFF” the remote controller's power, check that “NIS-Elements C” is not activated before switching “OFF” the controller.

10.2 Structure of NIS-Elements C with the Remote Controller

When “NIS-Elements C” and the remote controller are synchronized, the remote controller's icon appears in the Acquisition window. The icon is displayed for the channel currently adjustable with the remote controller.

Figure 10.2-1 Acquisition window

Remote controller icon

Chapter 10 Remote Control Function

95

10.2.1 Operation of Remote Controller and Display in the Window

10.2.1.1 Channel Select

Pressing the [+] or [-] button of Channel Select of the Remote Controller causes the Remote controller icon to move to another channel in the Acquisition window. The channel with the Remote controller icon can be controlled by the Remote Controller.

10.2.1.2 Remote Controller Operation and the Corresponding Window for Each Detector Mode

Items that can be controlled by the Remote Controller depend on the selected detector mode.

IR DU4-use

If the channel group is changed with the [+] button of Channel Select of the Remote Controller, channels are changed in the order of [1] to [4] in the Acquisition window of “NIS-Elements C.”

Figure 10.2-2 Acquisition window (IR DU4-use)

Table 10.2-1 Acquisition window's functions adjustable with the Remote Controller (IR DU4-use)

Name Function

(1) HV Sets the voltage to be applied to the PMT. The Remote Controller can adjust the HV values of the channels [1] to [4].

(2) Offset Sets the BL offset value of the PMT. The Remote Controller cannot adjust this value.

Table 10.2-2 Other functions adjustable with the Remote Controller (IR DU4-use)

Name Function

Scan Speed The Remote Controller can change the scan speed.

Zoom The Remote Controller can change the zoom magnification.

Start/Stop The Remote Controller can start or stop scanning.

(1) (2) [2] [1]

[4] [3]


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IR SD-use

The following items can be operated in the IR SD mode.

Figure 10.2-3 Acquisition window (IR SD-use)

Table 10.2-3 Acquisition window's functions adjustable with the Remote Controller (IR SD-use)

Name Function

(1) Si HV Adjusts HV of the Spectral Detector.

Table 10.2-4 Other functions adjustable with the Remote Controller (IR SD-use)

Name Function




(1)


97

IR VF-use

If the channel group is changed with the [+] button of Channel Select of the Remote Controller, channels are changed in the order of [1] to [4] in the Acquisition window of “NIS-Elements C.”

Figure 10.2-4 Acquisition window (IR VF-use)

Table 10.2-5 Acquisition window's functions adjustable with the Remote Controller (IR VF-use)

Name Function

(1) Gain Adjusts the laser PMT gain. The Remote Controller cannot adjust this value.

(2) Si HV Adjusts HV of the Spectral Detector. The Remote Controller can adjust the HV values of the channels [1] to [4] at a time.

Table 10.2-6 Other functions adjustable with the Remote Controller (IR VF-use)

Name Function




(1)

(2)

[4]

[2] [1]

[3]


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IR NDD-use

If the channel group is changed with the [+] button of Channel Select of the Remote Controller, channels are changed in the order of [1] to [4] ([1] to [8] when IR RT-NDD is selected) in the Acquisition window of “NIS-Elements C.”



Table 10.2-7 Acquisition window's functions adjustable with the Remote Controller (IR NDD-use)

Name Function

(1) HV Sets the voltage to be applied to the PMT. The Remote Controller can adjust the HV values of the channels [1] to [8].

(2) Offset Sets the BL offset value of the PMT. The Remote Controller cannot adjust this value.

Table 10.2-8 Other functions adjustable with the Remote Controller (IR NDD-use)

Name Function




(1) (2) [2]

[4]

[1]

[3]

(1) (2)

[2]

[4]

[1]

[3]

[6]

[8]

[5]

[7]

Channels of reflection-side NDD

Channels of transmission-side NDD


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10.2.1.3 Channel Assignment

You can change the channels assigned to the Remote Controller from the software side. Click on the blank area of another channel group. Then the Remote controller icon moves to that channel group and the selected channels are assigned to the Remote Controller.

Figure 10.2-7 Acquisition window

Click on the blank area.

Remote controller icon

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Fast Galvano Mode11 4

This chapter describes the Fast Galvano mode when the multi photon confocal microscope system A1 MP (A1 Multi photon) is used.

When a model that supports the Fast Galvano mode is in use, the scan speed of the Galvano scanner can be higher than the normal high-speed mode.

Turning on the [Fast] button makes the scan speed ultra-high.

Figure 11.1-1 Fast Galvano mode

Identification of Models When the model in use supports the Fast Galvano mode, “A1plus” is displayed on various windows.

* In the examples below, “A1 Settings” and “A1 Scan Area” are displayed for models that do not support the Fast Galvano mode.

Figure 11.1-2 Identification of models

Fast button

Chapter 11 Fast Galvano Mode

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Notes on using the Fast Galvano mode

Note the following restrictions when using the Fast Galvano mode.

• Only 256 and 512 are available for Scan Size.

• When Scan Area rotation has been set, turning on the [Fast] button resets the rotation angle.

• The Fast Galvano mode cannot be used together with the Spectral Detector mode (SD), Virtual Filter mode (VF), IR VF or IR SD. (If the detector is changed to [SD], [VF], [IR SD] or [IR VF] while the Fast Galvano mode is turned on, the Fast Galvano mode is automatically turned off.)

• When Piezo setting has been made, turning on the [Fast] button resets the Piezo setting.

Table 11.1-1 Details of Fast Galvano mode

Available Scan Size 256 512

Unidirectional 9 fps 5 fps Scan Speed

Bidirectional 19 fps 10 fps

Available Detector DU4/VAAS/IR DU4/IR NDD

Scan Zoom x8 to x1000

Scan area rotate function Unavailable

CROP scan area Unavailable

ROI scan area Unavailable

Free line scan Unavailable

AUX Mode Unavailable

CLEM Mode Unavailable

Use of Piezo Unavailable

Photo activation experiment Unavailable

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