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 Immunology
Unit Number:  Unit 21.1
DOI:  10.1002/0471142735.im2101s48
Online Posting Date:  May, 2002
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The light microscope is a basic tool for the cell biologist, 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.

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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 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
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
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