3d surface inspector version 7.0 for windows user guide · 3d surface inspector™ version 7.0 for...

3D Surface Inspector™

Version 7.0 for Windows

User Guide

Media Cybernetics, Inc. 4340 East-West Highway, Ste 400 Bethesda, MD 20814 (301) 495-3305, FAX (301) 495-5964 http://www.mediacy.com MAN 29N7000 20100108

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Contents

v

Contents 3D Surface Inspector Introduction & Installation .................................................... 1-1

Introduction ............................................................................................... 1-1 What’s New in Version 7.0 ....................................................................... 1-1 System Requirements ................................................................................ 1-2

Before You Begin ........................................................................ 1-3 Installation ................................................................................................. 1-4 Registration and Technical Support ......................................................... 1-10

Register as a User ...................................................................... 1-10 Technical Support On Line ....................................................... 1-10 Image-Pro on the Web ............................................................... 1-10 Contacting Technical Support ................................................... 1-11

Working with 3D Surface Inspector ........................................................................ 2-1 Capturing Input Images for 3D Surface Inspector ..................................... 2-1 Some Notes on Capturing Images from Stereo Microscopes .................... 2-4 Acquiring Images Using the Prior Scientific Z Stage. ............................... 2-6

The 3DSI Manual Acquire tab .................................................. 2-11 The 3DSI Auto Acquire tab....................................................... 2-13 The 3D SI Focus Settings tab .................................................... 2-16 The 3D SI Focus Settings tab .................................................... 2-16 The 3DSI Measurement tab ....................................................... 2-18

Manual Acquire and Focus ...................................................................... 2-21 The Generate Montage and Topo. Map Option ........................ 2-24

Processing Image Data and Calculating 3D Information ........................ 2-25 Creating a 3D Display ............................................................................. 2-31

Functions of the 3D Surface Inspector View Window .............. 2-33 Analyzing Surface Area and Volume of Objects in the Image . 2-39

Performing Cross-Sectional Measurements ............................................. 2-41

Contents

vi

Introduction

1-1

3D Surface Inspector Introduction & Installation

Introduction 3D Surface Inspector is a plug-in module for Image-Pro Plus/Image-Pro Express which adds the following features:

Create an Extended Depth of Field (EDF) image based on a Z-stack where profiles in the X and Y directions are displayed.

Measure the surface area and volume of each separate feature in the image.

View the features of the image in 3D using the surface plot tool.

Rotate and tilt the 3D display in any direction.

What’s New in Version 7.0 Supports Prior's Z axis motorized stage to capture a Z image stack

directly

Manual focused image synthesis

3D Profile function implementation Number of passes options for smoothing added.

Improved 3D output

Ability to create *.AVI movie files from Surface Plot output

3D Surface Inspector

1-2

System Requirements Before installation, please be sure that your system meets the following requirements:

Pentium IV CPU, running at 2.0 Ghz or higher configured with Windows™ XP Professional (service pack 2), or Windows Vista™.

A minimum of 1 GB of RAM recommended

60 GB disk drive with available disk space for installation with additional space to accommodate four times the image size with four bytes/pixel

An SVGA (or better) high color, 16-bit resolution graphics card with DirectX 9.0c or above graphics support, a hardware accelerator, and a minimum of 32Mb memory that supports 2D texturing

A Windows-compatible mouse, keyboard, and CD-ROM.

Media Cybernetics' Image-Pro Plus v. 7.0 or Image-Pro Express 6.3

The following equipment/software is optional, however it is strongly recommended for capturing images for 3D Surface Inspector: A microscope equipped with a camera supported by Image-Pro

Plus/Image-Pro Express and a Prior motorized Z stage (you can also use a stereo microscope or a video microscope for capturing input images for 3D Surface Inspector)

A motorized microscope with focus drive (Nikon, Olympus, Leica, Zeiss), equipped with a camera supported by Image-Pro Plus/ Image-Pro Express, with Media Cybernetics' Scope-Pro ver. 6.0 or above (for controlling the camera and Z stage)

System Requirements

1-3

Before You Begin Before you install 3D Surface Inspector on your computer, please ensure that: Image-Pro Plus 7.0 or Image-Pro Express 6.3 is installed on your

PC.

OS: Microsoft WindowsTM

2 XP Professional with Direct X9.0c or later is installed on your PC

Note: About DirectX 9.0c or above

To use 3D Surface Inspector ver. 7.0, Microsoft DirectX9c or above is required.

To find out if your PC has DirectX 9.0c or above installed, click the "Run" command in the "Start" menu. Enter "dxdiag" in the "Name" field and click "OK" to display the "DirectX Diagnostic Tool".

Use this tool to check if your version of DirectX is 9.0c or above.

If the version installed is not 9.0c or above, connect your PC to the Internet, and run "Windows Update" from the "Start" menu to download and install DirectX version 9.0c or above.


1-4

Installation To install 3D Surface Inspector on your computer, please follow the steps below:

STEP PROCEDURE

1. Attach the copy protection plug to an operable parallel (LPT1, LPT2). or USB port. Without this plug, 3D Surface Inspector will only operate in demo mode. More information about the plug appears in the following section.

2. Place the Image-Pro Plus product CD in your CD-ROM drive. Within a few seconds, the Setup program will start running automatically. The Selection screen will appear. Indicate that you are installing 3D Surface Inspector.

3. If the Setup program does not start automatically, click the Start button on your Windows desktop, and choose Run. Use the Browse button to find the your 3D Surface Inspector CD. Select the Start program. The path is: CD Drive letter:\start.exe

4. After clicking Install 3D Surface Inspector on the Autostart dialog, 3D Surface Inspector will ask you to wait for a few minutes to prepare the Install program.

Note that this may take some time, depending on your operating system.

Installation

1-5

STEP PROCEDURE 5. The installer will ask you which language you wish to use.

Select your language from the drop-down list and click OK.

6. You will see the 3D Surface Inspector Welcome screen. After reading it, click Next to continue installing 3D Surface Inspector.

7. The next screen asks if you want to install, modify, or remove the selected program. This screen will not appear if you are installing 3D Surface Inspector for the first time. Select one of the options, and click Next.

8. Choose the application for 3D Surface Inspector. If more than one imaging application is installed on your system, you will be given a choice of which application is the destination. Click the appropriate radio button, and then click Next to continue the installation process.


