Microscopy Techniques for Cell Analysis PDF

Differential interference contrast (DIC) microscopy uses a polarizer to create two distinct beams of polarized light (light in single plane) gives structures such as nuclei, endospores, vacuoles, and inclusions a three-dimensional appearance (Figure 1.21)  1.7 Imaging Cells in...

Differential interference contrast (DIC) microscopy uses a polarizer to create two distinct beams of polarized light (light in single plane) gives structures such as nuclei, endospores, vacuoles, and inclusions a three-dimensional appearance (Figure 1.21)  1.7 Imaging Cells in Three Dimensions Pseudomonas aeruginosa experimentally introduced into the biofilm Confocal scanning laser microscopy (CSLM) uses a computerized microscope coupled with a laser source to generate a three-dimensional image (Figure 1.22) Computer can focus the laser Filamentous cyanobacterium on single layers of the specimen. Different layers can then be compiled for a three- dimensional image.  Electron Microscopy Electron microscopes use electrons instead of visible light (photons) to image cells and structures (Figure 1.23) electromagnets function as lenses operates in a vacuum camera takes a picture = electron micrograph Two types: transmission electron microscopes (TEM) scanning electron microscopes (SEM) Figure 1.23  Transmission electron microscopy (TEM) much greater resolving power (0.2 nm) than light microscope (200 nm) enables visualization of structures at the molecular level Specimen must be very thin (20–60 nm) and stained with high atomic weight substances that scatter electrons well and improve contrast. Negative staining allows direct observation of intact cells/components.  TEM of a dividing bacterial cell TEM of negatively stained molecules of hemoglobin Scanning electron micrograph (SEM) Figure 1.24  Cryo-electron microscopy (cryo-EM) for dissecting protein structure Vu Q Nguyen, …, Andres E Leschziner, Cell, 2013 A modern cryo-EM specimen consists of an aqueous film that spans holes in a thin carbon film. The grid is rapidly plunged into liquid ethane. Frozen by liquid nitrogen. Then the cryo-samples are visualized at -170 °C, and 3D structures can be determined by analyzing thousands of individual protein particle images by sophisticated computer algorithms. Can be used for very large protein complexes e.g. of hundreds of kDa http://faculty.washington.edu/lw32/cryoem_home.php  Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) Specimen is coated with a thin film of heavy metal (e.g., gold). An electron beam scans the object. Scattered electrons are collected and projected to produce an image. (Figure 1.24c) Even very large specimens (centimeters) can be observed. magnification range of 15–100,000x only surface visualized 2. Growth media and laboratory culture Culture media (Table 5.1) nutrient solutions used to grow microbes in the laboratory typically sterilized in an autoclave Two broad classes: defined media: exact chemical composition known. complex media: composed of digests of microbial, animal, or plant products (e.g., yeast, meat extracts, serum), blood cells  Yeast extracts: cell contents of yeast Peptone: enzymatic digest of animal meat Tryptone: digestion of casein (milk protein) by the protease trypsin.