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Light Microscopy Reflection Focusing Cells and Coverglass
  • George McNamara, M.D. Anderson Cancer Center

Light Microscopy Reflection Focusing Cells and Coverglass 20150520Wed

Many light microscopes have the capability of using camera based brightfield, phase contrast, or fluorescence to find the most contrasty specimen focus. When I worked for UIC (1992-1997) we offered this with a combination of Ludl MAC2000 controller, Z-drive, video card thingy, and video camera (most customers at the time used analog video cameras).

Many automated light microscopes now have clever -- and pricey -- autofocus systems based on an NIR LED structured illumination reflecting light off the bottom of the coverglass.

I encourage a simpler approach - my hardware details are in the text file inside the ZIP download. Summary:

1. LED epi-illuminator (SOLA with 6 LEDs, MetaMorph enables my first gen SOLA to have single LED on control -- equally important, can control the light level, since reflection is a lot brighter than fluorescence).

2. filter cube (mine is Semrock Cyan-Yellow-HcRed)

- without an exciter filter

- dichroic beamsplitter (mine is triple pass)

- emission filter (mine is triple pass).

3. camera (FLASH4.0 sCMOS).

4. microscope (mine is Leica DMI6000 inverted scope).

5. imaging system - mine is MetaMorph 7.8.10.

6. imaging dish - Mattek glass bottom dish.

The TIFF image series inside the ZIP shows the results of focusing from below the coverglass, into the coverglass, past the cells, into the mounting medium. This experiment used fixed adherent cells, mounted in 80% glycerol:20% PBS. Live cells have better contrast.

I also included a MetaMorph Stack Arithmetic "Best Focus" showing for most pixels, the cells are best focus. This is probably calculated from "local variance" calculations (I first heard of this from Matt Batchelor and Rob Meyer of Meyer Instruments - has been around in photography for a long time and several free software can do it). I do not think MetaMorph currently has this as a easy to access command currently, but it was able to find the "best focus" for every pixel, so could be made to provide the user an image with each pixel intensity representing the plane number (simplest to make a 16-bit image where pixel value is the source plane number). In the case of MetaMorph, not really necessary (usually) for the user to have the "best focus pixel" image, what matters is that MetaMorph can drive the microscope to go to the best focus.

Back to my narrative: a reflection light Z-series can be used to:

1. find the underside of the coverglass (easiest if dirty, but best for experiment if clean).

2. find the coverglass-media interface.

3. find the last plane of interest (last cell of adherent layer) - or user can specify something like: "acquire coverglass to +42 um".

Upshot: reflection focus is easy to obtain on a fluorescence microscope. Simplest if one of the filter cubes (my scope has 6 cubes) is exciter-less, simply use the LED light source to choose what wavelength(s) to use (also intensity).

  • Light microscopy,
  • best focus
Publication Date
Spring May 19, 2015
Data is not subject to copyright (the two images). The text inside the zip, the text of the abstract and this copyright notice, are Copyright (C) 2015 George McNamara. If you recycle this work, please cite this site's web address,
Citation Information
George McNamara. "Light Microscopy Reflection Focusing Cells and Coverglass" (2015)
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