From Lasers and Current Optical Techniques in Biology: Comprehensive Series in Photochemistry and Photobiology, Volume 4

Chapter List

Chapter 13: Optical Microscopy
Chapter 14: Wide-Field Autofluorescence Microscopy for the Imaging of Living Cells
Chapter 15: Scanning Probe Microscopy
Chapter 16: Confocal and Multiphoton Microscopy
Herbert Schneckenburger

Optical microscopy, including wide-field microscopy, scanning microscopy and micromanipulation, has become an indispensable tool in cell biology and photo-biology. The present article is concentrated on wide-field microscopy and covers transillumination and fluorescence microscopy. Basic principles are described as well as advanced techniques, e.g. fluorescence lifetime imaging (FLIM), energy transfer spectroscopy (FRET) and total internal reflection fluorescence microscopy (TIRFM). A few applications are depicted, which may give some insight into a fascinating microcosmos.

13.1 Introduction

The theory of optical microscopy was established in the second half of the nineteenth century by Ernst Abb (1840 1905), although the first microscopes had been built much earlier. Usually, a microscope is characterized by a highly magnifying objective lens used for imaging a sample with a half-angle a of the incident cone and a numerical aperture A = n sin ?, where n is the refractive index of the medium between the sample and the objective lens. The numerical aperture is an important parameter for the quantity of light taken up by a microscope (which is proportional to A 2), for the lateral resolution ? x = ?/ A and for the depth of focus ? z = n ?/ A 2, with ? being the wavelength of optical radiation.


Products & Services
Scanning Probe Microscopes
Scanning probe microscopes form images of surfaces by using a physical probe that scans the specimen. Examples include AFMs (atomic force microscopes), MFMs (magetic force microscopes), and STMs (scanning tunneling microscopes).
Microscope Lenses and Objectives
Microscope lenses and microscope objectives include eyepieces or oculars, optical filters, objectives, adapters or mounts, condensers and other optical components for microscopy.
Optical and Light Microscopes
Optical and light microscopes use the visible or near-visible portion of the electromagnetic spectrum to magnify images of objects.
Measuring Microscopes
Measuring microscopes are used by toolmakers for measuring the properties of tools.  These microscopes are often used for dimensional measurement with lower magnifying powers to allow for brighter, sharper images combined with a wide field of view.
Metallurgical Microscopes
Metallurgical microscopes are used for metallurgical inspection including metals, ceramics, and other materials.

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