Figure 9–7 Contrast in light microscopy. (a) the stained portion of the cell will absorb light of some wavelengths, which depends on the stain, but will allow other wavelengths to pass through it. a colored image of the cell is thereby obtained that is visible in the normal bright-field light microscope. (B) in the dark-field microscope, oblique rays of light focused on the specimen do not enter the objective lens, but light that is scattered by components in the living cell can be collected to produce a bright image on a dark background. (C) Light passing through the unstained living cell experiences very little change in amplitude, and the structural details cannot be seen even if the image is highly magnified. the phase of the light, however, is altered by its passage through either thicker or denser parts of the cell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interference-contrast microscope.
Phasecontrast, differentialinterferencecontrast, and darkfield microscopy make it possible to watch the movements involved in such processes as mitosis and cell migration. Since many cellular motions are too slow to be seen in real time, it is often helpful to make timelapse movies in which the camera records successive frames separated by a short time delay, so that when the resulting picture series is played at normal speed, events appear greatly speeded up.
In recent years, electronic, or digital, imaging systems, and the associated technology of image processing, have had a major impact on light microscopy. Certain practical limitations of microscopes relating to imperfections in the optical system have been largely overcome. Electronic imaging systems have also circumvented two fundamental limitations of the human eye: the eye cannot see well in extremely dim light, and it cannot perceive small differences in light intensity against a bright background. To increase our ability to observe cells in these difficult conditions, we can attach a sensitive digital camera to a microscope. These cameras detect light by means of chargecoupled devices (CCDs), or high sensitivity complementary metaloxide semiconductor (CMOS) sensors, similar to those found in digital cameras. Such image sensors are 10 times more sensitive than the human eye and can detect 100 times more intensity levels. It is therefore possible to observe cells for long periods at very low light levels, thereby avoiding the damaging effects of prolonged bright light (and heat). Such lowlight cameras are especially important for viewing fluorescent molecules in living cells, as explained below.
Because images produced by digital cameras are in electronic form, they can be processed in various ways to extract latent information. Such image processing makes it possible to compensate for several optical faults in microscopes. Moreover, by digital image processing, contrast can be greatly enhanced to overcome
Figure 9–8 Four types of light microscopy. Four images are shown of the same fibroblast cell in culture. all images can be obtained with most modern microscopes by interchanging optical components. (a) Bright-field microscopy, in which light is transmitted straight through the specimen. (B) phase-contrast microscopy, in which phase alterations of light transmitted through the specimen are translated into brightness changes. (C) Differential-interference-contrast microscopy, which highlights edges where there is a steep change of refractive index. (D) Dark-field microscopy, in which the specimen is lit from the side and only the scattered light is seen.
the eye’s limitations in detecting small differences in light intensity, and background irregularities in the optical system can be digitally subtracted. This procedure reveals small transparent objects that were previously impossible to distinguish from the background.
intact tissues are Usually Fixed and sectioned Before Microscopy
Because most tissue samples are too thick for their individual cells to be examined directly at high resolution, they are often cut into very thin transparent slices, or sections. To preserve the cells within the tissue they must be treated with a fixative. Common fixatives include glutaraldehyde, which forms covalent bonds with the free amino groups of proteins, crosslinking them so they are stabilized and locked into position.