1-6

STEP PROCEDURE 9. If you have chosen to install or modify 3D Surface Inspector, you

will see the 3D Surface Inspector license agreement.

Click Yes to continue the installation process.

10. Choose the location for the 3D Surface Inspector files:

The default destination for 3D Surface Inspector is the C:\ drive in the directory IPWIN7.

Installation

1-7

STEP PROCEDURE 11. If you want to install 3D Surface Inspector in another folder, use

the Browse button to select a folder. Highlight the name of the folder and click OK. Click Next to continue installing 3D Surface Inspector.

12. Indicate which Image-Pro Plus menus should display the 3D

Surface Inspector:

Check the correct box, or click Select All. Then click Next to continue.


1-8

STEP PROCEDURE 13. Follow the instructions on the 3D Surface Inspector setup screen.

The Progress Indicator shows that the 3D Surface Inspector files are being loaded onto your computer.

14. When the installation process is complete, you will see the final screen. Click Finish.

Installation

1-9

Note: If an error occurs while invoking 3D Surface Inspector from Image-Pro Plus, you should first try switching to a 16-bit true color display mode using the Display Properties dialog.

If you get this error while in a 16-bit display mode, it may indicate that the graphics adapter does not meet the minimum system requirements for 3D Surface Inspector. This error is unlikely to occur on more recent computer systems.

Once 3D Surface Inspector is installed on your computer, a new menu item, 3D Surface Inspector, will appear on the Process menu of the Image-Pro Plus menu bar.

The first time that you select 3D Surface Inspector from the Advanced menu, you will be asked for your authorization code:

Click the top radio button and then click Continue to enter your authorization code, as shown here:

Click the Authorize button. 3D Surface Inspector should start automatically.

If your authorization code does not work, click the Contact Info button for the information about contacting Media Cybernetics for the correct information.


1-10

Registration and Technical Support

Register as a User Please complete and return the enclosed Warranty and Product Registration cards. As a registered user, you are entitled to customer support services during the warranty period. You will also receive advance information about other Media Cybernetics products and updates to the 3D Surface Inspector system.

You can also register your product electronically. Visit our website: http://www.mediacy.com. Then choose Support. Click on Register Product to see our online registration form.

Technical Support On Line Please refer to the Technical Support page, (accessible from the Media Cybernetics’ home page on the World Wide Web at http://www.mediacy.com) where valuable technical information is available to you. This information includes:

Answers to frequently asked questions

A collection of technical notes, including ‘How To...’ tips, sample macros, and more

Technical information on Media Cybernetics and third-party video acquisition hardware and software

A Technical Support E-mail form.

Image-Pro on the Web Media Cybernetics now has a presence on YouTube: http://www.youtube.com/user/MediaCybernetics We have a new Facebook Group for Image-Pro Plus Users. We plan to add Events, Recent News & Videos to this group. Join us! If you don't have a Facebook account, you may not be able to see this group.

Image-Pro Plus Users' Facebook Group: http://www.facebook.com/home.php?#/group.php?gid=44536513482

http://www.mediacy.com/�

http://www.mediacy.com/�

https://owa.piacton.com/owa/redir.aspx?C=efbddc249bc34c60aeb716639c343b99&URL=http%3a%2f%2fwww.youtube.com%2fuser%2fMediaCybernetics�

https://owa.piacton.com/owa/redir.aspx?C=efbddc249bc34c60aeb716639c343b99&URL=http%3a%2f%2fwww.facebook.com%2fhome.php%3f%23%2fgroup.php%3fgid%3d44536513482�

Registration and Technical Support

1-11

Contacting Technical Support Before you contact Media Cybernetics’ Technical Support for further assistance, please be prepared to provide the following information:

Your registration information: the product serial number (*), your name, phone number, organization name, mailing address, and e-mail address.

The software version number (*) and system information.

A description of the problem, and any relevant information regarding prior technical support assistance.

(*) These numbers are displayed in the ‘About’ box:

You may contact Media Cybernetics’ Technical Support in either one of the following ways:

To speak with a technical support representative, please call 301-495-3305, and ask for Technical Support. The Technical Support line operates on regular business days, between 9:00 a.m. and 5:00 p.m. EST.

To use e-mail, you may fill out the e-mail form available from the Technical Support page on the World Wide Web (see above); or e-mail your message directly to: mailto:[email protected].

To send a fax, address your fax to 3D Surface Inspector Technical Support, and send it to 301-495-5964.

Note: Technical support is available only to registered users, during the basic or extended warranty period. Please remember to return your registration card.

mailto:[email protected]�

Capturing Input Images for 3D Surface Inspector

2-1

Working with 3D Surface Inspector


3D Surface Inspector requires a set of sequential images (individual consecutive images or the frames in a sequence file, .SEQ) which have been captured while moving the microscope stage in the Z direction in equal steps. The amount the stage is moved in one step is called delta Z.

Camera Focus planes (Z positions) Stage

Delta Z


2-2

When capturing images to be processed by 3D Surface Inspector, please note the following points:

Please select your lens magnification so that each captured image has both in-focus and out-of-focus areas. Images which are completely out- of- focus and do not have any in-focus portions, or low magnification images with a long focus depth which have no out-of-focus portions, cannot be processed properly by 3D Surface Inspector.

To decrease focus depth, open the aperture of the camera fully (if applicable).

To prevent the pixels in the image from overflowing, reduce the lighting so that the saturated portions (which for example have the pixel value 255 in 8-bit images) in the image do not exceed 1% of the total image area.

To monitor the changing pixel values in the image on a real-time basis, you can use the Workspace Preview capture mode in Image-Pro Plus. If your camera has the gamma correction feature, please use it to enhance dark areas in the image.

Please capture the images in equal steps while changing the focus position. When capturing or saving the images, please ensure that the image files are given sequential numbers (for example, "Image001.tif", "Image002.tif", "Image003.tif", etc.).


2-3

You can also use the Acquire:Sequence Tools :Merge Images command in Image-Pro Plus to create an image sequence from your images, and save it as a sequence (.SEQ) file.

If you are working with a motorized Z stage and Scope-Pro, please be sure to check the Acquire multiple Z planes and Acquire as Sequence options on the Acquire tab of Scope-Pro before capturing images. For details, please refer to the Scope-Pro Users Guide.

NOTES: If the sample object has polished (mirrored) surfaces, sometimes it will be difficult for 3D Surface Inspector to find in-focus portions in the image (For example, this will be the case if you are capturing printed circuit boards.) In such a case, capture the polished surfaces at the lowest Z position of the stage.

