CHOOSE THE OPTIONS THAT APPLY TO YOUR EXPERIMENT - This methods file was meant to be edited for your specific experiment so includes ALL options. You need to document which filter setup you used, etc for your image collection from these options.   METHODS AND MATERIALS CONFOCAL MICROSCOPY  

Physiology and Biophysics Facility

The confocal microscope used is a BioRad MRC 600 Laser Scanning Confocal Microscope equipped with an Argon laser with filter cubes setups for excitation at 488nm wavelength using a 488 DF10 excitor, a 510 LP dichroic relector, and a 515 LP emission filter; or excitation at 514 using a 514 DF10 excitor filter. a DR 540 LP dichroic reflector, and a 550 LP emission filter. Simultaneous two color excitation was accomplished using a filter cube setup with a 514 DF 10 bandpass filter, a DR 527 LP dichroic in the first cube and a DR 565 LP dichroic, and emission barrier filters for the green signal of 540 DF 30 and for the red signal of EF 600 LP. The confocal device is attached to a Zeiss Axiovert microscope equipped with a 10x PlanNeoFluar NA 0.3, a 20x PlanNeoFluar NA 0.5, a 40x PlanNeoFluar NA 0.75, and a 63x Plan-NeoFluar NA 1.25 infinity objectives, using a zoom factor of 1 giving a final magnification of "n" x. (Consider a 10X magnification in the neck of the confocal attachment - the same as through the eye pieces - so a 10X at zoom 1 is 100X mag) Images were collected by averaging "n" images using the Kalman (or Expotential) filter. Z Series slices were collected using Kalman filter averaging "n" scans at intervals of "n" microns.