Compound Light Microscopy This is what we use in the laboratory Microscopy Chapter 3 BIO 440 A series of finely ground lenses is used to form a magnified image Specimen is illuminated with visible light Things to remember... Magnifying power ratio of the object s apparent size when viewed through a lens compared to the appearance of the object when viewed with the naked eye from a distance of 25 cm How to calculate the total magnification of a specimen Fig. 3.1 1
Things to remember... Resolution ability of the lenses to distinguish fine detail and structure (or, to distinguish between objects that are close together) Our microscopes can distinguish between objects that are as little as 0.2 μm apart Resolution is dependent on Wavelength of light (shorter the λ, greater the res) Numerical aperture (ability of lens to gather light) Fig. 3.3 Things to remember... Contrast refers to the differences in color and light between the parts of an object or between an object and its environment An unstained cell or organism has little contrast with its surroundings and thus is difficult to see Brightfield illumination This is what we use in lab FOV is brightly illuminated Dark specimen on bright background Fig. 3.4a 2
Darkfield Microscopy Specimen appears light on a dark background Handy in situations where we want to see live cells, cells are hard to see in light or do not stain well Treponema pallidum causative agent of syphilis Phase-Contrast Not necessary to fix or stain specimens, so allows us to see living microbes Better cellular detail Separate illuminating light from light refracted off of specimen Light travels faster through thinner areas and structures appear brighter Light travels slower through thicker areas and structures appear darker Fig. 3.4b Fig. 3.4c Differential Interference Contrast (DIC) Uses two beams of light which pass through prisms Resolution better than standard phasecontrast Image brightly colored and appears 3D Fluorescence Microscopy Takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light (UV) and give off light at a longer spectrum Some organisms fluoresce naturally under UV light, those that do not can be stained with fluorochromes (i.e. fluorescein isothiocyanate - FITC) Bright microbes on dark background Fluorescent antibody (FA) technique (aka immunofluorescence) Fig. 3.5 3
Immunofluorescence Fluorescent antibodies can be used to detect the presence of specific antigens Animal is injected with a specific antigen, and then antibodies are harvested, then Confocal Microscopy Specimens are stained with fluorochromes Short wavelength (blue) laser light passes through specimen Planes of the specimen are captured by the microscope, and can then be used by a computer to construct a 3D image Fig. 3.6 Electron Microscopy A beam of electrons, rather than beam of light, is used to eliminate specimen Can be used to visualize objects smaller than 0.2 μm (viruses or internal cell structure) λ of electrons is much shorter than λ of light, so MUCH higher resolution Uses electromagnetic versus glass lenses to focus light on specimen 4
Transmission electron microscopy Visualize viruses or internal intrastructure Ultrathin sections of specimen (100 nm) Image produced on fluorescent screen or photographic plate 10,000X 100,000X magnification High resolution (10 pm) Can use stains (mineral salts) to enhance contrast Black and white images, 2D Scanning electron microscopy Visualize surface features of cells and viruses Image produced on viewing screen or photographic plate 1,000X 10,000X magnification High resolution (10 nm) Black and white images, 3D Preparation of microscopic specimens Staining helps us localize and visualize small, colorless microbes on microscope slides But before any staining occurs, we must first fix the specimen Fixing the specimen Adhesion of the specimen to the slide Kills the specimen Preserves specimen with minimal distortion 5
Make a smear Air dry/slide warmer Heat fixation Pass the slide (specimen side up) through the flame of a Bunsen burner several times Once the heat fixation is completed, you can go ahead and apply stain to the slide Stains Stains are salts that are composed of a cation and anion One of those ions is colored, and is referred to as the chromophore Basicdyes are cationic, meaning that the chromophore has a positive charge Crystal violet, methylene blue, safranin Acidicdyes are anionic, meaning that the chromophore has a negative charge Eosin, nigrosin Simple stains An aqueous or alcohol solution of a single basic dye Good for visualizing cell shape and arrangement Differential stains Use more than one stain to differentiate cellular components Used to visual structural differences between different types of bacteria Gram stain Acid-fast stain Mycobacterium tuberculosis Mycobacterium leprae 6
Gram stain Primary stain Crystal violet Mordant Gram s iodine Decolorizer Counterstain - Safranin Special stains Used to color parts of microorganisms Capsule stain Endospore stain Flagellar stain 7