Olympus FluoviewFV500 Training

Welcome to...

Presented by Leeds Precision Instruments, Inc.and Cancer Center Confocal Core

Before Using the Confocal

• Fill in access form with user information, proof of

Hazardous Chemical Waste training and ID card

number.

• Turn in to the confocal administrator (Margaret, 470

CCRB, 322 CR or MMC 806).

• Read manual and policy documents. Handbook soon.

• Complete advanced training course given by Angela

Mortari or otherwise demonstrate familiarity with the

FV500 system. You Are Here

The goals of a shared instrument grant are to provideaccess to state-of-the-art technology that will facilitateinteraction and enhance scientific productivity.

A confocal microscope typifies that technology, whichbecause of cost is usually beyond the scope of a singlelaboratory to either purchase or maintain.

NCRR SHARED INSTRUMENTATION GRANTNCRR SHARED INSTRUMENTATION GRANT(#1 S10 RR16851)(#1 S10 RR16851)

UNIVERSITY OF MINNESOTA

BONE TUMOR BIOLOGY LABORATORY

DEPARTMENT OF ORTHOPAEDIC SURGERY

AND NCI DESIGNATED COMPREHENSIVE

CANCER CENTER

Room 506 CCRB

System Components:1. BX61 Upright Motorized Research Microscope2. FV500 Laser Scan Head3. Laser Combiner and Argon, Gr HeNe and Red HeNe

lasers4. Fiber Optic Delivery System5. Transmitted Light Detector6. BX61 Handswitch, Prior Motorized Stage Control

Joystick, Transmitted Light Source7. Microscope Control Unit8. Argon, Green HeNe and Red HeNe Laser Power

Supplies9. Prior Motorized Stage Controller10. Mercury Burner Power Supply11. FV500 Control Unit and Power Supply12. FV500 Computer13. Computer Monitor and Surge Protected Power Outlet

Principle and Advantages ofConfocal Imaging

Confocal microscopy offers severaladvantages over conventional opticalmicroscopy, including controllabledepth of field, the elimination of imagedegrading out-of-focus information,and the ability to collect serial opticalsections from thick specimens. The keyto the confocal approach is the use ofspatial filtering to eliminate out-of-focus light flare in specimens that arethicker than the plane of focus.

A confocal imaging system achieves out-of-focus rejection by twostrategies: a) by illuminating a single point of the specimen at anyone time with a focused beam, so that illumination intensity dropsoff rapidly above and below the plane of focus and b) by the use ofblocking a pinhole aperture in a conjugate focal plane to thespecimen so that light emmitted away from the piont in thespecimen being illuminated is blocked from reaching the detector.

FV550 Visible lasersArgon [10mW, 488 nm] yields good excitation for the following flurochromes:

• GFP – green fluorescent protein

• FITC – antibody labeling

• YOYO-1 – DNA

• Calcein – bone growth

Green Helium Neon (HeNe) [1mW, 543 nm] yields good excitation for thefollowing flurochromes:

• Propidium Iodide (PI) – DNA, RNA

• TRITC – antibody labeling

• Cy3 –antibody labeling

• Rhodamine-Phalloidin – actin fibers

Red HeNe [10mW, 633 nm] yields good excitation for the followingflurochromes:

• Cy5 –antibody labeling

• Alexa Fluor 633 –antibody labeling

DYE [excitation/emission] Laser DYE [excitation/emission] Laser

Ca++ Crimson [590/615] Red HeNe eGFP [488/509] ArgonCa++ Green-1 [506/531] Argon FITC [494/518] ArgonCa++ Green-2 [503/536] Argon Fluo-3 [506/526] ArgonCa++ Green-5N [506/532] Gr HeNe Fura Red [472/657] Red HeNeCa++ Orange [549/576] Gr HeNe MitoTracker [490/516] ArgonCa++ Orange-5N [___/580] Gr HeNe PI [536/617] Gr HeNeCy 3 [550/565] Gr HeNe Rhodamine-Phalliodin [542/565] Gr HeNeCy 3.5 [581/596] Gr HeNe SNARF-1 (pH !) [488/530] Red HeNeCy 5 [650/670] Red HeNe Texas Red [595/615] Red HeNeCy 5.5 [675/695] Red HeNe TRITC [496/520] Gr HeNeDil [549/565] Gr HeNe YFP [514/527] Gr HeNeDiO [484/501] ArgonNOT on list:

AlexaFluor-1 [632/647] Red HeNe Calcein [494/517] ArgonAPC [650/660] Red HeNe DS Red [558/583] Red HeNeAPC/Cy7 [650;755/767] Red HeNe YoYo-1 [491/509] Argon

Table Listing the Dyes in the <Available Dyes>List and Some Popular Dyes Not on the List

System Start-up [1]

Turn on the Surge Protected Power Outlet (Computer, monitor, FV500Control Unit & LG-PS2). Press the POWER button on the tower to turnon the computer.

System Start-up [2]

Turn on Laser Power Supplies ONLY as NEEDED (Argon, Green HeNe, RedHeNe) and “ON” for the Argon laser. The recommended warm-up is 10 min forthe Argon and 30 min for the Gr HeNe.

Red HeNe

Green HeNe

Argon

Turn on the Mercury Burner

System Start-up [3]Turn on the BX-UCB(Microscope Control Unit) Turn on the Prior Stage

Controller Power Supply

Turn the red lever on the nitrogen tank 90° counter clockwise to power the air table

Wait at least 2 min for the microscope systems to initializethen double-click the Fluoview Icon to launch the confocalprogram. Note: it takes about 2 minutes to launch.

Logon to the computer using USER NAME and PASSWORD

After clicking on the Fluoview Icon:

You will see the Main Fluoview Software Window:

DO NOT move objectives by hand on the microscope, as thiswill cause a software error

Using the Automated BX61 Microscope

Motorized Features of the Microscope:

1. Objective Nosepiece

2. Fluorescent Mirror Cube Turret

3. Fluorescent Light Shutter

4. Condenser Turret

5. Condenser Top Lens

6. Reflected Light Aperture

7. Reflected Light Field Diaphragm

Configuring the Microscope

The microscope and scan unit (FV500) canbe configured on the FLUOVIEW software.Use the following procedure to configurethe microscope:From the panel page tabs shown on thebottom right of the [acquire] panel, selectthe [settings] sub-panel.Select the<scope control> button at thebottom of the panel. The window as shownwill appear.

[Light Path] box

selects the light path <BI> is for directobservation, <LSM> is for scanning,<TV> not used.

[Mirror Unit] box

clicking the cube automatically switchesthe turret.

[Shutter] box

click to open (9:00) / close (12:00) theEPI shutter.

[Nosepiece] box

click to choose the objective.

[Top Lens] box

clicking the engages (centered icon) ordisengages (to side) the lens into thelight path.

[Condenser] box

selects the condenser used.

[Aperture Iris] box

changes the AS value.

Graphic User Interface (GUI)

Why the 20x objective was not here for 9 months

Escape the stage BEFOREBEFORE changing objectives

This software usespanel-type windows.Functions are executedby selecting the panelpage tab (at the edge ofthe panel) of thefunction to be executedand the panel will moveto the front.

The FLUOVIEWsoftware is organizedby two kinds of panels,the function panels(left, down side) anddisplay panel (big oneon right)

Using the FLUOVIEW Software

The panel structure of the software is set-up according to the tree below

Focusing on the Specimen –Combination with BX61

1. From the panel page tabs shown on the bottomright of the [acquire] panel, select the [settings]sub-panel.

2. Select the <BI> button in the [light path] groupbox. The <BI> button looks pushed in to indicatethat it is selected.

3. Push the cube button (3) of the hand switch (2) toengage the optimum cube for specimen dyd. Inthe cube display window (1), the selected cube isdisplayed.