To obtain calibrated surface area and volume values, use Image-Pro's Spatial Calibration tool to apply a “pixels per unit” calibration to the input images. Always use mm (millimeters) as the calibration unit.


2-4

Some Notes on Capturing Images from Stereo Microscopes

Due to some limitations in their optic systems, many stereo microscopes generate multi-focus images in which the sample objects appear to drift slightly from frame to frame.

3D Surface Inspector has an option to correct this: Checking the Pre-Align Images option enables 3D Surface Inspector to measure the amount of drift in the sample objects on each frame, which is then taken into account when an X-Y image (EDF image) and a Z image (topographic map image) are generated.

However, following limitations should be considered:

The maximum allowable amount of drift for an object between two adjacent frames is 2.5% of the image size. For example, if you capture 10 images while changing the Z position of the stage, the amount of drift between an object on the first frame and the same object on the last frame must be less than 25% (= 2.5% X 10 frames) of the image size (image width and height, respectively). If the frames have been captured using an analog camera (NTSC, 640 X 480 pixels), 2.5% of the image width will correspond to 16 pixels (=0.025 X 640), and 2.5% of the image height to 12 pixels (=0.025 X 480).

Object on frame 1 on frame 2 on frame 3...

Less than 2.5% of image size

The drift of the objects can only be compensated if their movement is uni-directional. If the objects move in a zigzag fashion, or back and forth, etc., 3D Surface Inspector will not be able to correct their positions.

The resulting images may be blurred or contain improper 3D information near the ends (seen along the direction of the drift of the objects). On the resulting images (X-Y image and Z image), the sample objects appear at the same size and position as they do on the frame in

Some Notes on Capturing Images from Stereo Microscopes

2-5

the middle of the image stack. However, since the objects drift from frame to frame, they may appear blurred or in improper orientation near the edges of the image. These areas have been generated from less image data than the middle of the image. For example, if the sample objects drift by 25% of the image size over the range of 10 frames in the horizontal direction, only 50% of the total image area, located in the middle of the image, will be calculated from all the data throughout the stack, while the remaining 50%, near the right and left edges of the image, will only be generated from some of the frames. In this case, areas near the right and left edges may contain noise or inaccurate 3D information.

Important Note: Do not use a spotlight! 3D Surface Inspector cannot detect in-focus areas in very bright spots or in dark shadows.


2-6

Acquiring Images Using the Prior Scientific Z Stage.

To acquire images using the motorized Z Stage from Prior Scientific, Inc., follow the procedures below:

1. Turn on the stage controller's power switch.

2. Launch Image-Pro Plus and check that the live image displays and images from the camera can be acquired

3. Run the 3D Surface Inspector command from the Process menu to launch 3D Surface Inspector.

4. When the Three Dimensional Observation dialog opens, click the Auto Acquire tab to display it at the front.

The following steps below are required when the Prior motorized Z Stage is used for the first time. These procedures are not necessary for subsequent use.


2-7

5. Click the Configure button at the upper right to open the PRIOR Settings dialog.

6. In the Communication port No. [COM] field, select the serial port (COM port) connected to the stage controller using RS232C and click OK. (Select 1 if the cable is connected to the PC's COM1 port, or 2 if it is connected to COM2.)

7. Other options are configured as required.

Resolution [μm]: Specify the resolution for the Z focus step motor. For effective values that can be set, refer to the stage manual.

Delta Z per Revolution [μm]: Specify the travel distance of the stage going up or down every time the focus knob rotates once.

Reverse rotation direction: Selecting this option reverses the Z focus motor's rotation direction. Select this option when the direction of the stage's up-and-down movement is the opposite of the defined direction.

Note: If a Device connection error is displayed in the Status field of the Acquire tab, first check the controller's power source and connection. Then click the Configure button (shown above), and click OK in the Configure dialog. If Paused is displayed in the Status field, it is running correctly.

8. Place a sample in the microscope.

9. (Optional) Click the Set Z Origin button on the Acquire tab to set the current height (Z position) as the stage's Z origin.


2-8

Important: Make sure that the stage has completely stopped before clicking the Set Z Origin button. Clicking while the stage is still operating could lead to recording with an incorrect position as the origin.

10. (Optional) In the Z Step Size [μm] field of the Acquire tab, specify the travel distance per step (the distance traveled every time the Up or Down button is clicked once) in μm units.

11. (Optional) In the Z Travel Speed field of the Acquire tab, specify

the travel speed.

12. At this time, run the Video/Digital command in the Acquire menu of 3D Surface Inspector Plus to open the Acquire Image dialog,

Set the current Z position as the origin

Move the stage up or down

Specify the travel distance per step

Move the stage up or down

Specify the travel speed


2-9

and click the Start Preview button to display the live preview image from the microscope camera.

13. While you watch the live preview, move the stage to the position so that the uppermost part of the object is in focus, using either the joystick or the Up and Down buttons in the Acquire tab. Wait until the stage has stopped, then click the Set Z Upper Limit button (shown below).

ライブプレビュー画面を見ながら、ステージを徐々に移動します対象物の最上部にピントを合わせて、「現在の位置を上限位置とする」ボタンをクリックします最下部にピントを合わせて、「現在の位置を下限位置とする」ボタンをクリックします

14. Using either the joystick or the Down button, move the stage to the position so that the lowermost part of the object is in focus. Wait till the stage has stopped, then click the Set Z Lower Limit button.

While watching the live preview screen, move the stage gradually

Focus on the uppermost part of the object, then click the "Set Z Upper Limit" button

Focus on the lowermost part of the object, then click the "Set Z Lower Limit" button

Specify the number of images to acquire

Specify the upper limit and lower limit positions for acquiring

Delta Z is displayed

Start the Acquire process


2-10

15. In the Number of Images field, specify the number of images to acquire (number of focal planes).

The delta Z value is determined by the above operation, and the value is displayed in μm units in the Delta Z [μm] field.

16. In the Direction field, select one of the following options.

Bottom Up: Acquires from the lower limit toward the upper limit.

Top Down: Acquires from the upper limit toward the lower limit.

Click the Acquire button to start the acquisition process.

17. (Optional) To measure the surface area and volume of the object in the image, and have the measurement results displayed in actual values, spatial calibration must be performed on the image just acquired.

