The centralized Tulane Imaging Center (CTIC) was created by Dr. Fermin in 1997 to aid staff unable to secured their individual digital microscopy setup. As part of the CTIC ongoing workshops to Pathology Residents, fellows and trainees, Dr. Fermin prepare a series of lectures that are posted here when appropriate. The lectures address these topics:
Microscopy, Electron and Photons
Microscopy and Digital Technology
Organelles, Enzymes and Substrates
Additional information about photography and operational details of cameras is available from Photography Tutorial and Introduction to Photography. Additional information is found from the Kodak home page. An excellent microscopy resource is Integrated Microscopy Resources.
For assistance with general imaging needs you might want to check this site, which in turn has links to microscopy and imaging resources 
Meyer Instruments.
Microscopic techniques offer many advantages to the non morphological researcher: 1) visualization of variables, 2) comparison of cell and tissue viability after experimental treatments, 3) estimation of volume change associated with osmotic pressure changes, 4) topographical distribution of markers in question, and 5) estimation of the relative concentration of markers in cells and tissues among other (Fermin and DeGraw, 1995). Microscopy however is a science of its own and its casual use without a complete understanding of at least its basic operation usually lead to unpredictable results. While a microscope appears to the eye as a simple machine, its components are not. Proper functioning of a research microscope depends on the fine tuning of hundreds of parts with a very narrow margin for error. The physical properties of glass, and the precision of metal milling greatly affect the performance of any good microscope. The technical specifications of this microscope, its workings, design, operation and application to research can not be covered in a short course of this type. Interested readers are directed to the accompanying reference materials extracted primarily from (Olympus, 1994b, Olympus, 1994c, Olympus, 1994d) and (Abramowitz, 1985, Abramowitz, 1987, Abramowitz, 1990, Abramowitz, 1993) . More details on microscopic techniques are found in (Inoue, 1987, Shotton, 1992) , and for confocal microscopy (Pawley, 1989).
The compound research and the stereo dissecting microscopes can be operated efficiently after a thorough review of the manuals provided by the manufacturers. A good compound upright research microscope is modular, permitting interchange of components and parts to obtain bright field, phase contrast, polarizing, Normanski interference contrast, Hoffman modulation contrast, fluorescent, and dark field microscopy. Each format has specific applications. Inverted microscopes that are used to visualize objects from under the surface of a container are less flexible in accommodating all formats above.
The main purpose of microscopy is to amplify objects. Aside from the technical specifications of a microscope components, which as mentioned above are dependent upon the physical properties of the materials used, the quality of an image depends greatly on the nature of the light used. The maximum resolution that photons of light can provide is 0.2µm. Any required enhancement of an image below this resolution is easily accomplished today with inexpensive computers and affordable video imaging systems (Fermin, 1995, Fermin and DeGraw, 1995, Fermin et al., 1992) . In light microscopy ctual (as opposed to empty) resolution below 0.2µm is provided by the shorter wavelength of electrons used to generate images in the electron microscope (EM). Unfortunately, for EM specimens must be thin enough to permit electrons to pass across the sections, the tissue must be generally included in hard media like epoxy, methacrylate plastics, etc. and can not contained water, water vapors or grease, except in cryo-electron-microscopy. A separate workshop addresses the differences between light and electron microscopy and their requirements.
Light microscopic images can be enhanced by many microscopic formats. Confocal (CM), Scanning Tunneling (STM) and Atomic Force Microscope (AFM) are some of the most recent formats that greatly enhance images without the requirements imposed by electron microscopy. Confocal microscopy is based on the ability of this format to reject out-of-focus portions of the specimen (Pawley, 1989, Shuman et al., 1989) . The STM and the AFM utilizes atomic size tips to resolve atoms, and contrary to the Scanning Electron Microscope (SEM), the STM and AFM, do not require conductive samples for generating the image (Drake et al., 1989, Hansma et al., 1988).
The fluorescent microscope utilizes a mercury lamp to generate ultraviolet irradiation instead of the standard tungsten or halogen light sources. The light emitted by the mercury lamp is not visible to the human eye without filtration. For this reason a fluorescent labeled object will have little contrast and appear unstained if viewed with regular light. Generally, a phase condenser and objectives are incorporated into fluorescent microscopes in order to view unstained specimens with regular light. Thus, in fluorescent microscopy, besides the power output of the lamp (watts), filtration is probably the most important consideration. Filters are however not perfect nor do they always permit the precise separation needed by most researchers. There are exciter and barrier filters, and as their names imply they enhance and block light respectively. A combination between these exciter and barrier filters, in conjunction with a bright lamp and a good quality fluorescent probe, allows restricting the bandwidth that reaches the film needed to produce high quality photomicrographs. Consequently, fluorochromes must be matched with the available filters set (Johnson, 1991, Olympus, 1994a) , or additional filters purchased to match each fluorochrome.
In this respect, recording of images obtained with a microscope requires a good knowledge of film exposure and development. To acquire the knowledge necessary to understand film and filter specifications refer to (Kodak, 1980, Kodak, 1981a, Kodak, 1981b, Kodak, 1981c) , which are excellent references. Suffice to remember that black and white films sensitive to all colors are called panchromatic, whereas those sensitive to one color or region of the spectrum are called ortochromatic. Thus, panchromatic films are the choice for recording objects of graded density (white-gray-black), whereas orthochromatic films are the best choice for line drawing (charts and tables). Both are available in rolls and professional thick based emulsions. Exposure of black and white films does not depend on the temperature of the light (intensity) but rather on the sensitivity of the film and filters used.
