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Confocal Microscope Basics
(BioRad MRC 1024 Laser Scanning Confocal Microscope)

 

Turning on

1.  Fluorescent bulb - switch and press button
2.  Laser-turn key one quarter turn clockwise
3.  Computer tower - one switch
4.  Monitor - one button
5.  Wait for blue screen to appear.
6.  Scan head-one switch (green light on scan head when on)
7.  Start "BioRad Acquisition" program-double click icon
8.  Login Password Same Name
9.  Set up filters METHOD, Triple Labeling etc.
10. Set up display - Load grayscale or Load SetCol
11. Set up Box Size - usually 768 X 512
12. Set Laser Power - program starts at 1%; usually use 10% or 30%
13. Find sample using epifluoresence or transmitted light
14. Open light path to scan head - move filters rotate microscope head
15. Start collecting images

Collecting Images
Check out Chapter 1 in Steve's book

Remember to use lowest laser power possible but do not take the gain above 1300 on any of the channels.
Remember that you can use the Slow Scan Mode with low signal set for dim specimens.

Transferring Files

Step 1: From the Confocal Computer to the Bock Server

1. Exit BioRad software to "blue" screen
2. Click FTP on program list.
3. Click O.K. in "Session profile"
4. Change to "d" drive - click on -d-, and change directory
5. Click on LS_USER
6. Locate your personal directory, and click on it.
7. Click on "IMAGES"
8. Highlight files, and click on arrow in direction of right box.
9. Files will be transferred to the Bock Server

Step 2: From the Bock Server to a lab. Mac

1. Use Fetch on any of the Lab. Macs.
2. Go to "CONFOCAL IMAGES" Shortcut
3. Access your image directory by changing MANAGER to YOUR DIRECTORY in the path.
4. Click O.K. and Click binary
5. Highlight Images to be transferred.
6. Get files
7. Save to a folder on the Mac.

Converting Files to TIFFs

1. Open GRAPHIC CONVERTER
2. Click on Convert More-FILE; Convert More
3. Highlight Files in Left Hand Box and set filter to BioRad
4. Make new folder in Destination Box and set Destination Format to TIFF
5. Click on CONVERT

Opening Files In PhotoShop

1. Open PhotoShop
2. Find files and open using OPEN AS. ** If you double click on the icon at this point the files will open Graphic Converter
3. When the file is open immediately save it as a Photoshop file using SAVE AS, and replace the old file. This will change the icon, and now when you double-click on it PhotoShop will start up.
4. Manipulating images-check out Chapter 21 in Steve's book

Turning Off

** Check schedule to see if someone is signed up after you, and go find him or her. Also do not turn off the computer if you have to transfer files to Macs. The laser is on standby mode unless it is actually scanning a specimen so itŐs OK to leave the system running. Rule-of-thumb turn it off if it is not being used for an hour or so.
1. Exit program FILE, LOGOUT and follow computer prompts to Shut Down
2. Turn off computer-are you transferring files?
3. Turn off monitor
4. Turn off scan head
5. Turn off laser - key quarter turn anti-clockwise (the cooling fan will continue running until the laser tube has reached a safe temperature).
6. Turn off fluorescent bulb - single switch.
7. Clean up your mess; wipe oil from lenses using lens tissue.

 

Three Color Imaging
Methods and Applications of Three-Color Confocal Imaging
BioTechniques 22:120-126 (January 1997)

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(Copyright by Eaton Publishing: publishing,
reproduction and/or distribution for commercial purposes is not allowed.)

 

Fluorescence probes and filters

http://FLUORESCENCE.BIO-RAD.COM/

http://www.cyto.purdue.edu/flowcyt/software/Spectra.htm
http://www.probes.com
http://www.aecom.yu.edu/aif/instructions/probes.htm
http://www.jacksonimmuno.com/
http://www.clontech.com/
http://www.chroma.com/
http://www.omegafilters.com/