Principles and application of light, phase constrast and fluorescence microscope

light microscope, phase contrast microscope, fluorescence microscope, phase contrast ring

1. Presented by… Thakor Maitri M . M.Sc. (Botany) Department of life sciences, H.N.G.U ., Patan . Principles and application of light, phase contrast, fluorescence microscope 1

2. Contents Introduction Light microscope ◘ Principle ◘ Applications Phase contrast microscope ◘ Principle ◘ Applications Fluorescence microscope ◘ Principle ◘ Applications 2

3. Introduction 3 A microscope is an instrument which used to see object that are too small to be seen by the naked eye. The object of magnification of cells and their components was achieved by the lenses of various type or a combination of lenses which could magnify the minute objects upto a particular limit, and therefore so many lenses were combined together to form an instrument known as the microscope . In Greek : micros = small ; skopein = to see , to look .

4. 4 Credit for the first microscope is usually given to Zacharias Janssen , in Middleburg, Holland, around the year 1595 . Eyeglass crafts – Magnify object 3-10x . Objective Body tube Eyepiece

5. The Light microscope 5 The most important scientific tool for a student of biology is the light microscope . The light microscope consists of various components which gather light and redirect the light path so that a magnified image viewed object can be within a short distance. The student’s microscope or the compound microscope of twentieth century is the microscope of much improved and modified type.

6. 6 Types – Simple dissecting M , compound M , stereomicroscopes. Sources: www.Biologydiscussion.Com/microscope/ light microscope

7. Principle of light microscope 7 In the light microscope, light is produced from either an internal or external light sources and passes through the iris diaphragm, a hole variable size which controls the amount of light reaching the specimen. The main components of the compound light microscope include a light sources that is focussed at the specimen by a condenser lens.

8. 8 The slide is held on the stage at 90° to the path of light which next travels through the specimen. The objective lens magnifies the image of the specimen before the light travels through the barrel of the microscope. The light finally passes through the eyepieces lens and into the viewer’s eye which sends impulses to the brain which in turn interprets the image.

9. How does it work ? 9 Light microscope are compound microscope, which means they contain at least two lenses. Lenses are curved pieces of glass or plastic that bend rays of light and can magnify objects, making them appear bigger than they actually are. Light microscope shown here magnifies an object in two stages. Sources : dr-monsrs.net

10. 10 Light from the mirror is reflected up through the specimen, or object to be viewed, into the powerful objective lens, which produce the one magnification. The image produced by the objective lens is than magnified again by the eyepiece lens, which act as a single magnifying glass. The magnified image can be seen by looking into the eyepiece lens. Important factor in light microscopy include: Magnification , Resolution , Contrast .

11. Application of the Light microscope 11 Light microscopes play a large role in today’s biology. Handy in use. Biologists use the microscopes to observe objects and details at a cellular level to learn more about the building blocks of all organisms. Microscopes are also used to observe real time movement in cells and organisms. Lastly, microscopes are used in forensics to help solve many crimes.

12. 12 Microscopes provide the students with an understanding of real cells and their supporting structures. Also, microscopes provide students who are inclined towards the medical field a more intense look at the career choice and devlop basic skills. Lastly, microscopes are used in biology to study diseases like cancer and AIDS to help diagnose the disease in patients and to help find a cure for them.

13. 13 Microscopes are used when studying light and optics to learn how light refracts through converging lenses and how a combination of different lenses with varying focal lengths affects the properties of the image. Often times, there will be human evidences left on the crime scene. This allows forensic scientists to examine the evidence under a microscope and match the results with a database to find the culprit.

14. 14 Minerologist’s also use light microscopy, typically with a special preparation of a sample called thin section. As the name imlies , thin section are very thin slices of a rock. The sample needs to be thin enough for light to travel through from the light sources to the user’s eye. The thin section will allow the shape of different crystal grains to be seen. The microscope can be used with different techniques, like epifluorescences and phase contrast.

15. The Phase contrast microscope 15 Phase contrast microscopy first described in 1934 by Dutch physicist Fritz zernike , Whom awarded by Nobel prize in physics in 1953. A phase con trast microscope makes it possible by utilizing two characteristics of light, diffraction and interference, specimens based on brightness differences. It requires additional specialized structure annular diaphragm and phase contrast ring .