Important: The unit of calibration must be "mm" at all times.


2-11

The 3DSI Manual Acquire tab The Manual Acquire tab allows you to set the parameters to control the acquisition of Z-stack images

Initial Position: Use the spin buttons to indicate the starting position of your microscope in micrometers.

Z Spacing (um): Enter the Z spacing (in micrometers) between the images you will capture, or use the spin buttons to adjust the spacing.

Direction: Click the radio button to indicate if the Z acquisition is going from the top of the stack down or from the bottom up.

Generate Montage and Topo Map: Check this box if you want to use the Montage and Topographic Map feature.

If the Generate Montage and Topo. Map option is selected before you click the Acquire button, focusing starts as soon as the acquisition process is completed. In addition, the value displayed in the Delta Z [μm] field of the Acquire tab automatically goes into the ΔZ field of the Focus Settings tab to be processed. This feature is convenient when you measure the surface area and volume of the object in the image after focusing.

Important:When you intend to use theGenerate Montage and Topo. Map option, configure theTopographicOptionssettings in theFocus Settingstab in advance, as shown below:


2-12

Step-by-Step Capture: This option allows you to move the microscope by rotating the focus knob and then clicking Acquire to capture each image.

Continuously Capture: To use this option, enter the number of images to aquire, and the range between each step. Then click Acquire.


2-13

The 3DSI Auto Acquire tab Auto Acquire lets you set up the Z stage parameters for automated acquisition, as shown here:

Z Stage: This group indicates the current position of the Z stage.

Status: This message box indicates if your stage is properly connected, or not.

Configure: Click this button to see the stage configuration dialog. In this example, we see the settings for a Prior stage:


2-14

• In the Communication port No. [COM] field, select the serial port (COM port) connected to the stage controller using RS232C and click OK. (Select 1 if the cable is connected to the PC's COM1 port, or 2 if it is connected to COM2.)

• Resolution [μm]: Specify the resolution for the Z focus step motor. For effective values that can be set, refer to the stage manual.

• Z Spacing per Revolution [μm]: Specify the travel distance of the stage going up or down every time the focus knob rotates once.

• Reverse rotation direction: Selecting this option reverses the Z focus motor's rotation direction. Select this option when the direction of the stage's up-and-down movement is the opposite of the defined direction.

Note: If a Device connection error is displayed in the Status field of the Acquire tab, first check the controller's power source and connection. Then click the Configure button (shown above), and click OK in the Configure dialog. If Paused is displayed in the Status field, it is running correctly.

Set Z Origins: (Optional) Click the Set Z Origin button on the to set the current height (Z position) as the stage's Z origin.

Z Travel Speed: (Optional) specify the travel speed.

Set Upper/Lower Z Limits: Follow these instructions to set the Upper and Lower Z limits.

• Run the Video/Digital command in the Acquire menu of Image-Pro Plus to open the Acquire Image dialog, and click the Start Preview button to display the live preview image from the microscope camera.

• While you watch the live preview, move the stage to the position so that the uppermost part of the object is in focus, using either the joystick or the Up and Down buttons in the Acquire tab. Wait until the stage has stopped, then click the Set Z Upper Limit button (shown on the next page).

• Using either the joystick or the Down button, move the stage to the position so that the lowermost part of the object is in focus. Wait till the stage has stopped, then click the Set Z Lower Limit button.


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Number of Images: Specify the number of images to acquire (number of focal planes).

Z Spacing: This field displays the Z spacing determined by the Upper/Lower Limit settings.

Direction: In the Direction field, select one of the following options.

Bottom Up: Acquires from the lower limit toward the upper limit.

Top Down: Acquires from the upper limit toward the lower limit.

Acquire: Click the Acquire button to start the acquisition process.

(Optional) To measure the surface area and volume of the object in the image, and have the measurement results displayed in actual values, spatial calibration must be performed on the image just acquired.

Important: The unit of calibration must be "mm" at all times.


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The 3D SI Focus Settings tab After capturing the images, use the Focus Settings tab to process them and to create an X-Y image (EDF image) and a Z image (topographic map image). 3D Surface Inspector needs these images to display objects in 3D or to measure their surface area and volume. The Focus Settings tab is shown here:

Available Images: This list shows the images that have been acquired and are available for processing.

Selected Images: This list displays the images that you will use to create the X-Y and Z images.

Add: To add a group of images to the Selected Images list, hold down the <Ctrl> key or the <Shift> key to select each image, then click the Add button to add them into the Selected Images window.

Add All: To add all the available images into the Selected Images window, simply click the Add All button.

Note: If a sequence file (.SEQ) is opened in Image-Pro, just select it in the Available Images window and click Add to add it into the Selected Images window, or double-click on the list of all available image files. In this case, all the frames contained in the sequence file will be used as input images for 3D Surface Inspector.

Remove: To unselect a file, select it in the Selected Images window and click Remove.

Clear: To unselect all files at once, use the Clear button.


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Move Up/Move Down: Confirm that the images in the Selected Images window are displayed in the correct order. To change the order of the files, select a file in the Selected Images window and use the Move Up or Move Down buttons.

Stack Order: In the Stack Order box, select Bottom Up if the first image in the Selected Images window represents the focus plane at the lowest Z position, or Top Down if it represents the plane at the highest Z position.

Z Spacing: Enter the Z spacing (in micrometers) between the images you will capture.

Sensitivity: Specify a value between 1 and 10 for proper results.

Smoothing: Specify the grade of smoothing between 0 and 29. The higher the value, the smoother the 3D object surfaces in the image will be.

To increase smoothing, set the value in the Passes box. The numerical value in this field specifies the number of times the filter is applied to an image to remove noise. If there is a lot of noise, increase the numerical value in this field. The values that can be specified are from 1 to 9.

Out of Focus Correction : Check this option if you captured sample objects with polished or mirroring surfaces or holes which make it difficult for 3D Surface Inspector to find out in-focus portions in the image.

Stereo M. S: If the images have been captured using a stereo microscope, check the Stereo M. S. option.

High Magnification Mode: Check this box if the images have been captured using a high magnfication.

Create: Click the Create button to start processing.

Once the input images have been processed, the results will appear in Image-Pro's workspace.


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The 3DSI Measurement tab Once a pair of the focused focal point and topographic map images have been generated, the object in the image can be displayed in 3D and measurements made.

Image Pair to be measured: An X-Y and Z pair of images will appear in this list.

