Biophotonics_Concepts_to_Applications

7. Special measurement devices such as grids, scales, and pointers that are intended
   to be in focus and registered on the specimen are placed at the locations of a set of
   field conjugate planes, which are simultaneously in focus and are superimposed
   on one another when observing specimens through the microscope
   The illumination methods illustrated in Fig.8.1include both transmission and
   reflection techniques, which are described in the following paragraphs.
   The two fundamental viewing and illumination techniques used in microscopes
   for examining different specimens are the following:

(1) Transmitted light microscopyis the simplest illumination and viewing tech-

nique. The general concept is shown in Fig.8.1. First a thin slice of a sample

is encapsulated in a specimen holder such as a thin glass or plastic microscope

slide. Standard glass slides have smoothed edges and typically are 2.5 cm by

7.5 cm (1×3 in.) in size with 1 to 1.2 mm thicknesses. After a prepared slide

is placed on the movable stage, it can be illuminated from below with white

light and observed from above, as shown by the bottom lighting configuration

in Fig.8.1. The image is formed through the absorption of some of the

transmitted (also calleddiascopic) illuminating light by dense areas of the

sample. This method creates a contrast pattern consisting of a dark sample on a

bright background. The advantage of transmitted light microscopy is that it is a

simple setup that requires only basic equipment. This simplicity results in

some limitations, which include a very lowcontrast(i.e., a low ability to

distinguish differences in color or intensity) of most biological samples, a low

optical resolution due to a blur caused by some areas being out of focus, and

the fact that the sample often needs to be stained (colored) to enhance

Diverse optical

elements

Movable stage

Specimen holder

Turret with

objective lenses

Reflecting mirror

Dichroic mirror

Viewer or camera

Reflection setup:

Lamp or LED

Transmission setup:

Lamp or LED

Condenser lens

Fig. 8.1 Basic transmission and reflection setups for a microscope

8.1 Concepts and Principles of Microscopy 235