Wong Lab Protocols: - Boston University



Wong Lab Protocols:

Setting up the Axiovert s100 microcope:

1. Turn on green toggle switch for brightfield illumination (turn dial to middle intesity setting)

2. Turn on Atto Arc Variable Intensity lamp control for fluorescence

*Do not turn either the fluorescence or brightfield illumination on and off repeatedly. This will cause the bulbs to blow up.

3. Turn on Uniblitz shutter controller (switch in back of smaller black box)

4. Turn on MicroMax digital camera (larger black box to left of microscope)

5. Turn on the Filter wheel controller located under monitor in the computer rack

6. Turn on Ludl Z-motor controller located above monitor

• If the Ludl controller is turned on DO NOT use the the fine focus of the microscope. The fine focus controller is now adjusted using the knob on the joystick located to the right of the microscope. The coarse adjustment can still be used on the microscope.

7. Once everything is turned on open Metamorph

8. Open the “Main Task bar” in the journal menu

9. Use the task bar to open bright field and fluorescence shutters

10. To view things through the eyepiece of the microscope push in the metal pin on the left side of the eyepiece.

11. To view things on the computer screen push the metal pin on the right side of the microscope in.

• This just closes a shutter so that light does not enter through the eyepiece to the camera

12. Rotate objective turret to the desired objective

13. Rotate the condeser turret to the appropriate Phase setting:

• 10 x objective ------Phase I

• 40 x objective ------Phase II

• 63 x (oil immersion)----Phase III

14. Set up Koehler illumination:

15. Focus on specimen to be viewed with coarse adjustment

• This can be made easier by moving the stage back and forth while focusing

16. Adjust with fine focus knob located on joystick

17. Close the diaphram field stop on the condensor until there is a small diameter of light through the eyepiece

18. Move the condensor up and down until the edges of the view are crisp and sharp

19. Open the diaphram to so that the entire view field is lite

Saving Images:

2 options:

1. With stack in metamorph, go to File->Save As and select "tif" as type.

It is one option above stk. This will then save the stack as a series of

tif images. For example, test.stk becomes test1.tif and test2.tif. This

saves bitdepth as-is - so if it's a 16bit image, it'll be a 16bit tif. Many programs do not accept 16bit tifs.

2. In Scale Image -> More section. You can adjust the scale of the image

and then copy the stack in to an 8 bit stack. You can then follow #1 to save

the 8 bit tif images.

Making movies:

To make a movie that can be played on any computer with a media player such as Microsoft Media Player:

1. In Metamorph, open image stack.

2. Choose "Stack", "Make AVI file".

3. Choose images to include in stack and playing time.

4. Save file using Radian Cinepac compression option, 85% compression, options--black and white images.

5. View movie in Media Player.

Calibration:

1. Go to Measure->Calibrate Distances, select new, draw line on image of stage

micrometer, and tell the software the length and units of the line.

2. Once you have the calibration defined, write a journal to apply it to future acquired images with

1. Journal->Start Recording

2. Measure->Calibrate Distances: highlight calibration, select Video

device=PI, and hit Apply

3. Journal->Stop Recording and save this as something like

"Apply40xCalibration

3. You can create these for each set of optics you use, put the journals on a

taskbar. The user would only need to hit the appropriate button before

acquiring the images and all the images will have the calibration stored with

the file.

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