Phase contrast and Fluorescent microscope

bioinstrumentation, microscopic studies, phase contrast microscope and Fluorescent microscope

1. PHASE CONTRAST MICROSCOPE Dr.P.Nithiya

2. PHASE CONTRAST MICROSCOPE Phase contrast microscope was first discovered by Professor Fredrike Zernick and he was awarded Nobel prize. This microscope widely used in biological and medical research .

3. Principle 1 . When light rays pass through the specimen , they undergo different phase changes ( direct rays or undeviated rays and diffracted or deviated rays or reflected rays) due to different refractive indices and thickness of the cell contents. ( refractive index is defined as the ratio of the velocity of light in vacuum to its velocity in the transmitted medium. N= c/v)

4. Light wave

5. PHASE CONTRAST MICROSCOPE 2. If the direct and diffracted rays are of same frequency combined together in a same phase ( the crest or trough of both light waves coincide), the amplitude or brightness will doubly increased. This is called coincidence and the subject looks very bright. 3. When the rays are in out of phase, the amplitude or brightness decreased. This is called as interference. Both these phenomena (interference and coincidence) are used in phase contrast microscope.

6. Phase contrast microscope When light passes through a living cell the phase of the light wave this change according to the cell refractive index light passing through a relative or dense consequently is shifted relative to light that has passed through and adjacent in region of the cytoplasm. The phase contrast microscope export the interference effect produced when these two sets of specimen appears as different degree of brightness and contrast. It is used for the study of live and unstained cells which are in general transfer and to light.

7. Instrumentation: The basic construction of phase contrast microscope is like a light microscope (ocular, objective & condenser) with annular diaphragm and annular phase plate. 1. Annular diaphragm: it has annular stop, which allows only hallow cone of light to pass through it. 2. Annular phase plate: it is a special optical disc with annular groove. This plate is fitted in such a way that direct rays pass through the groove and diffracted rays pass outside of the groove. 3. Both annular diaphragm and annular phase plate are optically aligned.

8. Working procedure: • A hollow cone of light travels through the condenser and enter in to the specimen. Some rays are diffracted (reduced ¼ wavelength) and some are direct ray. • Direct rays are pass through grooves of annular plate and the wavelength is enhanced ¼ (1 ¼ wavelength). It forms background of the image. • The diffracted ray travel through the thicker part of annular plate and the wavelength is ¾). • These two rays are joined together with high amplitude and pass then through ocular lens which forms image.

9. • Based on the image formation and configuration and properties of the phase ring, there are two types. 1. Dark phase or positive contrast microscope- image is dark and back ground is bright. 2. Bright phase or negative phase contrast microscope- image is bright than the surroundings.

10. Phase contrast microscopic images

11. Limitations Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so phase contrast is often not used in teaching labs except perhaps in classes in the health professions. To use phase-contrast the light path must be aligned. Generally, more light is needed for phase contrast than for corresponding bright-field viewing, since the technique is based on the diminishment of the brightness of most objects.

12. Applications: it is used for studying  Live unstained specimens.  Microbial mobility.  Cell division.  Shape, bands and contents of chromosomes.

13. FLUORESCENCE MICROSCOPE Dr.P.Nithiya

14. FLUORESCENCE MICROSCOPE Ernst Abbe invented the fluorescence microscope in 1873 its magnification is up to 100x.

15. Principle 1 . It is based on the phenomenon of fluorescence. 2. Certain chemicals absorb short wave length light (Ex. UV rays 290-490 nm), after short time(less than 10-5 seconds) visible light of long wave length is reemitted. It is called fluorescence.

16. 3. The substance which emits fluorescence is known as fluorophore . 4. The specimens are coated with fluorescent dye (Ex. Fluorescein isothiocyanate , Auramine O, Acridine orange etc.) and illuminated by blue light or UV rays (short wave length with more energy). 5. After few seconds specimens emits long visible green light .

17. Instrumentation

18. Light source Lamp: mercury or xenon or tungsten or halogen.

19. Optical system 1 . Heat filter: this filter absorbs the heat generate by the lamp. 2. Exciter filter: it is placed between the UV source and specimen and absorbs longer wave length light. 3. Barrier filter: located between the objective lens and eye piece and absorbs all short wave length rays. 4. Diachronic mirror: a) It is located between barrier filter and objective lens. b) This mirror reflects short wave length (below 500nm) but transmit longer wave length (above 500 nm) in a single direction.

20. Fluorescence microscope

21. Observation system The objectives and oculars are used for formation of image. The lens is made up of quartz or fluorite.

22. Working procedure:  Lamp source produce UV rays to exciter filter.  The exciter filter passes the UV rays on the diachronic mirror.  The mirror reflects shorter rays to the objective condenser which focus into the fluorescent coated specimen.  The specimen emits longer visible rays. This ray again reaches the mirror.  The diachronic mirror absorbs shorter waves and transmits only longer ones.  The barrier filter absorbs all shorter waves which is harmful to eye.  The long fluorescent light to the ocular lens.  Thus the final fluorescent image with black background is formed.

23. Limitations of Fluorescence Microscope Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching . Photobleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons excited during fluorescence. Cells are susceptible to phototoxicity , particularly with short-wavelength light. Furthermore, fluorescent molecules have a tendency to generate reactive chemical species when under illumination which enhances the phototoxic effect. Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of the specific structures which have been labeled for fluorescence.

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