Add Pair: Click this button to select a pair of images from the list or available processed images.

Release Pair: Click this button to remove a pair of images from the list.

Surface Plot: Click this button to display the Surface Plot of the pair of images. The 3D Surface Inspector View window will open, and the topographic map image will be displayed in 3D:


2-19

The 3D image can be tilted or rotated freely. The window size can be made larger or smaller by dragging a corner of the 3D Surface Inspector View window. More information about the Surface Plot appears in the following pages.

Show Marker: With the Show Marker option on the Measurement tab checked, place the cursor within the X-Y image to display a cross-hair cursor with an X and Y line profiles. By looking at these profiles you can check the 3D shapes in the image.

Cursor Coordinate: The current position of the cursor is displayed in the Cursor Coordinate box

Height: The corresponding height (Z) information appears in the X Axis and Y axis boxes.

Show 3D Profile: Click this box to display the image in 3D.

The Measurement group box contains the following options:

Fill area: Check this box to determine the area to be measured. To cancel the measurement mode, uncheck the Fill Area option.

Move the slider to determine the Z plane above which the objects are to be measured.


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You can measure the objects in the image one by one, as far as they are separated from each other on the bottom surface which has been selected using the slider.

The slider scale corresponds to the entire range of the Z positions divided into 100 equal parts, where the lowest Z position corresponds to 0 and the highest Z position to 100. As the slider is moved, the corresponding Z plane area is displayed in red. (The current Z position is displayed in the "Bottom Position" box.) The Z plane which has been specified by moving the slider represents the bottom surface of the objects to be measured, i.e. their surface area and volume are measured between this bottom surface and their respective tops.

Click on one of the objects to measure its surface area and volume. Once clicked, the object is displayed in blue, and the measurement results are shown in the Surface Area and Volume boxes.

Clear: To unselect a currently selected object, click the Clear button.

Manual Acquire and Focus

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Manual Acquire and Focus Using the Manual focus function enables focusing simply by turning the fine focus knob of the microscope slowly, although accuracy is compromised to a degree. Furthermore, the results processed with 3D Surface Inspector can be previewed before the Z stack is acquired using Scope-Pro. To perform the Manual focus, follow the procedures below:

Note 1: For performing the Manual focus, your PC should have a processing speed of 2 Ghz or more.

Note 2: Before you perform the Manual focus, check to see if the focus can be adjusted smoothly to view the live image in the workspace preview. High-resolution cameras which require a lot of time for image acquisition, and the high-definition mode in which one image is captured with several shots, are not suitable for the Manual focus. Make sure the Workspace Preview option is unchecked once the above operation has been checked.

Note 3: The Manual focus cannot be used when the position of the image shifts with the focus position change, as in a stereoscopic microscope.

1. After launching Image-Pro Plus, and checking that the camera's live image displays and images can be acquired, run the 3D Surface Inspector command in the Advanced menu .

2. Go to the Manual Acquire tab.


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3. In the Manual Acquire group, indicate the intial position of the microscope.

4. Indicate the Delta Z (amount of change to move the the focus).

5. Indicate the direction of movement (Up or Down)

6. To display the live image from the camera, while checking that the object for the focus will not touch the object lens, focus on the lowermost surface of the object. After it is done, turn off the camera's live image.

Note 4: To perform the Manual Focus and Acquire while the camera's live image is displayed, move the live image to the upper right hand of the 3D Surface Inspector workspace.

7. To use the Step-by-step Capture, rotate the focus knob and then click the Acquire button.

8. To use the Continuously Capture option, indicate the number of images and the range of movement.

9. Click the Acquire button. You will see the Capture Count Down dialog:

When the numerical value shown in the Capture Count Down reaches 0 sec, the Capture Count Down dialog disappears. The image acquisition starts; and the focus image is displayed at the upper left of the 3D Surface Inspector workspace.

10. Every time the numeral value of the Capture Count in the Manual focus dialog increases, turn the microscope's focus knob little by little, to maintain a specific rotation angle. Continue this operation until you reach the uppermost surface of the object for the focus.

11. As soon as you reach the uppermost surface, click the Pause button.

The focus results remain in the upper left of the Image-Pro workspace. When you place the cursor within this image, a

Manual Acquire and Focus

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cross-hair cursor will appear, and the cross-sectional profiles of Direction X and Direction Y will be displayed.

12. To check the 3D shape in the 3D Surface Inspector View window, stop the live image display, then click the 3D Surface Inspector View button. You will see the 3D Surface Inspector View window, as shown here:

Note 5: From the time when the Focus Start button is clicked until the Pause button is clicked, the 3D shape is output on the assumption that the focus surface (Z axis) moved from the lowest position to the highest position at a specific speed.

Note 6: If the PC's display screen is large enough, the 3D Surface Inspector View window can be displayed simultaneously during the Manual focus. To choose this option, check the Show 3D Surface Inspector View simultaneously option before performing the focus. Make sure consideration is given to the placement of the 3D Surface Inspector View window etc. in advance if this option is checked.

13. To end the Manual focus mode, click the Exit button.


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The Generate Montage and Topo. Map Option If the Generate Montage and Topo. Map option is selected before you click the Acquire button, focusing starts as soon as the acquisition process is completed. In addition, the value displayed in the Delta Z [μm] field of the Acquire tab automatically goes into the ΔZ field of the Focus Settings tab to be processed. This feature is convenient when you measure the surface area and volume of the object in the image after focusing.

Important: When you intend to use the Generate Montage and Topo. Map option, configure the Topographic Options settings in the Focus Settings tab in advance, as shown below:

Processing Image Data and Calculating 3D Information

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After capturing the images, perform the following steps to process them and to create an X-Y image (EDF image) and a Z image (topographic map image). 3D Surface Inspector needs these images to display objects in 3D or to measure their surface area and volume.

1. Open the saved images (individual sequential image files or a sequence file) in the Image-Pro Plus workspace.

2. Select 3D Surface Inspector from the Advanced menu to open the 3D Surface Inspector dialog box. Click the Focus Settings tab.


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3. All the images currently open in Image-Pro are listed in the Available Images window. Select the images to be processed from the Available Images list and add them into the Selected Images window.

NOTES: To add a group of images to the Selected Images list, hold

down the <Ctrl> key or the <Shift> key to select each image, then click the Add button to add them into the Selected Images window.

To add all the available images into the Selected Images window, simply click the Add All button.

