Microscopy
A typical video microscope
What do you see?
Integument pigmented skin
Staining• Increases contrast and resolution by coloring specimens
with stains/dyes• Smear of microorganisms (thin film) air dried to slide
and then fixed to surface by heat or chemical fixation• Microbiological stains are usually salts composed of
cation and anion and one is colored (chromophore)• Acidic dyes stain alkaline structures; basic dyes stain
acidic structures and are used more commonly
Staining• Simple stains • Differential stains – Gram stain– Acid-fast stain– Endospore stain
• Special stains – Negative (capsule) stain– Flagellar stain– Fluorescent stains
• Staining for electron microscopy
Simple Stains
Figure 4.16b
Immunohistochemistry
Fluorescence Microscopy
Fluorescent Microscopes
• Direct UV light source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength
• UV light increases resolution and contrast • Some cells and molecules are naturally
fluorescent, while others must be stained • Used in immunofluorescence to identify
pathogens and to locate and make visible a variety of proteins
Immunofluorescence
Figure 4.10a
Polarized Microscope
Phase Contrast Microscope
Four Kinds of Light Microscopy
Figure 4.8a
Four Kinds of Light Microscopy
Figure 4.8b
Four Kinds of Light Microscopy
Figure 4.8c
Four Kinds of Light Microscopy
Figure 4.8d
Laser scanning confocal microscopy
Electron Microscopy
• Light microscopes cannot resolve structures closer than 200 nm because shortest wavelength of visible light is 400 nm
• Electrons have wavelengths of 0.01 nm to 0.001 nm, so electron microscopes have greater resolving power and greater magnification
• Magnify objects 10,000X to 100,000X• Provide detailed views of bacteria, viruses, internal cellular
structures, molecules, and large atoms• Two types
– Transmission electron microscopes– Scanning electron microscopes
Transmission Electron Microscope
Figure 4.11
Transmission Electron Microscope (TEM) : Philips CM120
Applications:
1. Atomic structure determination (including phases distribution).
2. Particle size and shape determination.
3. Local elemental analysis.
O QUE SE VÊ?Electron micrograph of a thin section taken through an exocrine cell of the monkey pancreas. G, Golgi Apparatus; L, lumen of Acinus; M, mitochondrion; N, Nucleus, Nu, Nucleolus; RER, rough Endoplasmic Reticulum; S, secretory granule; S', secretory granule pouring its contents into lumen of Acinus; arrow, pair of Plasma membranes of two adjacent cells. 12,000 X. Inset: high-magnification electron micrograph of region indicated by arrow in which two Plasma membranes, running parallel to one another, are cut in cross section; micrograph shows the trilaminar appearance of each of the two Plasma membranes. 129,000 X
TEM Image
Figure 4.11c
Scanning Electron Microscope Philips XL-30
SEM Image
Figure 4.13a
SEM Image
Figure 4.13b
SEM Image
Figure 4.13c
SEM Image
Figure 4.13d
Bug in a leg and Human glomerulus with tuft of capillaries (C) in scanning electron microscopy.
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