4. Focus on the specimen by looking into theeyepiece.

Switching to scan mode:

Select the <LSM> button in the [light path]group box. The <LSM> button lookspushed in to indicate that it is selected.

NOTE: when scanning is started while the<BI> button is selected, the LSM light pathis selected automatically. It is switchedback to the visual observationautomatically when scanning completes.

Selecting the Dyeing Method

1. From the page tabs on the bottom right of the [Acquire] panel, select the [Dyes] sub-

panel.

2. Select the specimen dyeing method by

dragging [FITC] and [TRITC] in the [Available

Dyes] list box in the [Selected Dyes] group box to the field immediately above the list box.

3. Click the <Apply> button to apply the selected

dyeing method to the [Ch] group box

on the upper part of the [Acquire] panel.

Selecting the Dying Method

Setting the Channels

In the [Ch] group box, make sure that the check boxes showing the applicable dyeing methods are check -marked to

indicate that the channels are ready for image acquisition. If the check box of a channel is not check -marked, check it to

make the channel ready.

Setting the Channels

Setting the Scan Speed

Set the scan speed by using the scale in the [Scan Speed] group box in the [Acquire] panel

Setting the XY Observation Mode

1. In the [Mode] group box in the [Acquire] panel, select

the [Surface] option button. 2. In the [Mode] group box in the [Acquire] panel, select

[800 by 600] from the [Size] drop-down list.

3. In the [Acquire] panel, select the XY observation

mode option button.

Select the <XY Repeat> button. The acquired image

will be displayed in the [Live] panel.

Setting the Scan Speed

Setting the XY Observation Mode

Setting the Cross-section to be Observed

While acquiring image, move the Z stage to select the cross-section to be observed.

1. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Z Stage] sub -panel

2. Check the [Locked] check box in the [Z Stage] sub-panel. Do not turn the fine focus adjustment knob while the [Locked] check box is checked, for this may damage the Z motor. 3. While observing the image in the [Live] panel, locate the plane to be observed by displacing the stage using the <Z stage coarse adjustment> and <Z stage fine adjustment> buttons in the [Z Stage] sub-panel.

Setting the Cross-section to be Observed

Stopping Repeated Scanning

After the brightness and gain have been adjusted, select the <STOP SCAN> button in the [Acquire] panel to stop

scanning temporarily

Acquiring Image

Select the <Once> but ton. The acquired image will be

displayed in the [Live] panel.

Stopping Repeated Scanning

Acquiring Image

Saving Image

1. Display the [File I/O] panel.

2. When saving images acquired

with more than one channel, it is possible to select whether images

from more than one image are saved simultaneously or only one

of the images is saved. Use the <Display channel switch>

buttons to select the images to be

saved. The selected images will be saved

under the conditions set for each channel.

Example)When only the image of Channel 1 is displayed, only the

image of Channel 1 will be saved.

3. Click <Experiment> button in

the [Save] group box. The [Save

Experiment As] dialog box will open.

Saving Image [1]

4. Enter the file name in the [File name:] text box.

5. Select “FLUOVIEW MultiTif” from [Save as type:]

6. Click the <Save> button

Saving Image [2]

Clicking the <Link Setting> button displays the [Link

Setting] dialog box as shown below.

Saving, Opening and Shredding Images

Use the [File I/O] panel to save, open or shred an image.

Display the [File I/O] panel at the front

Saving, Opening and Shredding Images

Shredding Images

Shredding an image refers to removing it from the objects of processing including display. Shredding does not

actually deletes the image saved in the disk .

1. Click the <Experiment List> button in the toolbar at the bottom of the [File I /O] panel. The [Experiments in Memory]

dialog box appears as shown below.

2. In the [Experiments in Memory] dialog box, select the file

name of the image to be shredded and click the <Shredder> button. The file can also be shredded by

placing the mouse pointer on it and dragging it to the <Shredder> button. The mouse pointer tr ansforms to an

image icon during dragging.

