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Menu for Instructions for use of the BioRad Radiance 2000 Confocal at the Analytical Imaging Facility at AECOM

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The confocal microscope is for 1.) thin optical sections of fluorescence stained material, 2.) thin optical sections of reflectance stained material or 3.) reflectance imaging of relief surfaces or thin spaces such as the cell-substrate interface.

The confocal microscope is not for convenient multiple color imaging; we have widefield microscopes for this.  The confocal microscope is not for low light imaging; we have widefield microscopes for this.

Probes for BioRad Radiance 2000 Confocal Microscopy:
There are a large number of fluorescent
probes for use in microscopy and for your convenience we have broken many of them into groupings and listed them here.
For the BioRad confocal microscope, you may not use any of the stains from group blue which require excitation at 360 nm.
You may use any of the dyes from the green, red, or near infra red categories.   The confocal microscope has lasers that excite dyes at 488 nm, 568 nm and 637 nm only.

lambda.gif (698 bytes) is the new button for "Lambda Scan" mode.  It replaces the button that looked like a Ven diagram of red, green and blue which was next to the laser intensity slider.

Main Manual  Checklist for Startup and Shutdown
Recommendations for slide preparation for the confocal.

Information for methods sections:

Nikon Eclipse 200 modified laser safe (Melville, NY).  Infinity corrected 10X, 20X, 40X, 60X objectives.  

BioRad Radiance 2000 laser scanning confocal microscope (CA).  Kr/Ar laser for excitation at 488 and 568 nm and diode laser for excitation at 633 nm.
Standard filters with narrow emissions for imaging green and red channels.  Far red imaged with 650LP filter.

Absence of bleed through from one wavelength to a longer wavelength is achieved using one of these methods:
1.   balancing of laser intensities with gain settings;
2.  "lambda scanning" which is scanning each line rapidly in sequence with individual excitations; or
3.  sequential imaging of each dye by excitation with a single laser line.


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