Microscopy Textbook Notes 2.

2.1: Some Principles of Light Microscopy o Light microscopes used to examine cells at relatively low magnifications o Electron microscopes used to look at cells and cell structures at very high magnification o The Compound Light Microscope Uses visible light to illuminate cell structures Bright-Field Microscope: o Specimens are visualized because of the slight differences in contrast that exist between them and their surrounding medium o Bacterial cells are difficult to see well because the cells themselves lack significant contrast with their surrounding medium o Pigmented microorganisms are an exception because the color of the organism itself adds contrast Highest magnification: Highest resolution: 2000x 0.2 m

Improving Contrast in Light Microscopy o Staining: Increasing Contrast for Bright-Field Microscopy Basic dyes bind strongly to negatively charged cell components Because cell surfaces tend to be negatively charged, these dyes also combine with high affinity to the surfaces of cells Very useful general purpose stains Staining kills cells and can distort their features Two forms of light microscopy improve image contrast without the use of stain and dont kill cells: Phase-Contrast Microscopy: o Widely used in teaching and research for the observation of wet-mount (living) preparations o Based on the principle that cells differ in refractive index from their surroundings o Light passing through a cells differs in phase from light passing through the surrounding liquid 1

o Phase-Contrast and Dark-Field Microscopy

This subtle difference is amplified by the phase ring resulting in a dark image on a light background

Dark-Field Microscopy: o A light microscope in which light reaches the specimen from the sides only o Only light that reaches the lens is that scattered by the specimen, and thus the specimen appears light on a dark background o Better resolution than by light microscopy o Excellent way to observe microbial motility bundles of flagella are observable

o Fluorescence Microscopy Used to visualize specimens that fluoresce Used in clinical diagnostic microbiology and also in microbial ecology for enumerating bacteria in a natural environment or in a cell suspension

2.3:

Imaging Cells in Three Dimensions A form of light microscopy that employs a polarizer in the condenser to produced polarized light (light in a single plane) Interference effect visibly enhances subtle differences in cell structure Typically used for observing unstained cells because it can reveal internal cell structures that are nearly invisible by the bright-field technique Has advantage that specimen does not have to be treated with fixatives or coatings Allows living specimens to be viewed not possible with electron microscopes A computerized microscope that couples a laser source to a fluorescent microscope Generates a 3D image and allows the viewer to profile several planes of focus in the specimen

o Differential Interference Contrast Microscopy

o Atomic Fore Microscopy

o Confocal Scanning Laser Microscopy

Using CLSM is has been possible to improve on the 0.2 m resolution of the compound light microscope to a limit of about 0.1 m Useful anywhere thick specimens are assessed for microbial content with depth

2.4:

Electron Microscopy

o Use electrons instead of visible light to image cells an cell structures o Electromagnets function as lenses o Whole system operates in a vacuum o Transmission Electron Microscopy Used to examine cells and cell structures at very high magnification and resolution Enabling one to view structures at the molecular level o This is because the wavelength of electrons is much shorter than the wavelength of visible light and wavelengths affect resolution o Individual protein and nucleic acid molecules can be visualized in the TEM Unlike visible light, electron beams do not penetrate very well Even a single cell is too thick to reveal its internal contents directly by TEM

o Scanning Electron Microscopy Specimen is coated with a thin film of heavy metal An electron beam then scans back and forth across the specimen Even fairly large specimens can be observed, and the depth of the field is very good

o Electron micrographs taken either by TEM or SEM are black and white images

Microscopy Lab Notebook Bright-Field Microscope o Contains two-lens system o Specimen is illuminated by a beam of tungsten light focused on it by a condenser Result: Specimen appears dark against a bright background o Major Limitation: The absence of contrast between the specimen and the surrounding medium Making it difficult to observe living cells o Mostly performed on nonviable, stained preparations Dark-Field Microscope o Condenser system is modified so that the specimen is not illuminated directly Condenser directs the light obliquely so that the light is deflected or scattered from the specimen, which then appears bright against a dark background

o Living specimens may be viewed readily Phase-Contrast Microscope o Possible to observe unstained microorganisms discern cellular components Fluorescent Microscope o Used most frequently to visualize specimens that are chemically tagged with a fluorescent dye o Used primarily for the detection of antigen-antibody reactions Electron Microscope o Provides magnifications up to 1 million x o TEM: o SEM: Used for visualizing surface characteristics Make internal structures visible This permits the visualization of submicroscopic cellular particles as well as viral agents