Compound microscope

Bioinstrumetation, microscopy, light microscopy, compound microscopy, microscope

1. Compound microscope Dr. P. Nithiya Assistant Professor, Department of Botany, Seethalakshmi Ramaswami College, Tiruchirappalli

2. Microscopy Microscopy is a technique for making very small things visible to the unaided eye. An instrument used to make the small things visible to naked eye is called a Microscope

3. Light microscope Light or optical microscope uses visible light as a source of illumination. Light travel through the specimen, this instrument can also be called a T ransmission light microscope

4. Working principle of light microscope Light microscope creates a magnified image of specimen which is based on the principle transmission, absorption, diffraction and refraction of light waves.

5. Transmission Transmission of light  is the moving of electromagnetic waves (whether visible  light , radio waves, ultraviolet, etc.) through a material .

6. absorption Light absorption is a process by which light is absorbed and converted into energy . Absorption depends on the electromagnetic frequency of the light and object’s nature of atoms. Absorption of light is therefore directly proportional to the frequency. If they are complementary, light is absorbed. 

7. diffraction Diffraction: Diffraction is the slight bending of light as it passes around the edge of an object. The amount of bending depends on the relative size of the wavelength of light to the size of the opening.

8. Refraction Refraction is the bending of a wave when it enters a medium where its speed is different. The refraction of light when it passes from a fast medium to a slow medium bends the light ray toward the normal to the boundary between the two media. The amount of bending depends on the  indices of refraction  of the two media and is described quantitatively by  Snell's Law .

9. Transmission and absorption

10. COMPOUND MICROSCOPE The simplest form of light microscope consists of a single lens, a magnifying glass. Microscope made up of more than one glass lens, in combination is termed compound microscope A compound microscope is an optical microscope with multiple lenses (objectives and eye piece).

11. Light microscope

12. Mechanical parts Base Pillar Arm Stage Body tube Coarse and fine adjustment screw Draw tube

13. Mechanical parts A. Base : It is a supporting stand resting on the table. It bears weight of the microscope. B. Pillar : it stand vertically on base. C. Arm : It is curved, solid piece. It can be adjusted with the base at the inclination joint.

14. Mechanical parts D. Stage : ( i ) It is a rectangular platform. (ii) It has a circular hole at the centre with two clips. E. Body tube: It is a metal tube. It has revolving nosepiece with objective lens.

15. Mechanical parts F. Coarse and fine adjustment screw: The coarse adjustment screw moved the tube vertically very fast. The fine adjustment screw moves it slowly. G. Draw tube: The upper part of the body tube is called draw tube. Its length can be adjusted.

16.   Optical parts Nose piece : A metal disc with 2 or 3 holes. Objectives : The lens system nearer to the specimen is called objectives It magnifies the specimen for several times. There are three objectives namely low power (10X), high power (100X) and oil lens (100X).  

17. Optical parts Eye piece : The lens system nearer to the eye is called eye piece. It magnifies the real image of the object. There are 3 types of eye piece- a) Huygenian eye piece b) Compensating eye piece c) hyper plane eye piece.

18. Eye piece Huygenian eye piece was invented by Christian Huygens and first compound eye piece. It consists of two Plano- convex lenses separated by an air gap and used to correct chromatic aberrations Ramsden eyepiece An eyepiece consisting of two  plano convex  glass lenses of equal focal length, placed with the convex sides facing each other and with a separation between the lenses of  about  two-thirds of the  focal length  of each. I t was discovered by Jesse Ramsden  in 1782.

19. Optical parts D. Condenser : Condenser & iris diaphragm and fitted below the stage. It consists of two or more lenses. It condenses light rays from mirror and converges and focuses the light into the specimen. E. Mirror: A Plano concave round mirror and fitted below the stage.  

