Microscope is an optical instrument consisting of lens for making enlarged or magnified images of minute objects. Robert Hook (1635 – 1703) made and used a first compound microscope. The basic difference between simple and compound microscope is that simple microscope uses only one lens whereas, compound microscope uses more than one lenses to further magnify the object.
The ability of microscope to distinguish two adjacent object as separate and distinct images rather than single blurred image is called resolving power of microscope. Theoretically, size of image can be increased by adding lenses. Magnification of object is only useful if the enlarged image is clearly visible. So, effective magnification depends on resolving power of microscope.
A microscope’s resolving power is dependent on wavelength of beam for illumination and optical quality of lenses. Shorter wavelength gives better resolution. Resolution or shortest distance (d) can be calculated by using the formula, d = λ/2nsinϴ where, d = resolution, λ = wavelength of light, n = refractive index and ϴ = angle subtended.
Note: Oil is used with oil immersion lenses because it has higher numerical aperture than air and also higher refractive index and this greatly improves resolution.
Types of microscope:
Bright field microscope: it is most commonly used in elementary biology and microbiology courses and consist of two series of lenses; objective and ocular (eye piece) lenses. These two lenses function together to resolve the image. Microscopic specimens are visualized because of difference in contrast that exist between them and surrounding medium. The contrast difference arises because cell absorb or scatter light in varying degree. It has maximum useful magnification of X1000 time. Specimens are sometimes stained or unstained but bacterium are generally stained and appear color of stain. It is commonly used for observing stained specimen and counting microbes.
Advantage of this microscope is, It is very easy to use, readily available and relatively inexpensive than other microscope. Similarly allows staining reaction for bacteria. Disadvantage of this microscope is, it lacks contrast so that it is unable to resolve very thin bacteria and viruses.
Dark field microscope (DFM): dark field microscope is light microscope in which the lightening system has been modified to reach the specimen from the sides only. Here the background is dark and the object is illuminated because of special kind of condenser that transmit hollow cone of light from the source of illumination. DFM is used for observing unstained cells that is not observable with bright field microscope. Its advantage is, it allows living microbes to be viewed such as spirochetes. However, it cannot be used for staining reaction for microbial cell.
Fluorescence microscope: fluorescence microscope is used to visualize specimens that emit light of one color when light of another color shines upon them. Fluorescence occurs either because of presence of naturally occurring fluorescence substances within the cell (such as chlorophyll). It can also be due to treatment of cells with fluorescent dye. Fluorescent microscope contains two filters. Exit filters transmits only blur light to pass to specimen and barrier filter block out blue light and allows green light only to eyes.
Fluorescent microscope is used for rapid identification of infectious microorganism. Furthermore, it is used for detecting specific infectious agents in tissue and detecting immunological reaction.
Phase contrast microscope (PCM): phase contrast microscope was developed to improve contrast differences between the cells the surrounding medium, making it possible to observe cells without staining them. It is based on the principle that cells differ in refractive index from their surrounding and hence bend some of the light rays that pass through them.
The effect is amplified by a special ring in the objective less of phase contrast microscope called phase shifting plate, leading to the formation of a dark image on a light background. The different cells in phases or wave front wave irregularities makes the object detectable by the eye.
Phase contrast microscope is widely employed in research application because it can be used to observe wet mount (living preparation) and observing intracellular structures. PCM enhances sub-cellular anatomy. It allows observation of motility, phagocytosis and biological activities. Disadvantage of PCM is that, it cannot be used to evaluate staining reaction.
Electron microscope: Bright field microscope, dark field microscope and phase contrast are the light microscope (use light rays as a source of illumination) and they look more or less alike in their structure. Electron microscope is totally different from light microscope in the sense that, electron beam is used as a source of illumination (with the employment of 60 – 80 KV electron).
Electron microscopes are widely used for studying the detail structure of the cell. To study the internal structure of cells, a transmission electron microscope (TEM) is essential, where electron are used instead of light rays and are focused by electromagnetic lenses. After being transmitted through specimen, the magnetic objective focuses the electron into a first real image of object which is enlarged (2000 times) and again magnetic projector lens magnifies a portion of first image producing magnification up to 240,000 times more. The final image can be reviewed by striking on a fluorescent screen which makes the object visible. In electron microscope, whole system operates in a vacuum. The resolving power of microscope is much greater than that of light microscope and thus the electron microscope enable to see many structure of even molecular size such as protein and nucleic acid. To obtain sufficient contrast the preparation are treated with a special stain such as osmic acid, permanganate, uranium or lead. Alternatively, one can use the scanning electron microscope (SEM). The specimen is coated with a thin film of heavy metal such as gold and electron beam is exposed down from the specimen for observing image on screen.
Electron microscope are used for examination of viruses and ultra-structure of cells, similarly for diagnosing certain viral diseases, cancer and detecting certain giant molecules. Disadvantage of electron microscope is, it is very expensive, needs sophisticated technology. Even a single cell is too thick to be observed directly so special technique of thin sectioning are needed to prepare a specimen.
Name : Pratiksha Shrestha
Ms. Shrestha holds masters degree in food engineering and bioprocess technology from Asian Institute of Technology (AIT) Thailand. She is currently working for Government of Nepal at Department of Food Technology and Quality Control (DFTQC), Kathmandu. She is also a teaching faculty in College of Applied food and Dairy Technology (CAFODAT) affiliated to Purbanchal university, Nepal.