Proper Alignment and Adjustment of the Light Microscope

Edward D. Salmon1, Kate von Lackum2, Julie C. Canman1

1 University of North Carolina, Chapel Hill, North Carolina, 2 University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2A.1
DOI:  10.1002/9780471729259.mc02a01s00
Online Posting Date:  October, 2005
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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, Fluorescence, and Dark‐Field Microscopy
  • Basic Imaging for Dark‐Field 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
  • Alignment for Dark‐Field Microscopy
  • Basic Protocol 6: Alignment for Low‐Power Magnification Dark‐Field Microscopy
  • Basic Protocol 7: Alignment for High‐Power Magnification Dark‐Field Illumination
  • 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, Dark‐Field, and DIC Microscopes Using a Squamous Cheek Cell Test Slide
  • Support Protocol 5: Testing Fluorescence Using a 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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