Microscopy

Microscopy Techniquesobjective

light source

“transmitted”

Light microscope - transmitting

Microscopy Techniqueslight source/objective

reflecteddissecting microscope

Stereo or dissecting microscope

Microscopy Techniqueslight source/objective

incident/epi-illuminationfluorescent

Fluorescence

• fluorescence = process of excitation, loss of energy & emission of light from a fluorophore

Fluorescence Microscopy

Contrast

• contrast = difference in color & light between parts of an object/image – required to see an object by microscope– can come from variations in• Intensity (DIC, Phase)• color (bright field, fluorescence)

• http://micro.magnet.fsu.edu/primer/techniques/dic/dicphasecomparison.html

Contrast

• cells typically are– transparent (not amplitude objects)– phase objects– low in contrast

• contrast-generating techniques turn phase differences into intensity differences so we can see unstained cells using transmitted light– DIC (Differential Interference Contrast) microscopy

– http://learn.hamamatsu.com/galleries/digitalvideo/spinningdisk/folu473laser/FoLu-EGFP-EB3-100x-1s.html

• In Figure 1(c), a thick section of murine kidney tissue imaged with differential interference contrast reveals a bundle of cells enclosed within a tubule. A phase contrast image (Figure 1(d)) of the same area is confusing and disturbed by the presence of phase halos outside the plane of focus.

• A human buccal mucosa epithelial (cheek) cell, revealing the nucleus, cytoplasmic inclusions, and numerous bacteria on the upper surface, is presented in Figure 1(a), imaged with differential interference contrast. The same viewfield with phase contrast illumination is illustrated in Figure 1(b). The phase contrast image features pronounced halos around the cellular periphery and nucleus, which are absent in the differential interference contrast image.

• Relatively high magnification views of an Obelia polypoid annulated stem perisarc in DIC and phase contrast are illustrated in Figures 1(e) and 1(f), respectively. In DIC, (Figure 1(e)) the annular structure appears hemispherical with internal rays that emanate radially from the stem. In addition, granular particles are visible within the stem structure, but anatomical detail is largely undefined. The phase contrast image (Figure 1(f)) is confused by halos around the annular rings and within the stem.