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3.3  Light Microscopy: The Basics

3.3.1  MAGNIFICATION

The prime function of a light microscope is to magnify features in a biological sample, which

are illuminated by VIS light, while maintaining acceptable levels of image clarity, contrast,

and exhibiting low optical aberration effects. Magnification can be performed most effi­

ciently using a serial combination of lenses. In a very crude form, a single lens is in effect

a very simple light microscope but offering limited magnification. In its most simple prac­

tical form, a light microscope consists of a high numerical aperture (NA) objective lens

placed very close to the sample, with a downstream imaging lens focusing the sample image

onto a highly sensitive light detector such as a high-​efficiency charge-​coupled device (CCD)

camera, or sometimes a photomultiplier tube (PMT) in the case of a scanning system such

as in confocal microscopy (Figure 3.2a). Most microscopes are either upright (objective lens

positioned above the sample stage) or inverted (objective lens positioned below the sample

stage).

The two-​lens microscope operates as a simple telescope system, with magnification M

given by the ratio of the imaging lens focal length to that of the objective lens (the latter typ­

ically being a few millimeters):

FIGURE 3.2  Light microscopy methods. (a) Magnification in the simplest two-​lens light

microscope. (b) Back focal plane detection (magnification onto quadrant photodiode is f2/​f1

where fn is the corresponding focal length of lens Ln). (c) Laser dark field. (d) Phase retardation

of light through a cell sample in phase contrast microscopy.