Applications of phase contrast microscopy: It is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed is widely used for examining such specimens as biological tissues. It is a type of microscopy that enhances contrasts of transparent and colorless objects by influencing the optical path of light. It is able to show components in a cell or bacteria, would be very difficult to see in an ordinary light microscope Figure. (a) organelles are nearly invisible in bright although they have different refractive indexes; (b) light is bent and retarded more by objects with a high refractive index; (c) in phase contrast a phase plate is placed in the light path. Barely refracted light passes through the center of the plate and is not retarded. Highly refracted light passes through the plate farther from center and is held back another one quarter wavelength.; (d) The microscope shows a darker background (in this case the cell cytoplasm has a higher refractive index than the contractile vacuole), with the organelles in sharp contrast
Figure 1: is a cut-away diagram of a modern upright phase contrast microscope, including a schematic illustration of the phase contrast optical train. Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser focal plane. Wave fronts passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures and phase gradients present in the specimen. Undeviated and diffracted collected by the objective is segregated at the rear focal plane by a phase plate and focused at the intermediate image plane to form the final phase contrast image observed in the eyepieces. Figure 2 is a comparison of living cells in culture imaged in both brightfield and phase contrast illumination. The cells are human glial brain tissue
microtome A microtome is used for cutting thin sections. It is just a sharp knife with a mechanism that allow the advancing of a paraffin block standard distances across it. Cut sections are floated on water in a water bath at 50 C, then picked on glass slides. Sections come as a ribbon paraffin sections picked on slide. The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide Staining of sections: H & E is commonly used to stain paraffin sections: Hematoxylin is a basic stain. It stains basophilic structures such as chromatin and ribosome's a deep purple or blue. The advantage of this stain is that it provides a clear stain of the cell nuclei. Eosin is an acidic stain that stains acidophilic structures red. It provides a good stain for the cell membrane Reagents: -Hematoxylin – (Sigma Accustain Harris Hematoxylin) -Acidic Alcohol (396 ml of 95% Ethanol, 4 mls concentrated HCl) -Bluing Agent (1 liter water, 1 gm sodium bicarbonate) -Eosin - Sigma Accustain Eosin Y -75%, 95%, 100% ethanol -Histo-clear (order No. HS 200) -Permount Staining procedure: Hematoxylin – 5 minutes o Rinse in tap water – 30 seconds, to remove excess stain. o Acidic Alcohol – 5 dips, to destain and differentiate. o Rinse in tap water – 30 seconds o Bluing Agent – 2 minutes. This step is optional. The sodium bicarbonate changes the color of the hematoxylin-stained nuclei from purple to blue.
The projection is two-dimensional against the view screen and relations in the z-axis between structures are lost. It is important that the samples need to be thin, or they will absorb too much of the electron beam The layout of the electron microscope: The main parts of the electron microscope: The gun (electron source) A wire is superheated by an electric current to emit electrons. The wire is usually tungsten or lanthanum hexaboride (LaB6). Electrons are collated into a tight beam and sent down the microscope column, across the sample, lenses, and apertures Lenses Electron lenses are magnetic coils that have been designed to focus and direct a passing electron beam. Three primary lenses are used to form and magnify an image: The objective lens is the topmost lens and does the first step of image magnification and focusing. The intermediate lens and its positioning and strength controls the magnification of the image or the diffraction pattern. The projector lens used to focus and project the image onto the imaging surface. The apertures Apertures are holes along the microscope column that can limit the size of the electron beam that passes through it. The first aperture, the condenser aperture is located near the top of the column. It is used to condense and maintain the coherence of the electron beam. The second aperture, the objective aperture, is located below the sample just after the objective lens. The objective aperture is used primarily to control contrast in the image Fluorescent viewing screen coated with a or material such as zinc sulfide