closely spaced points. Next, the functions of confocal microscopes,fluorescence
microscopy, multiphoton microscopy, Raman microscopy, light sheet microscopy,
and super-resolution fluorescence microscopy are given in Sect.8.3 through
Sect.8.8, respectively. The following two chapters then describe spectroscopic and
imaging techniques that make use of microscopic instruments and methodologies.
8.1 Concepts and Principles of Microscopy.
This section addresses the operational concepts of basic optical microscopes, which
are used widely to obtain both topographical and dynamic information from bio-
logical samples. The functions and parameters of interest include viewing and
illumination techniques, observation methods, numerical aperture,field of view,
and depth offield [ 1 – 8 ]. In basic microscopes the entire sample is illuminated
simultaneously with a light source such as a pure white mercury lamp. Thus these
instruments are known aswide-field microscopes. The light coming from the illu-
minated object is viewed with a device such as a CCD camera. A limitation of
wide-field microscopes is that light emitted by the specimen from above and below
the focal plane of interest interferes with the resolution of the image being viewed.
Consequently, images of specimens that are thicker than about 2μm appear blurry.
Finer resolutions can be obtained with other types of instruments, such as the
confocal microscopes described in Sect.8.3.
8.1.1 Viewing and Illumination Techniques.
Modern conventional microscopes show a magnified two-dimensional image that
can be focused axially in successive focal planes. This capability enables the
examination of thefine structural details of a specimen in both two and three
dimensions. The basic setup for a microscope is shown in Fig.8.1. The key
components are the following:
- A movable stage that includes clips for holding a specimen and a manual or
automated translation mechanism to accurately position and focus the specimen - A set of selectable objective lenses with different magnifications mounted on a
rotatingturret(also called thenosepiece) for magnifying the specimen - One or more lamps, lasers, or LED sources that can include optical elements
such as collector lenses,field diaphragms, heatfilters, and various lightfilters - One or more mirrors for directing light onto the specimen
- A viewing device such as an eyepiece, a photodetector array, a photomultiplier
tube, or a video camera - Various optical focusing elements that are strategically placed to achieve a
desired illumination intensity and contrast in the specimen image
234 8 Microscopy