How to Obtain Images for Stitching

 by Katherine Barlow

  1. Set up your sample the way you would set it up to be imaged normally, ensuring the correct lens, zoom, and resolution are chosen in WiscScan (If you are comfortable with this, skip to step 2)

    a. Turn off all lights

    b. Bring image into focus using eyepiece and lamp on microscope (may have to flip paddle on top of microscope to right position to see sample using eyepiece.

    c. Turn off lamp and flip paddle to left position on top of microscope

    d. Place the lever outside of the microscope box to “OUT” or “IMAGE”

    e. Log in to WiscScan

    f. Choose appropriate lens, zoom and resolution for your image

*If you only want your multiphoton image, uncheck Channel 0. Leaving Channel 0 checked will save both the brightfield image and multiphoton image, and your stitch in Fiji will default as a red and green color scale rather than grayscale.

uncheckChannel0.PNG

2. As a starting point, set pockel cell to 80.  You may have to decrease or increase this depending on your sample.

3. Increase gain until the multiphoton image, the image on right side of screen, becomes minimally saturated (typically between .300 and .500). Only a small amount of red should be seen on the screen.

4. Ensure your multiphoton image is in focus - may have to use z controller to bring into focus

5. Use the xy controller to choose your initial image frame

xyzcontrol.PNG

6. Click on “XY Stage” tab

XY stage.PNG

7. Define the start position in your metadata by selecting "set 0"

XY stage - Copy.PNG

8. Click on Generate.  Your grid can be generated three ways:

    1. Method 1 a:  Define a total image size.

                    a. Enter the size of the entire area that you would like to image (in microns) in the box labeled Total Size

                    b. Set the amount of overlap you wish to have between adjacent images, then press "calculate number of positions". Around 10% overlap seems to work well.  You can alternatively set your own step size and overlap amount and calculate the size of each image

                    c. Click on Calc # Pos from Total Size to display how many images you will acquire in the X and Y directions

                    d. Once you have calculated all the necessary positions, press “Ok”

  1. Method 1 b: Define the number of images to stitch together.

                    a. Choose number of columns you’d like stitched together, and place that number in the “# X” box

                    b. Choose number of rows you’d like stitched together, and place that number in the “# Y” box

                    c. Choose the amount of overlap you’d like to have.  Around 10% overlap seems to work well.  

                    d. Click on Calc Total Size from # Pos to display the total size of your image.

                    e. Press “Ok”

  1. Method 2:  Define a grid by selecting two corners of a rectangle.

                    a. Use the XY joystick to maneuver to any corner of the a rectangle that encompasses the area that you would like to image.  

                    b. Click “Set current post as START corner”.

                    c. Again use the joystick to find the diagonal corner of the rectangle.  Click “Set current position as END corner”.

Example:

grid.PNG

**If you get an error message when you try to generate a grid or select a point, something about the specified points being outside of the move range, all you have to do is increase the number in the box next to "Move Range:" Just throw a couple of more zeroes in there to make the range orders of magnitude larger.

9. Select “Z motor” tab

zmotor.PNG

10. Under 4d imaging, calculate Z bottom, Z top, and Z step (These numbers should be set as 0 unless you are imaging in more than one plane of your sample - see a-c below for more than one plane)

    1. Use controller to find top of area of interest. Click Set next to z top.

    2. Use controller to find bottom of area of interest. Click Set next to z bottom.

    3. Then choose i or ii:

      i. Decide how many images you want in a stack: enter that next to "Number of Sections". Then click "Calculate" next to Z Step, and it will tell you what your step size is. Only numbers greater than 1 are meaningful.

      ii. Decide what you want the spacing between z slices to be, and enter that as Z Step. Then click "Calculate" next to Number of Sections, and that will tell you how many images you'll have if you move from z bottom to z top with a spacing of z step.

useXY.PNG

12. Select "Use XY coordinates" and then “Start Sequence”

13. Press “OK” on the pop up window

14. Save your images as something specific that you can remember.  Each grid postion and z slice saves as its own image, so this can generate many files.  You may want to put it in its own folder.

 


How to Stitch Images and Create Z-Stacks in FIJI

Once WiscScan has run, your output will be a series of images that can be stitched together in FIJI

  1. Open FIJI, go to “Plugins>Stitching>Grid/Collection Stitching”

FIJI1.PNG

  1. For “Type”, choose “Positions from file”

  2. For “Order”, choose “Defined by image metadata”

fiji2.PNG

  1. For “Multi Series File”, choose the chronologically oldest .tiff file from your saved images

fiji3.PNG

  1. For “Fusion Method”, choose “linear blending”

  2. Uncheck “Compute Overlap”

  3. Press “OK”

  4. Voilà! You have a stitched image.

    a. If Z top, Z bottom, and Z step were used in WiscScan, you can collapse these images into one: Image>Stacks>z project.

    fiji4.PNG

    b. Choose your start slice and end slice. If you wish to stack all of your images, you do not need to change the numbers FIJI gives.

    c. For projection type, choose Max Intensity.

fiji5.PNG

*You can view this z-stack in 3D by using Image>Stack>3D Project.

fiji6.PNG

For “Axis of rotation”, choose the axis you prefer to rotate your stack around. Then press “Ok”.

fiji7.PNG

 

*If the image has seams (see images below), please see “How to Calibrate Microscope” section to fix or let

Josh Weber (jjweber3@wisc.edu) know so we can fix.

 

Good Image (no visible seams):  

 good.png

Bad Image:

bad.png

 

Precautions and Notes

  1. DO NOT attempt to adjust the focus of the image with the manual knob on the microscope while the xyz controller is engaged. You will wear down the gears.

  2. DO NOT turn on the detector until the main lights are off. You will damage the detector.

  3. The normal range for the gain is .300-.500. Exceeding .500 should be taken with caution.

  4. If a noticeable amount of overlay or seams can be seen in your images, there is a pixel to micron error within the microscope's initial setup. To fix this, recalibrate the microscope's objectives (See “How to Calibrate Microscope” section) or contact