Color films are either daylight sensitive (hot sun or 5500 Kş) or tungsten light sensitive to incandescent light from tungsten or halogen sources (indoor-cool or 3200-3400 Kş). Soft white fluorescent light is skewed toward the green portion of the spectrum and, photos taken under such lights without corrective filters appear greenish. A daylight film exposed outdoors under bright light produces a balance negative that does not require adjustments during printing. However, the same film exposed with the tungsten or halogen source found in most microscopes and copy stands needs an 80B (blue filter) or the photo will be too blue. Conversely, a tungsten film exposed to the bright sun light without an 80A filter will result in photos, that if printed without compensation, will be too reddish-yellow. Thus, if you exposed film to the wrong light source you must inform the processing laboratory so that prints are compensated for (sometimes this is not possible).
For recording fluorescent images, one must remember the properties of light and color films and also the variable time required for exposure of the film. First. fluorescent signals fade quickly even when mounted with antifading compounds. Second, the film emulsion is designed for the temperatures mentioned above which are able to penetrate evenly the color layers of the film. Generally, the speed of the film (ISO, old ASA) should be 400 or higher (except in unusual circumstances) and the reciprocity of the emulsion must be carefully adjusted. Reciprocity is the adjustment made to the automatic exposure device to compensate for: a) partial filling of the frame, and b) for the ideal time required to properly expose the emulsion. For instance, if a fluorescent signal occupies only 25% of the camera frame (field of view), then the exposure should be approximately 25% that shown on the automatic exposure meter. But in general the correct exposure can only be gauged by trial and error. The best remedy is to chose a subject that best represents the experimental condition, and to take a series of pictures at the indicated meter reading and several steps below and above. A good approximation for exposure compensation is that 2 seconds of exposure will generally compensate for one f-stop (e.g., from f8-11). Furthermore, a well exposed black and white negative of good density and contrast (e.g., ISO 100 film exposed outside at noon at f11 for 1/125 sec) will usually produce a good print on paper F-2 when exposed for 2 sec at f-8. These are not rules. They are only starting points from which to go on!
Abramowitz M. Contrast methods in microscopy. Transmitted light.New York: Olympus Co., 1987: (Abramowitz M., ed. Basic and Beyond; 2).
Abramowitz M. Reflected light microscopy. An Overview.New York: Olympus Co., 1990: (Abramowitz M., ed. Basic and Beyond; 3).
Abramowitz M. Fluorescence Microscopy. The essentials.New York: Olympus Co., 1993: (Abramowitz M., ed. Basic and Beyond; 4).
Drake B., Prater C.B., Weisenhorn A.L., et al., Imaging crystals, polymers, and processes in water with the atomic force microscope. Science. 1989, 243: 1586-1589.
Fermin C., Anatomy, histology and color thresholding. Microscopy Today. 1995, 95-5: 16.
Fermin C., DeGraw S., Color Thresholding in video imaging. J. Anat. 1995, 186: 469-481.
Fermin C.D., Gerber M.A., Torre-Bueno J., Colour thresholding & objective quantification in bioimaging. J. Microscopy. 1992, 167: 85-96.
Hansma P., Elings V., Marti O., Bracker C., Scanning tunneling microscopy and atomic force microscopy: Application to biology and technology. Science. 1988, 242: 209-216.
Inoue S. Video Microscopy.New York: Plenum Press, 1987:
Johnson I. Optical Properties of Fluorescent Probes. In: Larison K.D., ed. Handbook of Fluorescent Probes and Research Chemicals. 5th ed. Eugene, OR: Molecular Probes, Inc. (RP Haugland), 1991: 1-15. 1992-1994).
Kodak. Photography through the microscope. (8th ed.) NY: Eastman Kodak Company, 1980: 96 pages. (Delly J., ed. The Kodak Workshop Series; Publication P-2).
Kodak. Kodak Color Films. (8th ed.) NY: Eastman Kodak Company, 1981a: 95 pages. (Ferguson B., ed. The Kodak Workshop Series; Publication E-77).
Kodak. Kodak filters for scientific and technical uses. (3er ed.) NY: Eastman Kodak Company, 1981b: 95 pages. ; Publication B-3).
Kodak. Using Filters. (8th ed.) NY: Eastman Kodak Company, 1981c: 95 pages. (Doeffinger D., ed. The Kodak Workshop Series; Publication KW-13).
Olympus. Applications of fluorescence microscopy Microscopy userıs manual (M19ES-9409). Olympus Optical Co., LTD. New York, 1994a.
Olympus. Basics of the optical microscope Microscopy userıs manual (M135E-0992B). Olympus Optical Co., LTD. New York, 1994b.
Olympus. How to improve photograpy throught the microscope Microscopy userıs manual (M132E-0992T). Olympus Optical Co., LTD. New York, 1994c.
Olympus. The use of the olympus fluorescence microscope Microscopy userıs manual (M131E-0592T). Olympus Optical Co., LTD. New York, 1994d.
Pawley J., ed. The Handbook of Biology Confocal Microscopy. Madison, WI: IMR Press, 1989:201.
Shotton D. Electronic Light Microscopy. Techniques in modern biomedical microscopy. (First ed.) New York: Wiley-Liss, 1992:
Shuman H., Murray J., DiLullo C., Confocal microscopy: An overview. BioTechniques. 1989, 7: 154-164.
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