20. Oil immersion objective Objective lens used for oil immersion to get the maximum resolution of specimen under a compound microscope is called oil immersion objectives . A drop of cedar wood oil is placed on a specimen and gives maximum resolution . Disadvantage of oil immersion : it absorbs blue and ultraviolet light, damage  objectives  with repeated use, and Cedar oil must be removed from the objective immediately after use. While removing , hardened cedar oil can damage the lens. In modern microscopy synthetic oil is used

21. Oil immersion lens In the case of an oil-immersion objective of x100, the magnification of the primary image at the plane of the eyepiece diaphragm is x100, and the intensity of the magnified image is therefore one ten thousandth of the illumination intensity at the plane of the specimen. The primary image is further magnified by the eyepiece, which projects it (with or without a focus achromat ) an appropriate distance to fill the film gate of the camera in use. Since this distance is also subject to the inverse square relationship between projection distance and image intensity, it is not surprising that it takes a much longer time to expose a 10" x 8" photomicrographic plate than a frame of 16mm film.

22. https://www.youtube.com/watch?v=baFTRZN_fQU

23. Oil immersion objective

24. Working procedure Compound microscope has two sets of lenses , the objective and the eyepiece. The basic principle – 1. When the beam of light passes through the specimen and then convex lens of objective lens, it forms a real inverted. 2. The enlarged image of the object in the focal plane of eyepiece. This image now acts as object for the eyepiece. 3. Eyepiece lens finally forms a further enlarged virtual image of the object. 4. Magnifying power of a compound microscope = M ×M e ). (M 0- magnification of objective lens , M e - magnification of eye piece.)

25. Magnificatio n M=M ×M e M 0- magnification of objective lens A’B’/AB M e - magnification of eye piece . A”B”/A’B’

26. Magnification of Objective lens = size of an real image A’B’ / size of a specimen AB AB= 1mm A’B’=5mm Magnification of Objective lens =A’B’/AB= 5/1=5X Magnification of Ocular lens = size of an second image / size of a real image A’B’=5mm A”B”=50mm Magnification of Ocular lens =A’B’/AB= 50/5=10X

27. Working procedure A compound microscope consists of two convex lenses: an objective lens O of small aperture and an eye piece E of large aperture. The lens which is placed towards the object is called objective lens, while the lens which is towards our eye is called eye piece. These two convex lenses i.e. the objective and the eye piece have short focal length and are fitted at the free ends of two sliding tubes at a suitable distance from each other.

28. Although the focal length of both the objective lens and eye piece is short, but the focal length of the objective lens O is a little shorter than that of the eye piece E. The reason for using the eye piece of large focal length and large aperture in a compound microscope is, so that it may receive more light rays from the object to be magnified and form a bright image.

29. Focal length and focal point Focal length (shown in red) is the distance between the center of a convex lens or a concave mirror and the focal point of the lens or mirror — the point where parallel rays of light meet, or converge.

30. FORMATION OF IMAGE IN COMPUND MICROSCOPE The ray diagram to show the working of compound microscope is shown in figure. A tiny object AB to be magnified is placed in front of the objective lens just beyond its principal focus fo ’. In this case, the objective lens O of the compound microscope forms a real, inverted and enlarged image A’B’ of the object .

31. FORMATION OF IMAGE IN COMPUND MICROSCOPE

32. Now A’B’ acts as an object for the eye piece E, whose position is adjusted so that A’B’ lies between optical centre C2 and the focus fe ’ of eye piece. Now the eye piece forms a final virtual, inverted and highly magnified image A”B”. This final image A”B” is seen by our eye hold close to eye piece, after adjusting the final image A”B” at the least distance of distinct vision of 25 cm from the eye. This image is called ‘ Ramsden’s disc’ . The lens of the eye casts an image of Ramsdens disc over the whole surface of retina now fills the eye.

33. Ramsden disc The circular spot of light formed at that distance above the eyepiece The small disk of light visible in the back focal plane of an eyepiece. A n eyepiece consisting of two plano -convex crown-glass lenses of equal focal length, placed with the convex sides facing each other and with a separation between the lenses of about two-thirds of the focal length of each .

34. Ramsden disc The diagram shows the concept of the Ramsden circle, and is a key to understanding the problems of film exposure in light micrography . The Ramsden circle, also called the exit-pupil of the microscope, is a circular area through which pass all the image-forming rays leaving the microscope. It is itself an image of the back-lens of the microscope objective, demagnified in proportion to the power of the eyepiece, and is usually located a few millimetres above the eye lens of the eyepiece.

35. Thank you