Bright field microscopy, Principle and applications

Introduction
History
Basic Component of Microscope
Light Microscopy
Types of Light Microscopy
What Are Bright Microscopy
Principle of Bright Microscope
Advantage
Disadvantage
Application
Conclusion
Reference

1. 1 Principle And Applications Of Bright-field Microscopy By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. CONTENT Introduction History Basic Component of Microscope Light Microscopy Types of Light Microscopy What Are Bright Microscopy Principle of Bright Microscope Advantage Disadvantage Application Conclusion Reference 2

3. General Introduction Originated from Greek word Micros=small, scopien= to watch Generally human eye feels difficulty in seeing an object smaller then 1mm; therefore the microscope is used to observe smaller then this size. Microscopy plays a critical role in a majority of life sciences. Microscopes have contributed significantly in the fields of cell biology and histology where great discoveries have been made over the years. The discovery of blood cells in the human body paved the way for advanced studies in cell biology 3

4. History Hans and Zacharias Jansser (1590) First compound microscope (2 lenses),tube with lenses at each end. Antonie Van Leeuwenhoek (1632-1723) Simple microscope (1lenses) Discovered microorganism and spermatozoa. Robert Hooke(1635-1703) Compound microscope improvement(two lenses, objective and ocular lenses). Coined the biological term cell. 4

5. Classification Depending on number of lenses Simple microscope- uses a single lens to magnify an object and cannot reach high magnification.   Compound microscope-use two sets of lenses - an objective lens and an eyepiece - to produce images Depending on number of eyepiece Monocular microscope- microscopes have one eyepiece. Binocular microscope- microscopes have two eyepieces and reduce eye strain. Depending on source Light microscope Electron microscope 5

6. Light microscope Light microscope are simplest of all microscope. A light microscope uses focused light and lenses to magnify a specimen, usually a cell. Most cells (bacterial or otherwise) are both small and transparent, and so light can easily pass through them. A light microscope is much like a telescope, except that instead of the object being very large and very far away; it is very small and very close to the lens. Great importance in the study of microorganisms and biomolecules. 6

7. Types of light microscope Bright-field microscope- color-stained, high –contrast sample. Dark-field microscope- find structure and tiny sample in dark background. Phase-contrast- low-contrast, transparent sample. Differential interference contrast- low-contrast sample, for surface observation. Bright-field microscope Dark-field microscope Phase-contrast 7

8. Bright–field microscope Dark sample on a bright background. In bright-field microscopy, the sample illumination is transmitted white light and contrast in sample is caused by absorbance of some of the transmitted light in dense areas of the sample and this contrast allows us to see the specimen. High-contrast sample. Staining is often required to increase contrast. Bright-field illumination is useful for samples which have an intrinsic color, for example chloroplasts in plant cells. 8

9. A lgae with visible cells 9

10. Basic Component of Microscope 10 Mechanical part B ase- support the microscope. Arm- joins the body tube and base. Body tube (Head)- connects the eyepiece to the objective lenses Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the nosepiece to select different objective lenses. Stage: The flat platform where the slide is placed. Coarse adjustment: Brings the specimen into general focus. Fine adjustment: Fine tunes the focus and increases the detail of the specimen Optical parts- Eyepiece: The lens the viewer looks through to see the specimen. The eyepiece usually contains a 10X or 15X power lens. Objective lenses: they are the lenses closest to the specimen. Iris diaphragm: Adjusts the amount of light that reaches the specimen. Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed.

11. Principle It has a series of two lenses; (i) the objective lens close to the object to be observed and (ii) the ocular lens or eyepiece, through which the image is viewed by eye. Light from a light source (electric lamp) passes through a thin transparent object. The objective lens produces a magnified ‘real image’ first image) of the object. This image is again magnified by the ocular lens (eyepiece) to obtain a magnified ‘virtual image’ (final image), which can be seen by eye through the eyepiece. As light passes directly from the source to the eye through the two lenses, the field of vision is brightly illuminated. That is why; it is a bright-field microscope. 11

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14. Appropriate magnification 14

15. Digital Imaging Options Although a basic method of microscopy, bright field as a technique is well suited to mating with new technologies. Digital imaging systems can make high resolution images of properly stained microorganisms using this technique. Three-dimensional imaging accessories can be used with the bright field method and newer technologies will allow real time viewing in 3D. Also suited to video imaging, this enhancement will allow the user to view motile organisms interacting with their environment. 15

16. Advantages Bright field microscopy is very simple to use with fewer adjustments needed to be made to view specimens. Some specimens can be viewed without staining and the optics used in the bright field technique don’t alter the color of the specimen. It is adaptable with new technology and optional pieces of equipment can be implemented with bright field illumination to give versatility in the tasks it can perform. 16

17. Limitations Very low contrast of most biological samples. The practical limit to magnification with a light microscope is around 1300X. Although higher magnifications are possible, it becomes increasingly difficult to maintain image clarity as the magnification increases. Samples that are naturally colorless and transparent cannot be seen well, e.g. many types of mammalian cells. These samples often have to be stained before viewing. 17

18. Application Widely use in pathology to view fixed tissue sections or cell films/smears. Important for hematology, microbiology, TB and malaria testing. Microscope magnifies the blood samples, so, the doctor can see the malaria parasites attacking the red blood cells. Used in bacteriology, biology and medicine to examine minute objects such as bacteria, other unicellular organisms and plant and animal cells and tissue. Advances in flurochrome stains and monoclonal antibody techniques caused growth in use of analysis and cell biology. 18

19. Conclusion In conclusion, it is safe to say that microscopes have played a central part in life sciences. This has positively contributed to the enhancement of quality of life since a lot of discoveries directly contributed to the development of drugs and cures used in the treatment of diseases and conditions that were previously misunderstood or not well understood. A cell is the single unit of life, and to understand and study it, the microscope is necessary. The discovery of cells and genes were major milestones in the medical sciences and were a great influence to the development of new effective cures and a reduction of mortality cases among populations. 19

20. Reference Cell and Molecular Biology,6 th Edition- Gerald Karp Internet www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.htm https://www.microscopemaster.com/life-sciences.html http://www.open.edu/openlearn/science-maths-technology/science/biology/introduction-microscopy/content-section-7 http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html 20