Very few research instruments have as widespread a usage in science as do microscopes. I will present a very brief and readily understandable description of microscopy and the many different types of microscopes in this short series of articles. These are not in-depth discussions, but rather are designed to provide a good introductory background about microscopy for teachers, technicians, students, parents, and other beginning users. Since I want to keep everything concise and suitable for non-experts, I will not utilize any of the usual optical equations and mathematics, ray path diagrams, or standard instructions about using microscopes!
The initial article in this series gave an overview of the most fundamental concepts and terms for using microscopes and understanding microscopy (see: “Part 1: Fundamentals for Beginners” ). This second Part examines light microscopes and light microscopy; all beginners are urged to first study Part 1. The fun of microscopy is pointed out throughout the entire series!
Different kinds of light microscopes: the dissecting light microscope.
The dissecting light microscope uses white light reflected from the surface of solid specimens to achieve a more moderate level of magnification and resolution than are obtained with compound light microscopes. The light source can be a ring lamp along the optical axis, giving illumination from all directions, or, a single focused light (i.e., a spotlight) that can be moved to shine onto soecimens at different angles and orientations. For examination of whole mounts or cut surfaces with a dissecting light microscope, the natural specimen thickness often is good. Only natural colors are observed (i.e., biological samples usually are not stained). The dissecting light microscope is valuable for showing finer details on natural surfaces or those produced by grinding or cutting. It provides higher magnifications and better resolution than are given by magnifying glasses, but gives lower magnifications and poorer resolution than are produced by compound light microscopes. It is always valuable for scientists to look at whole specimens with a dissecting light microscope before examining thin slices or polished specimens from the same sample at higher magnifications with a compound light micoscope.
Different kinds of light microscopes: the common compound light microscope.
The major parts of standard compound light microscopes are shown in the schematic diagram given above under the title. The mixture of many different colored waves within white light is used for the most common light microscopes. Better resolution is obtained if only a single wavelength of light is used (i.e., monochromatic waves, such as only green or only blue); the light waves with the smallest wavelength (e.g., ultraviolet waves) give the best optical resolution. Unlike the single lens in a magnifying glass, the common light microscopes have several compound lenses, each of which includes a group of several different single lenses arranged to function coordinately. This design with grouped lenses serves to increase useful magnifications, decrease optical aberrations, and provide ready resolution of small details in specimens.
The lenses in compound light microscopes are constructed from special glasses by industrial lensmakers. Typically, waves from a light source (e.g., a lamp bulb or LED) are focused onto the specimen by a condenser lens (assembly). Focus for all compound lenses is adjusted by moving either the assembly or the specimen up or down the optical axis; this is the same action used to focus a magnifying glass. Specimens commonly are mounted onto thin glass slides, which are held over a hole in the stage. Lateral movements of the slides are produced by the stage controls. After passing through a thin or transparent specimen where some absorption takes place, the transmitted waves then are focused by the objective lens (assembly) onto either the eyepiece lenses (monocular or binocular), or a viewing screen. The focused images are recorded by a detector using either photography or a digital camera. Magnifications typically run up to around 1,000 times natural size; the several objective lens assemblies mounted on a turret provide different amounts of magnification.
Preparation of specimens for light microscopy
Samples for a compound light microscope must be sufficiently thin to allow light waves to be transmitted through them (i.e., several micrometers thick). Typical specimens are several micrometers thick. Biological samples from organs typically are chemically fixed (e.g., buffered formalin), dehydrated to dryness, embedded into paraffin or a polymer, sectioned with a microtome (i.e., a special cutting machine), mounted onto glass slides, and finally stained with a general or specific chemical procedure; the added coloration enhances recognition of different tissues, cell types, and substructural elements. Physical materials often are ground into a thin layer or crushed into small particles; these then are mounted onto glass slides.
