MICROSCOPY Interaction of probe used (photons: light, phase contrast, polarizing & fluorescence microscopy; electron beams: EM), and tissue components produce image. Considerations in microscopic analysis: that the probe being utilized must not be larger than the detail to be seen that the probe and object being investigated must interact it must be possible to observe and interpret this interaction Units for measuring microscopic dimensions:
IMPORTANT TERMS IN MICROSCOPY Magnification – increases the apparent size of the specimen; a property of both ocular & objective lenses Numerical aperture – a measure of the size or angle of the cone of light delivered by the illuminating condenser lens to the object plane and of the cone of light emerging from the object. Resolving power – a measure of linear distance of the smallest degree of separation at which 2 details can still be distinguished from each other; dependent on quality of objective lens; R also varies according to the refractive index at the interface of the media used Resolution becomes a problem in microscopes with high magnification Powerful microscopes have higher numerical aperture resulting to improved resolution.
TYPES OF MICROSCOPES Anton van Leeuwenhook1.Light microscopes- (1632-1723) compound, dissecting, brightfield, and phase-contrast Best resolution is 0.2 µm. Maximum magnifications are between 1000X and 1250X.
Compound microscopes bring small objects "closer" to the observer by increasing the magnification of the sample. Since the sample is the same distance from the viewer, a "virtual image" is formed as the light passes through the magnifying lenses.
Phase contrast microcopy uses a lens system that changes light speed as it passes through structures with different refractive indices The phase of the light is altered by its passage through the cell, and small phase differences can be made visible by exploiting interference effects Phase-contrast and differential interference optics produce 3-D images of transparent living cells, tissues
2.Fluorescence microscopy– uses strong UV light source that irradiate substances dyed with fluorescent stains, e.g. acridine-orange These appear as brilliant, shiny particles on a dark background; useful for identifying & localizing NA in cells Fluorescence spectros- copy analyzes light emitted by fluorescent compounds in a micro- spectrophotometer This permits highly sensitive assays of cellular substances such as catecholamines
The confocalmicroscope produces opticalsections by excludingout-of-focus light
3. Polarizing Compact microscopy– bone birefrigent substances rotate direction of polarized light emerging from polarizing filters Useful for visualizing substances with repetitive, oriented molecular Collagen fibers, structures polarizing microscopy
4.Electron microscopy– uses high energy electron beams (between 5,000-109 electron volts) focused through electromagnetic lenses. Interaction of electrons deflected by lenses beamed on tissue components permits high resolution (0.2 - 1 nm) and 400x greater magnification than light microscopes The increased resolution results from the shorter wavelength of the electron beam Disadvantages of EM: requirements of a vacuum-enclosed system, high voltage, mechanical stability; special treatment & sample preparation make it highly complex and costly; requires the services of well-trained personnel
Specimen Preparation for EM Fixation in osmium tetroxide/ dichromate, acrolein and glutaldehyde. Since registration of color is not possible with the EM system, staining with colored dyes is not done in EM studies.
Scanning vs. Transmission EM: In the TEM, the image is formed directly on the image plan In the SEM, the image is formed indirectly by accumulation of information from the specimen point by point There is no need to cut ultra thin sections because the beam of the SEM does not pass through the specimen. The resolution of the SEM is about 100 Angstrom vs. 4-5 Angstrom achieved by the transmission type. The SEM has great depth of field making it possible to obtain 3-D images. TEM magnifications are commonly over 100,000X SEM displays images on high resolution TV monitors.
SEM: T-lymphocyte, E.coli TEM: mitochondria &attacked by macrophage chloroplast
Freeze-cleaving, Freeze- etching or Cryofracture methods Used with EM; replicas are made of surfaces of frozen aqueous materials at very low temperatures in vacuo The use of chemical fixatives, dehydrating and embedding agents are avoided by using a freezing microtome/cryostat which permit sections to be obtained without embedding
Freezing does not inactivate most enzymes, hinders diffusion of small molecules, eliminates dissolution of tissue lipids by solvents The tissue is impregnated with a 25% glycerol solution chloroplast thylakoid membranes before rapid freezing in liquid nitrogen or Freon12 at 1000C to 1550C. Not entirely free of artifacts; valuable in the study of membranes and their junctional specializations. vesicles
ISOLATING CELLSAntibody is coupled to a fluorescent dye to label specific cells. Labeledcells are then sorted out in a fluorescence activated cell sorter.Individual cells traveling single file in a fine stream pass through a laser beam and the fluorescence of each is rapidly measured. Fluorescent cells are deflected by a strong electric field into an appropriate container.
In laser capture microdissection, selected cells isolated from tissue slices are coated with a thin plastic film, and a region ofinterest is irradiated with a laser beam. This melts the film, and captured cells are removed. A related method uses a laser beam to directly cut out a group of cells and catapult them into a container. Cells can be cultured, cytoplasm and organelles extracted, or specific molecules purified for analysis.
CELL FRACTIONATIONHomogenization/ differential centrifugation separate organelles based on their sedimentation coefficients. The latter depends on its size, form and density, and viscosity of the medium.
A mixed organelle fraction can be further separated by equilibriumdensity gradient centrifugation. Pure fractions of organelles can be biochemically studied and analyzed for purity under EM.
Electron micrographs of 3 cell fractionsisolated by density gradient centrifugation. A: Mitochondrial fraction, contaminated with microsomes. B: Microsomal fraction. C: Lysosomal fraction.
CELL AND TISSUE CULTURE• Cells/tissues are dispersed mechanically or chemically (treatment with trpysin or collagenase), & grown in chemically defined synthetic media to which growth factors, hormones and serum components are added.• Cell and tissue culture Epithelial cell culture: keratin techniques permit direct (red). DNA (green) analysis of cell behavior.• Used also for studies of cellular parasites, metabolism of normal and cancerous cells; cytogenetic research, molecular biology and recombinant DNA technology, stem cell research.
Because they contain rapidly growing cells, embryos ortumors are frequently used asstarting material. The cells are dispersed into a suspension and added to a culture dishcontaining nutrient media. The cells in a primary culture usually grow until they cover the culture dish surface.Normal human fibroblasts can usually be cultured for 50 to100 population doublings, after which they stop growing and die. In contrast, cells derived from tumors frequently proliferate indefinitely in culture and are referred to as immortal cell lines.
MICROSURGERY Cultures provide cells free of CT and spread out on a glass surface, so that they are accessible to microsurgical procedures. Extremely minute instruments as microneedles, microhooks, and micropipettes are devised. These are positioned within an operating chamber on the stage of the compound microscope by mechanical micromanipulators capable of achieving controlled movements in various planes.
Reproductive cloning Donor cell Nucleus from donor cell Implant embryo in Clone of surrogate mother donor is born Therapeutic cloning Remove Grow in culture Induce stem Add somatic cell Remove embryonic cells to form nucleus nucleus from to produce an stem cells from specialized cellsfrom egg adult donor to early embryo embryo and grow for therapeutic cell enucleated egg in culture use cell Transplantation & explantation techniques used in grafting experiments as well as embryo transfer utilize this method.
IRRADIATION Selective irradiation of small areas of living cells using microbeams of protons, UV beams, & high power organ lasers produce discrete lesions in chromosomes or other cell components without previous sensitization with a vital dye. By irradiation, it is possible to achieve the selective destruction of specific cell organelles and to assess its effect upon the cell as a whole.