If a sequence file (.SEQ) is opened in Image-Pro, just select it in the Available Images window and click Add to add it into the Selected Images window, or double-click on the list of all available image files.

In this case, all the frames contained in the sequence file will be used as input images for 3D Surface Inspector.

To unselect a file, select it in the Selected Images window and click Remove. To unselect all files at once, use the Clear button.

4. Confirm that the images in the Selected Images window are displayed in the correct order. To change the order of the files, select a file in the Selected Images window and use the Move Up or Move Down buttons.

5. In the Stack Order box, select Bottom Up if the first image in the Selected Images window represents the focus plane at the lowest Z position, or Top Down if it represents the plane at the highest Z position.

6. In the Topographic Options box, specify the delta Z value (in mm) in the "Delta Z" input box.

7. In the Sensitivity box, specify a value between 1 and 10 for proper results.


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NOTES: If the images have been captured using Scope-Pro, this value

will be automatically calculated based on the Z position information which has been stored in each image by Scope-Pro.

As the measurement unit of calibrated delta Z values, only mm (millimeters) are accepted. If your input images are calibrated in a unit other than mm, please re-calibrate them in mm.

If the input images are not calibrated, the unit of the delta Z value will be automatically set to pixel.

If your main purpose in using 3D Surface Inspector is creating surface plots (3D Surface Inspector View), we recommend that you set the delta Z value to 5 or more (However, in this case, the area surface and volume measurements would be inaccurate since this setting does not correspond to the real delta Z value).

You may want to start with 10 as a sensitivity value, and if you are not satisfied with the processing results, decrease the value by 1 or 2, and try again.

If decreasing the sensitivity value does not significantly improve the processing results, use Image-Pro's BCG tools to apply a gamma correction (within the range of 1 - 1.4) to all the input images, then process again.

8. In the Smoothing box, specify the grade of smoothing between 0 and 29. The higher the value, the smoother the 3D object surfaces in the image will be.

To increase smoothing, set the value in the Passes box. The numerical value in this field specifies the number of times the filter is applied to an image to remove noise. If there is a lot of noise, increase the numerical value in this field. The values that can be specified are from 1 to 9.

9. If you captured sample objects with polished or mirroring surfaces or holes which make it difficult for 3D Surface Inspector to find out in-focus portions in the image, check the Out of Focus Correction option. Otherwise, leave it unchecked.

10. If the images have been captured using a stereo microscope, check the Stereo M. S. option.

11. Click the Create button to start processing.


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Once the input images have been processed, two types of result images will appear in Image-Pro's workspace:

X-Y Image (EDF image): An all-in-focus image containing X and Y (2D) information. This image corresponds to Image-Pro's EDF (Extended Depth of Field) image.


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With the Show Marker option on the Measurement tab checked, place the cursor within the X-Y image to display a cross-hair cursor with an X and Y line profiles. By looking at these profiles you can check the 3D shapes in the image. The current position of the cursor is displayed in the Cursor Coordinate box, and the corresponding height (Z) information appears in the X Axis and Y axis boxes.

Z Image (topographic map image): A floating point image containing Z (height) information along with the X and Y information. This image corresponds to 3D Surface Inspector's EDF topographic map image.

XY Image Z Image


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Using the Z and X-Y images, 3D Surface Inspector can display the sample objects in the image in 3D (Surface Plot) and measure their surface area and volume.

Creating a 3D Display

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Creating a 3D Display Once a pair of the focused focal point and topographic map images have been generated, the object in the image can be displayed in 3D.

Follow the steps below:

1. Check that a pair of the focused focal point and topographic map images is displayed in the X-Y image (EDF image), and Z image (topographic map image) under the Measurement tab of the Three Dimensional Observation dialog box, and that their names are highlighted (shown below).

If the names of the images are not highlighted, select the names of the images by highlighting them.

Check that a pair of the EDF image and Topographic Map images are selected

Create the 3D display


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2. Click the Surface Plot button. The 3D Surface Inspector View window will open, and the topographic map image will be displayed in 3D:

The 3D image can be tilted or rotated freely. The window size can be made larger or smaller by dragging a corner of the "3D Surface Inspector View" window.


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Functions of the 3D Surface Inspector View Window Some of the functions of the 3D Surface Inspector View window include:

Tilt/Rotate the 3D Image: Drag the 3D image directly using the mouse.

Important: If you drag the outer edge of the 3D image with a somewhat bouncy motion, the 3D image will keep on rotating even when the mouse is stopped. To stop the rotation, click the 3D image.

Display the 3D Image with a Specific Tilt Angle: To specify the tilt angle of the 3D image, use the buttons in the View point box. The current tilt angles are displayed in the X=, Y=, and Z= fields between the range of -180 and +180° respectively.

• The X-Y/X-Z/Y-Z Buttons: Clicking these buttons will display the XY plane, XZ plane, and YZ plane of the 3D image respectively.

• The Keep button: This button saves the current tilt angles.

• The Restore button: Clicking this button will restore the tilt angles saved with the Keep button.

Drag the 3D image with the mouseto rotate


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• The Reset button: Clicking this button will restore the default tilt angles (X=60, Y=0, Z=45).

Adjust the Z direction size (height) of the 3D Image: Drag the Z Scaling slider to a value within the range of 0% to 1000% as required.

• Normalization: When this option is selected, and the Z

scaling is set to 100%, the height of the 3D image will become approximately 20% of the height of the displayed area (the actual height is ignored). Select this option when the 3D image is too flat.

• x1: The height of the 3D image is displayed with the original magnification (x1).

• x10: The height of the 3D image is enlarged to ten times the actual size.

Important: When the Normalization option is not selected, and the Z scaling is adjusted to 100%, the 3D image will show the actual (original) height.

Enlarge the 3D Image: To make the 3D image in the 3D Surface Inspector View window larger and smaller, place the cursor in the 3D Surface Inspector View window, then, while pressing the < Shift > key, drag the mouse up or down.

Important:

• If you use a mouse with a wheel, clicking the 3D image once then turning the wheel will make the image larger or smaller.

• To enlarge the entire 3D Display window, either drag the four corners of the window or click the Maximize button on the title bar of the window.

Pan the 3D image: To pan (not rotate) the 3D image in the 3D Surface Inspector View window, place the cursor on the 3D image, then, while pressing the <Ctrl> key, drag the image (or drag using the right mouse button).


