Microscopy Techniques (PDF)

1. Microscopy 1.5 Light Microscopy and the Discovery of Microorganisms 1.6 Improving Contrast in Light Microscopy 1.7 Imaging Cells in Three Dimensions 1.8 Probing Cell Structure: Electron Microscopy  Light microscopy enabled microbiology Robert Hooke (1635– 1703): first to desc...

1. Microscopy 1.5 Light Microscopy and the Discovery of Microorganisms 1.6 Improving Contrast in Light Microscopy 1.7 Imaging Cells in Three Dimensions 1.8 Probing Cell Structure: Electron Microscopy  Light microscopy enabled microbiology Robert Hooke (1635– 1703): first to describe microbes (Micrographia in 1665) illustrated the fruiting structures of molds https://micro.magnet.fsu.edu/primer/museum/hooke.html Antoni van Leeuwenhoek (1632–1723): first to describe bacteria (Figure 1.14)  Light microscopy parameters magnification: the ability to make an object larger resolution: the ability to distinguish two adjacent objects as distinct and separate Limit of resolution for light microscope is about 0.2 μm (half of the wavelength of the visible light). Several types bright-field phase-contrast differential interference contrast dark-field fluorescence  Compound microscope Two sets of lenses form the image objective lens (magnifies 10–100x) and ocular lens (magnifies 10–30x) total magnification = objective magnification x ocular magnification magnification of 1,000x needed for 0.2 μm diameter resolution (limit for most light microscopes) green alga Bright-field microscope: under normal light, specimens visualized Direct observation because of differences in contrast of colored microbes (density) between specimen and surroundings Purple phototrophic bacteria  Microscopy & Staining Staining improves contrast Dyes are organic compounds that bind to specific cellular materials. basic dyes: positively charged, bind strongly to negatively-charged cell components (e.g., nucleic acids, acidic polysaccharides, cell surfaces) examples: methylene blue, crystal violet, and safranin Simple stain uses dried cells. (Figure 1.17)  Differential stains: Different kinds of cells are different colors. example: gram stain (Figure 1.18) differences because of cell wall structure Bacteria can be divided into two major groups: gram-positive and gram-negative. Gram-positive bacteria appear purple-violet, and gram-negative bacteria appear pink. (Figure 1.18b)  Dr Huang’s gram staining a mixture of E. coli and S. aureus Pseudomonas aeruginosa (gram-negative, green) and Bacillus cereus (gram- positive, orange)  Phase-contrast microscopy (Figure 1.19) improves image contrast of unstained, live cells Phase ring amplifies differences in the refractive index of cell and surroundings. Resulting image—dark cells on a light background (Figure 1.19b) Bright field Phase-contrast Darkfield the yeast Saccharomyces cerevisiae  Dark-field microscopy Light reaches the specimen from the sides. Only light reaching the lens has been scattered by specimen. Image appears light on a dark background. (Figure 1.19c) better resolution than light microscopy excellent for observing motility (flagella) https://jcm.asm.org/content/jcm/4/3/258.full.pdf  Fluorescence microscopy used to visualize specimens that fluoresce (emit light after illumination with different wavelength) cells appear to glow on black background due to filters. fluoresce naturally (autofluorescence) or after they have been stained with a fluorescent dye such as DAPI widely used in microbial ecology for enumerating bacteria in natural samples Fluorescent staining of E. coli SYTO 9 cell penetrating dye showing live cells Propidium iodide (PI, a DNA dye) showing dead cells