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.