Zeiss LSM 800 MSB 694
S/N 2633000140

Advance scheduling:
https://nyumc.ilab.agilent.com/schedules/498939#/schedule/

Upright microscope with 2 GaAsP detectors and motorized stage.
DO NOT SATURATE IMAGES TO AVOID DAMAGING DETECTORS!!

Lenses:
63x/1.4 oil DIC Plan-Apochromat
63x/1.3 multi-immersion Plan-neofluor DIC adjustable collar must be set depending on immersion media
40x/1.3 oil =Plan-neofluar
20x/0.80 air Plan-Apochromat
20x/0.75 or 0.80 Plan apochromat may be on the instrument for better spectral registration
10x/0.30 Plan-Neofluar
Lenses are changed manually by turning them by hand.

Lasers:
405 nm
488
561
640

Detectors:
2 @ GaAsP

Click here for an example of two track settings for dapi, Alexa488, and Alexa568 (where the blue and red may be imaged simultaneously because the red channel is brighter then the blue channel).

Zen 2.3 (Zen Blue versions may vary slightly and are significantly different than Zen Black)
Only one detector at a time is displayed in Live mode.
Autosave feature may save time, but remember to change the file name before scanning.
Option in Z to collect only one plane of a track, main benefit on this scope to save time where you may only need on plane of a nucleaus, for instance, as a guide for segmentation.

If you need an explanation how to use the 63X lens to achieve full resolution, please ask Michael or Yan.
In brief:

• Pinhole 1 Airy unit (or smaller -- 0.7 is a sweet spot of higher resolution without excessive signal loss)
• Either increase the image size (number of pixels) or use Zoom to set pixel size to 0.1 um. 
• Slow scan or line average to reduce noise. (more info here)

General Confocal Best practices:

• The pinhole is what makes the confocal a confocal. 1AU (which means 1 Airy unit) or smaller (0.5 to 1.0 ok) and click the 1AU button each time you change lenses.
  If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead.
  Except in special case of live cell imaging where you understand that images are not confocal, this is NOT AN ACCEPTABLE WAY TO MAKE IMAGES BRIGHTER. You won't hurt the instrument, but when you write your methods, you won't be accurately describing your microscopy as "confocal".
• Offset. Always use at 0 or 1 (or greater than one in some rare cases).
  Negative numbers are wrong.
• Digital gain. The preset is 1. Leave it there.
• Zoom 1 or greater. Using a Zoom less than 1 may cause a hot spot &/or curvature at the edges.
• Avoid saturation. Saturating images 1.) makes features appear larger than they are, 2.) prevents quantification of intensities and 3.) may damage the detectors in the microscope.
  Use the Range Indicator button to make sure you have no saturated pixels. If you see red pixels, you need to turn down the Gain or Laser.
  Turning gain down will reduce noise. Less noise means you can scan faster. (More here.)
• Always save images in .czi format. This preserves each channel and all instrument metadata.
  Why take images at high resolution of a confocal and then throw away this information by saving as lossy jpg?

Highly recommended reading: Tutorial: guidance for quantitative confocal microscopy

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