16. 16 Sources: www.biologydiscussion .com/microscope/phase contrast-microscope/682.

17. Principle of Phase Contrast Microscope 17 It based on the wavelength (nature) of light rays and the fact that light rays can be in phase or out of phase. Different shade of grey are distinguished to our eyes due to differences in amplitude of light rays. PCM converts invisible small phase changes caused by the cell component in to visible intensity changes.

18. 18 In a Phase contrast microscope, one set of light rays comes directly from the light sources. The other set comes from light that is reflected or diffracted from a particular structure in the specimen. The images differences in refractive index of cellular structure. Light passes through thicker parts of cell is held up relative to the light that passes through thinner parts of cytoplasm.

19. How does it work ? 19 Light that does not interact with the speciman is collected by the objective passes through the phase ring, and is regarded exactly ¼ wavelength. The Phase shifted is not detectable by the eye so the resulting image on the image plane in the microscope appears as a normal bright background.

20. 20 Light passing through one material & into another material of slightly different refractive index or thickness will undergo a charge in phase. This charge in are translated into variations in brightness of the structures. Natural light vibrates in many directions but polarized light only one direction.

21. 21 Sources : https://www.bwoptics.com/newsend2asp ?id=3

22. Application of Phase Contrast microscope 22 Most commonly used to provide contrast of transparent specimens such as living cells or small organisms. Useful in observing cells cultured in vitro during mitosis. Phase contrast enables visualization of internal cellular components. It’s used in examination of growth, dynamics, and behaviour of a wide variety of living cells in cell culture.

23. 23 It applied for equipment from the study of the living biological specimens, medical applications, study of live blood cells, and other biological and science applications. It’s used in diagnosis of tumour cells.

24. The Fluorescence microscope 24 This microscope additionally requires an excitation filter, a barrier and a dichromatic mirror, fluorescent stain. Fluorescent microscope is much the same as a conventional light microscope with added features to enhance its capabilities. A specific wavelength of light is used to excite fluorescent molecule in specimen. Light of higher wavelength is then imaged.

25. 25 It is also used to visually enhance 3-D features at small scales. This is achieved by using powerful light sources, such as lasers, that can be focused to a pinpoint. This focusing is done repeatedly throughout one level of a specimen after another. Most often an image reconstruction program pieces the multi level image data together into a 3-4 D reconstruction of the targeted sample.

26. 26 Sources :Zeiss-campus.magnet.fsu.edu/articles/basics/fluorescence.html

27. Principle of the Fluorescence microscope 27 When certain compounds are illuminated with high energy light, they then emit light of a different , lower frequency. This effect is known as Fluorescence . In most cases the sample of interest is labelled with a fluorescent substance known as a fluorophore and then illuminated through the lens with the higher energy sources. Often specimens show their own characteristic auto fluorescence image, based on their chemical makeup.

28. 28 The key feature of fluorescent microscopy is that it empoys reflected rather than transmitted light, which means transmitted light techniques such as phase contrast and DIS can be combined with fluorescent microscopy.

29. How does it work ? 29 The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores , causing them to emit longer wavelength of light (or a different colour then the absorbed light ).

30. Application of Fluorescence microscope 30 Fluorescence microscopy is a critical tool for academic and pharmaceutical research, pathology, and clinical medicine. This method is used for demonstration of naturally occurring fluorescent material and other non- fluorescent substances or micro- organisms after staining with some fluorescent dyes. e.g.; Mycobacterium tuberculosis, amyloid, lipids, elastic fibers etc.

31. 31 Imaging structural components of small specimens, such as cells. Conducting viability studies one cell populations (are they a live or dead ?). Imaging the genetic material within a cell ( DNA & RNA ). Viewing specific cells within a larger populations with techniques such as FISH.

32. 32 1) Biophysics Author : Vasantha pattabhi , N. Gautham . Edition : 2009 ( Second ) 2) Basic Biophysics For Biologist Author : M. Daniel Edition : 2003 3) WWW.Slideshare.net References

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