Proper Alignment and Adjustment of the Light Microscope

Edward D. Salmon1, Julie C. Canman1

1 University of North Carolina, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3N
DOI:  10.1002/0471142905.hga03ns38
Online Posting Date:  November, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The light microscope is a basic tool for biologists, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase‐contrast, differential interference contrast, and fluorescence epi‐illumination), and how it should be maintained as required to obtain maximum image‐forming capacity and resolution. The principles of microscopy and step‐by‐step alignment and adjustment procedures are described in this unit.

PDF or HTML at Wiley Online Library

Table of Contents

  • Major Components of the Light Microscope
  • Basic Imaging and Köhler Illumination Light Paths for Bright‐Field and Fluorescence Microscopy
  • Basic Protocol 1: Alignment for Köhler Illumination in Bright‐Field, Transmitted Light Microscopy
  • Basic Protocol 2: Alignment of the Eyepieces
  • Basic Protocol 3: Alignment for Köhler Illumination in Epifluorescence Microscopy
  • Basic Protocol 4: Alignment for Phase‐Contrast Microscopy
  • Basic Protocol 5: Alignment for DIC Microscopy
  • Support Protocol 1: Matching Microscope Magnification to Detector Resolution
  • Support Protocol 2: Calibrating Image Magnification with a Stage Micrometer
  • Tests for the Optical Performance of the Microscope
  • Support Protocol 3: Testing Phase‐Contrast and DIC Using Diatom Testing Slide
  • Support Protocol 4: Testing Phase‐Contrast and DIC Using Squamous Cheek Cell Test Slide
  • Support Protocol 5: Testing Fluorescence Using Red, Green, and Blue Fluorescent Tissue Culture Cell Test Slide
  • Support Protocol 6: Care and Cleaning of Microscope Optics
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library


Literature Cited

Literature Cited
   Allen, R.D., David, G., and Nomarski, G. 1969. The Zeiss‐Nomarski differential interference equipment for transmitted‐light microscopy. Z. Wiss. Mikf. Microtech. 69:193‐221.
   Hinsch, J. 1998. Mating camera to microscopes. Methods Cell Biol. 56:147‐152.
   Inoué, S. 1989. Imaging unresolved objects, superresolution and precision in distance measurement with video microscopy. Methods Cell Biol. 30:112.
   Keller, H.E. 1998. Proper Alignment of the Microscope. Methods Cell Biol. 56:135‐146.
   Pluta, M. 1989. Advanced Light Microscopy Vol. II: Specialized Methods. Elsevier/North‐Holland, Amsterdam.
   Salmon, E.D. and Tran, P. 1998. High resolution video‐enhance differential interference contrast (VE‐DIC) light microscopy. Methods Cell Biol. 56:153‐183.
   Salmon, E.D., Shaw, S.L., Waters, J., Waterman‐Storer, C.M., Maddox, P.S., Yeh, E., and Bloom, K. 1998. A high‐resolution multimode digital microscope system. Methods Cell Biol. 56:185‐215.
   Taylor, D.L. and Salmon, E.D. 1989. Basic fluorescence microscopy. Methods Cell Biol. 29:207‐237.
   Zernike, F. 1942. Phase contrast: a new method for the microscopic observation of transparent objects. Physica 9:686‐693.
   Zernike, F. 1955. How I discovered phase contrast. Science 121:345‐349.
   Zernike, F. 1958. The wave theory of microscope image formation. In Concepts in Classical Optics (J. Strong, ed.) pp. 525‐536. W.H. Freeman, San Francisco.
PDF or HTML at Wiley Online Library