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Display the Coordinate Axes of XYZ: In the Drawing Type box,

select the Display Axis option.

Check this option

The coordinate axes are displayed (On the coordinate axes, the maximum values for X, Y, and Z are displayed respectively)

The 3D image can be moved up and down, left and right with the <Ctrl]>key + drag (or right drag)


2-36

Change the 3D Image's Display Type: In the Drawing Type box, select one of the following options:

• Wire Frame: The 3D image will be a wire frame display. The wire frame display can be rotated the fastest, and it is suitable for detailed examination of the surface shape of the 3D image.

• Pseudo color: The 3D image will be a pseudo color display. The color arrangement (in the Z direction) will be: the highest area = red → the lowest area = blue. The saturation and contrast of the pseudo color can be configured in the Saturation and Contrast fields in the Pseudo color box (they can be configured within the range of 0 to 100 respectively).

• Texture: When this option is selected, the focused focal point

image will be texture mapped (pasted) on the surface of the 3D image, creating a display closest to the actual object. This option is selected by default.

• Contrast Enhancement: When this option is selected, the contrast of the 3D image is enhanced.

• Shadow Enhancement: When this option is selected, the shadows of the 3D image are enhanced.

Adjust the Tilt and Direction of the Lighting: Use the Elevation and Rotate controls in the Lighting group box. These controls can be set to values in the range of -180 and +180°.

Wire Frame Display Pseudo color Display Texture Display


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Save the 3D image: To save the 3D image displayed in the 3D

Surface Inspector View window, use the following options:

Save As New Image: When this button is clicked, the 3D display is

output to 3D Surface Inspector Plus as a new image (opened as a new window). To save this image, use the Save As command in the File menu of 3D Surface Inspector.

Save Next#: When this button is clicked, the 3D image can be saved directly to a file (the image is saved directly to a file without being opened in 3D Surface Inspector Plus). The current date and time are automatically included in the name of the destination file.

To specify the file name and destination, click the Save Setting button to open the Save Next# Setting dialog, then specify the file name and destination in the File Name box.

For example, if FILE_ is specified in the File Name box, and the Save Next# button is clicked in the year 2004 on May the 1st at 15:37 55 seconds, the name of the file which the 3D image is to be saved to will be FILE_20040501_153755.tif .

After specifying the file name and destination, click the Save button. This completes the process, so display the 3D image you wish to save in the 3D Surface Inspector View window, then click the Save Next# button to save.


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To save your images as an animation sequence or *.AVI, use the Create Animation option:

Indicate the rotation direction, number of steps, and rotation angle.

Then click OK. The animation sequences will be created in the Image-Pro Plus workspace, where you can save them as *.AVI or other movie files.


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Analyzing Surface Area and Volume of Objects in the Image You can use 3D Surface Inspector to analyze the surface area and volume of objects in the image. Different objects in the image are separated based on the Z position of their deepest frames. To perform measurements, follow these steps:

Note: To obtain calibrated surface area and volume values, use 3D Surface Inspector's Spatial Calibration tool to apply a pixels per unit calibration to the X-Y image before you perform the following steps. As the calibration unit, always use mm (millimeters).

1. On the Measurement tab of the 3D Surface Inspector dialog box, select one of the X-Y images in the list of X-Y images, and the corresponding Z image in the list of Z images. This brings the X-Y image to the foreground (i.e. it becomes active).

2. Check the Fill Area option. Uncheck the Show Marker option.

3. Move the slider to determine the Z plane above which the objects are to be measured.

The slider scale corresponds to the entire range of the Z positions divided into 100 equal parts, where the lowest Z position corresponds to 0 and the highest Z position to 100. As the slider is moved, the corresponding Z plane area is displayed in red. (The current Z position is displayed in the "Bottom Position" box.) The Z plane which has been specified by moving the slider represents the bottom surface of the objects to be measured, i.e. their surface


2-40

area and volume are measured between this bottom surface and their respective tops.

Click on one of the objects to measure its surface area and volume. Once clicked, the object is displayed in blue, and the measurement results are shown in the Surface Area and Volume boxes.

You can measure the objects in the image one by one, as far as they are separated from each other on the bottom surface which has been selected using the slider.

NOTES: To unselect a currently selected object, click the Clear button.

To cancel the measurement mode, uncheck the Fill Area option. To add a new pair of X-Y and Z images, click the Add Pair button

and select the corresponding images. To cancel a pair of images, use the Release Pair button.

You can use Count/Size tool with a Z image to get volume data for multiple objects at one time. To do this, select IOD and subtract the corresponding volume from 0 to the bottom surface from the IOD value.

Performing Cross-Sectional Measurements

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Performing Cross-Sectional Measurements By using the Show 3D Profile option under the Measurement tab, you can generate the cross-sectional profiles of in arbitrary direction and of an arbitrary length in the image to perform cross-sectional measurements. To take cross-sectional measurements, follow these steps:

1. Select the focused focal point image by clicking it, then select the Show 3D Profile option under the Measurement tab

The cross-sectional profile is displayed in the image, and the pixel values on the profile line are simultaneously displayed as the numerical data in the 3D Surface Inspector: 3D Profile window.

Check this option


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The cross-sectional profile is a graphic plot of the pixel values on the profile line (Z = height).

If the cursor is placed on the endpoint of the cross-sectional profile, it turns into the shape of a four-way arrow ( ). Using this four-way arrow, you can make the profile larger or smaller or change its tilt by dragging the endpoint. In addition, the entire profile can be moved by placing the cursor in the center of the profile and dragging.

The origin of the profile is the end with the red marker. The origin corresponds to the position of Index 0 in the 3D Surface Inspector: 3D Profile window. The value of the Index is the distance (in pixel units) from the

profile origin.

The Z value is the height information (Z position).

X and Y are the positions for X and Y in the image (the position in relation to the image origin).

The end with the red marker is the origin (= Index 0)

Cross-sectional profile: The pixel values on this line is plotted as a graph, and simultaneously displayed in the chart below.

Performing Cross-Sectional Measurements

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The measurement data displayed in the upper part of the chart are: Start: The positions for X, Y, and Z of the profile origin (the end

with a red marker) are displayed.

End: The positions for X, Y, and Z of the profile endpoint are displayed.

Max.: The highest position (Z position) on the profile is displayed.

Min.: The lowest position (Z position) on the profile is displayed.

dz: The delta Z (thickness of the Z slice) is displayed.