3. Click the <Done> button in the [Experiments in Memory] dialog box to close it.

Multiple images displayed can be shredded simultaneously.

Shredding Images

Displaying an Image in Simulated Colors

1. Display the [Display] panel of the image to be colored at

the front. 2. Click the <LUT> button in the toolbar at the bottom left of

the screen. The [Color Tool] dialog box appears.

3. When the image was acquired from more

than one channel, select the channels to

be colored using the [Ch1], [Ch2], [Ch3] option buttons. (The [Ch1], [Ch2] and [Ch3] option

buttons are displayed only when an image

acquired from more than one channel mode is displayed (selected).

4. From the [Standard Color LUTs] group box, select the desired color button. The

selected LUT will be applied immediately to the

image in the [Display] panel.

5. Click the <OK> button.

Displaying an Image in Simulated Colors

Merging Image Channels

For instance, when a specimen is imaged with two scans

for 2-channel fluorescence observation and 1 -channel transmitted light observation, the transmitted light image

can be merged to the fluorescence images to create a 3 -channel image.

Merging Image Channels

Extracting Channels from Image Desired channels can be extracted from an image. Use this facility to extract only the images of required channels from

an image acquired from more than one channel

Extracting Channels from Images

Image Processing

Images can be processed using the [Process] panel. Display the [Process] panel at the front.

Filtering

Use the [Filters] sub -panel in the [Process] panel to apply filtering to images.

Image Processing

Filtering

Image AnnotationImages can be annotated by clicking the Annotationbutton in the toolbar

Image Annotation

Image Analysis

Images can be analyzed using the [Analyze] panel. Display the [Analyze] panel at the front.

Image Analysis

Transferring Analysis Data to Another Application Analysis data can be transferred to Excel. 1. Display the [Analyze] panel and executes analysis. After it, display the [Enhanced Profile Plot], [Intensity Map], [Enhanced Histogram Plot], {Average Intensity Trace] or [Integrated Intensity Trace].

Transfering Analysis Data to Another Application [1]

2. Click the <Save> button. When the [Save As] dialog box

appears as shown below, set the file name and click the <OK> button to save the analysis data.

Transfering Analysis Data to Another Application [2]

Viewing 3D Image Use the [Visualize] panel to view an image three -dimensionally.

Viewing 3D Image

System Shut-down [1]

• Clean objectives of any residual oil using lens paper

• Return microscope stage to non-escaped position(UP)

• Return objectives to 4x• Exit from the FV program. This may take awhile,

however if you turn off the microscope before exiting,a system error will occur

• Transfer your files to removable media. Only 1.5 GBof storage is allowed

• Turn off the Prior Stage Controller Power Supply• Turn off the BX-UCB (Microscope Control Unit)• Turn off the Mercury Burner.

• Turn off Laser Power Supplies

• Select <shut down> on the computer and select LOGOFF from the drop-down menu. The computer will logyour name out and return to the login menu box.

• Select the shut down button to shut down thecomputer. Note: power will go off.

• Turn off the Surge Protected Power Outlet. Do not turnthe monitor, FV 500 Control Unit or LG-PS2 off.

• Turn red lever on tank 90° clockwise to stop flow of gasto table.

• Sign out on the log sheet.• Report any problems.

System Shut-down [2]

On-line Reservations for Confocal Microscope [1]

Sign up using the on-line reservation schedule at:

<www.cancer.umn.edu/cgi-bin/confocal/calweb.cgi>

Reservations are on a first-come, first-serve basis.

If you must cancel, do so in advance.

Log in with ID and password, both case sensitive.The same ones are used for log-in to the FV500.

On-line Reservations for Confocal Microscope [2]

Select date and approximate time by clicking near time

On-line Reservations for Confocal Microscope [3]

Fill-in user information. NOTE: the USER ID will fill in the PIfield and appear on the weekly calendar in the appropriate time slot

Huh?!What?!

Hands-on session tofollow...