Specimen preparation is particularly important for determining what kinds of information can be produced by light microscopes. Thin samples usually are either whole mounts of very small objects (e.g., microbes, pollen, blood cells, microparticles produced by crushing, strands of polymers, etc.), or sections/slices through larger objects (e.g., slices 7 micrometers thick from larger fixed and embedded pieces of organs or other soft biomaterials; thin discs or wedges prepared from mineral or metallurgical samples). Various types of chemical treatments for specimens enable selective information to be acquired (e.g., precipitants, stains, immuno-cytochemistry, etc.). Recorded images of serial sections can be processed to show the third dimension of the specimen being examined.
Different kinds of light microscopes: more specialized light microscopes.
Phase contrast light microscopes contain objective and condenser lens groups with special optics so that changes in the phase of light waves passing through specimens are detected as a difference in contrast. By doing this, otherwise invisible parts of transparent objects (e.g., living cells, polymeric sheets, unstained sections of biomaterials, etc.) can be made visible and studied. The mounting of several different objective lenses upon a turret makes it easy to examine the same specimen alternatively with phase contrast or standard imaging. These microscopes are used widely with optically transparent specimens (e.g., cultured cells, protozoa, glasses, fibers, and polymers); staining usually is not utilized.
Polarizing light microscopes illuminate specimens with polarized light and have special lens systems that can detect smaller regions within larger specimens that have a different degree or orientation of ordering. With ordinary (i.e., unpolarized) white light, these differences are not visible. Polarizing light microscopes can observe ordered components within natural materials, sliced specimens, or thinned samples.
Fluorescence light microscopes image specimens treated wuth special dyes (e.g., stains) emitting fluorescence when illuminated by certain wavelengths. These compound light microscopes feature several optical filters designed to remove background intensity; the resultant images show one or several brilliant colors coming from the dyes utilized.
Confocal light microscopes utilize illumination from laser light source(s) with digital recording and computer processing to provide good images of small details within thicker volumes or slices.
Several other types of light microscopes use more optical arrangements to provide specialized detection of various properties in specimens, but will not be discussed here. An extensive range of accessories now are available for research use with light microscope instruments; these include chambers that keep living cells warm and well-fed during observation, and other chambers that enable catalyst particles to be observed within a monitored gaseous or wet environment.
Practical steps for using light microscopes.
A common sequence of operational steps is used with the several different kinds of light microscopes.
1. Preliminary preparation of the microscope. All compound lenses must be cleaned and aligned upon the optical axis.
2. Preparation of specimens to be examined. Specimen preparation aims to produce samples with the required small size or thinness, having a surface revealing the desired information, and retaining the structural properties found naturally.
3. Mounting of dried or wet specimens for study. Samples for light microscopy most commonly are mounted upon a clean glass slide. Some wet samples are dried by evaporation directly onto slides; others go through special procedures for dehydration and drying so as to avoid damage to fragile specimens. Addition of a mounting medium and a very thin cover glass provides a good stable environment for most samples.
4. Imaging. Images of samples are recorded at both low and higher magnifications.
5. Analysis of recorded images. The recorded image is a light micrograph; this is not simply a snapshot, but rather is a package of experimental data that can be analyzed qualitatively (e.g., condition of structural features) and/or quantitatively (e.g., numerical measurements of various features).
Recent advances in light microscopy.
Light microscope technology continues to advance! Light microscopes until recently were able to resolve very fine details only to a limit of around 0.2 micrometers, as first determined with optics by Ernst K. Abbe in classical times; after all the many following decades of light microscopy, new special approaches recently succeeded in surpassing that limit. Special optical arrangements recently developed for light microscopy now allow super-resolution to be obtained (see: “Press Release – 2014 Nobel Prizes in Chemistry” , and, “Explanatory Notes for 2014 Kavli Prize in Nanoscience” ). New types of illumination and new optical arrangements now are exciting brain researchers because they can allow individual nerve cells (neurons) to be imaged deep within the brain. Advances and new protocols for specimen preparation also continue to be developed, and serve to enlarge the types of information that can be retrieved by light microscopy.