The graph and data are automatically updated when you move or stretch the profile. 3. (Optional) To send the measurement data to an external file,

externally, you can use:

The Save the Data to a File command in the File menu: This command saves the measurement results to a file in the CSV format (comma-delimited format).

The Copy Data to the Clipboard command in the File menu: This command copies the measurement results to the clipboard.

The DDE Transfer to Excel command in the File menu: If the PC has Microsoft Excel installed, the measurement results are transferred to Excel.

Important: The path to Excel must be specified correctly in the DDE Options command in the File menu to transfer the data to an Excel spreadsheet.

The DDE Options command in the File menu: This command configures the settings to transfer the measurement results to Excel. For details on this command, refer to Appendix D in the Image-Pro Plus Reference Manual.


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Index

Index-1

Index

3

3D SURFACE INSPECTOR View button, 2‐23 3D SURFACE INSPECTOR View window, 2‐18, 2‐23, 2‐32, 2‐33, 2‐34, 2‐37

A

Acquire button, 2‐10, 2‐11, 2‐15, 2‐24 Acquire tab, 2‐3, 2‐6, 2‐7, 2‐8, 2‐9, 2‐11, 2‐14, 2‐24 Add Pair button, 2‐40 Advanced menu, 2‐21 analog camera, 2‐4 animation sequence, 2‐38 Authorization code, 1‐9

B

Bottom Up, 2-10, 2-15, 2-17, 2-26

C

Calibration, 2‐3, 2‐10, 2‐15, 2‐39 Capture Count Down, 2‐22 Capturing Input Images, 2‐1 Clear button, 2‐16, 2‐20, 2‐26, 2‐40 Communication port, 2‐7, 2‐14 Configure button, 2‐7, 2‐14 copy protection plug, 1‐4 Count/Size, 2‐40 cross‐sectional profile, 2‐41, 2‐42 Cursor Coordinate, 2‐19, 2‐29

D

DDE Options, 2‐43 Default destination, 1‐6 Delta Z, 2‐10, 2‐11, 2‐15, 2‐17, 2‐24, 2‐26 Delta Z per Revolution, 2‐7, 2‐14 Device connection error, 2‐7, 2‐14 Direction field, 2‐10, 2‐15 DirectX, 1‐3 DirectX9c, 1‐3 Down button, 2‐8, 2‐9, 2‐14 Drift, 2‐4

E

EDF image, 2-4, 2-16, 2-25, 2-28, 2-31 Exit button., 2-23 Extended Depth of Field, 1-1, 2-28

F

Fill Area, 2-19, 2-40 Focus depth,, 2-2 Focus Settings tab, 2-11, 2-24 Focus Start button, 2-23 Focus Settings tab, 2-11 Functions

EDF Window, 2-33

I

Installation, 1-4 Introduction, 1-1

K

Keep button, 2-33

Index

Index-2

L

License agreement, 1-6 Live image, 2-6, 2-21, 2-22, 2-23 Live preview, 2-9, 2-14

M

Manual Acquire, 2-21 Manual focus, 2-21, 2-22, 2-23 Measurement tab, 2-19, 2-29, 2-31, 2-39, 2-41 Millimeters, 2-3, 2-27, 2-39 Montage, 2-11, 2-24

N

Normalization, 2-34 NTSC, 2-4 Number of Images, 2-10

O

Out of Focus Correction, 2-17, 2-27

P

Pause button, 2-22, 2-23 Pause button., 2-22 Pre‐Align Images, 2-4 Prior, 1-1, 1-2, 2-6 PRIOR Settings dialog, 2-7 Process menu, 2-6 Pseudo color, 2-36

R

Registration and Technical Support, 1-10 Release Pair button, 2-40 Reset button, 2-34 Resolution, 2-7, 2-14

Restore button, 2-33 Rotate, 1-1, 2-33, 2-36 Rotation direction, 2-7, 2-14

S

Save As New Image, 2‐37 Save Next#, 2‐37 Save Setting, 2‐37 Save the 3D image, 2‐37 Scope‐Pro, 1‐2, 2‐3, 2‐21, 2‐27 Selected Images window, 2‐16, 2‐17, 2‐26 Sensitivity box, 2‐26 sequence file, 2‐1, 2‐16, 2‐25, 2‐26 Set Z Lower Limit, 2‐9, 2‐14 Set Z Origin, 2‐7, 2‐8, 2‐14 Setup program, 1‐4 setup screen, 1‐8 Show 3D Profile, 2‐41 Show 3D SURFACE INSPECTOR View simultaneously, 2‐23 Show Marker, 2‐19, 2‐29, 2‐39 Smoothing, 2‐17, 2‐27 Spatial Calibration, 2‐3, 2‐39 Stage controller, 2‐6, 2‐7, 2‐14 Start Preview, 2‐9, 2‐14 Stereo M. S, 2‐17, 2‐27 Stereo Microscopes, 2‐4 Surface area, 1‐1, 2‐3, 2‐10, 2‐11, 2‐15, 2‐16, 2‐20, 2‐24, 2‐25, 2‐30, 2‐39, 2‐40 Surface Plot, 1‐1, 2‐30, 2‐32 System Requirements, 1‐2

T

Technical Support, 1-10 Texture, 2-36 Three Dimensional Observation dialog, 2-6, 2-31 Tilt, 2-33, 2-36 Top Down, 2-10, 2-15, 2-17, 2-26

Index

Index-3

Topo. Map, 2-11, 2-24 Topographic map image, 2-4, 2-16, 2-18, 2-25, 2-29, 2-31, 2-32 Topographic Options box, 2-26

V

Version 7.0, 1-1 Video/Digital command, 2-8, 2-14 Volume, 1-1, 2-3, 2-10, 2-11, 2-15, 2-16, 2-20, 2-24, 2-25, 2-27, 2-30, 2-39, 2-40

W

Welcome screen, 1-5 Wire Frame, 2-36 Working with 3D Surface Inspector, 2-1 Workspace Preview, 2-2, 2-21

Z

Z axis, 1-1, 2-23 Z direction, 2-1, 2-34, 2-36 Z focus, 2-7, 2-14 Z stage, 1-2, 2-3 Z Stage, 2-6 Z Step Size, 2-8 Z Travel Speed, 2-8, 2-14

Index

Index-4

3d surface inspector version 7.0 for windows user guide · 3d surface inspector™ version 7.0 for...

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