The most general advantages of light microscopy.
Light microscopes usually are rather easy to use, applicable to an enormously wide variety of samples, and involve only a moderate cost. Sample preparation and preservation are well worked out in detail. Compound light microscopes can be readily adapted to image dynamic changes in samples (e.g., video or time lapse recordings of living cells) and to analyze the third dimension of structure. The extensive range of specimens that can be examined by light microscopy means that there are a gigantic number of ways for scientists to have fun applying these tools for their research studies.
Light microscopes in education.
More and more primary and secondary schools are adding light microscopy to their activities for science education. Light microscopes can be somewhat costly for schools to acquire, but they last a long time and each one can serve at least 3-6 students in a laboratory class; acquiring a viewing screen or a projection device permits much larger groups to observe results from only a single microscope. Working with light microscopes and studying their images can be used not only to teach students about optics, measurements, and scientific research, but additionally to instruct about the biology, chemistry, and physics of those specimens being examined.
Most students are inherently interested in observing a stained blood smear, first with a magnifying glass, then with a dissecting light microscope, and finally with a compound light microscope at increasing magnifications. Watching living protozoa (e.g., from local pond water) with a compound light microscope will be an amazing experience for all sudents, even those maintaining zero interest in science; for these samples, phase contrast microscopes are not needed, since defocusing usually will provide sufficient contrast to see the transparent unicellular creatures.
Many classroom exercises with light microscopy have been developed by Caroline Schooley and her colleagues working with Project MICRO at the Microscopy Society of America (see: http://www.microscopy.org/education/ProjectMicro/index.cfm ). Special sessions about Project MICRO are held during the annual meetings of this national science society (see: https://www.microscopy.org ); the same meetings usually also have special presentations about microscopy aimed at teachers and the general public.
Light microscopes continue to be very vital research tools for all 3 branches of science. Their large importance for research applications is matched by its importance for industrial uses in failure analysis and fabrication fidelity. One of its most special capabilities is the observation of living cells. In conjunction with old and newly developed methods for specimen preparation, light microscopy in 2015 is utilized extensively for the identification and localization of different components within larger specimens; this is true for pathology, minerology, metallurgy, materials science, and cell biology. Today, organized programs for teaching and using light microscopy in the classroom form a successful hands-on part of modern science education in primary and secondary schools.
Recommended for further information about light microscope images and videos.
After reading about light microscopy you now are ready to look at images! The internet provides an extensive variety of images from light microscopy; Dr.M recommends that you start by inspecting the following sources.
1. IMAGES: For galleries with a multitude of images and diagrams from light microscopy, look up “light microscope” or “light microscopy” in the image section of your favorite web browser. When you find something of interest among the many hundreds of collected panels displayed, click on its thumbnail and you will be taken to the explanatory details provided by its source.
2. WEBSITE ABOUT MICROSCOPY: A large variety of images, knowledge, and explanations is available at: http://microscopyu.com . Some commercial companies selling light microscopes also have a portion of their website devoted to explaining how these instruments work and what they can accomplish (e.g., Leica, Nikon, Olympus, Zeiss, etc.).
3. VIDEOS OF LIVING CELLS USING USING SPECIAL COMPOUND LIGHT MICROSCOPY: Some very wonderful movies of living cells taken by special light microscopes are available at: https://microscopyu.com/moviegallery/livecellimaging/index.html. These dramatically reveal an entire other world that most people never see!
4. LATEST NEWS: The specialist monthly journal, Microscopy Today, features all aspects of modern microscopy (i.e., news, methods, commercial offerings, meetings, teaching using microscopes, web Q & A, history, optics, etc.). Free subscriptions are available at: http://www.microscopy-today.com/jsp/common/home.jsf .
5. SCIENCE IS ART: For those either agreeing or disagreeing with me that science and art can be almost interchanged, please see “Small World Photomicroscopy Gallery” at: http://www.nikonsmallworld.com/galleries .
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