Microscope. Dr. Leena Barhate Department of Microbiology M.J.College, Jalgaon

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1 Microscope Dr. Leena Barhate Department of Microbiology M.J.College, Jalgaon

2 Acknowledgement y/neurons1_i.htm Google Images OU NanoLab/NSF NUE/Bumm & Johnson

3 The History Hans and Zacharias Janssen of Holland in the 1590 s created the first compound microscope Anthony van Leeuwenhoek and Robert Hooke made improvements by working on the lenses Anthony van Leeuwenhoek Hooke Microscope Robert Hooke

4 Light Microscope Microscope utilizing light as a source of illumination is called as light microscopes. Type of light microscopes Bright field microscope Dark field microscope Phase contrast microscope Fluorescence microscope

5 Bright field microscope Microscope that forms dark image against bright background is called as bright field microscope Widely used bright field microscope for basic study of microbes is the compound microscope

6 How a Microscope Works Convex Lenses are curved glass used to make microscopes (and glasses etc.) Convex Lenses bend light and focus it in one spot.

7 The History Zacharias Jansen The First Microscope

8 How a Microscope Works Ocular Lens (Magnifies Image) Body Tube (Image Focuses) Objective Lens (Gathers Light, Magnifies And Focuses Image Inside Body Tube) Bending Light: The objective (bottom) convex lens magnifies and focuses (bends) the image inside the body tube and the ocular convex (top) lens of a microscope magnifies it (again).

9 Ocular Lens Body Tube Nose Piece Objective Lenses Stage Clips Diaphragm Arm Stage Coarse Adj. Fine Adjustment Light Source Base

10 Body Tube The body tube holds the objective lenses and the ocular lens at the proper distance Diagram

11 It holds occular and objectives are lenses. It also provides sufficient space for image formation

12 Nose Piece The Nose Piece holds the objective lenses and can be turned to increase the magnification Diagram

13 A base in which objectives are fixed Simply rotating the nosepiece can rotate each objective into place

14 Objective Lenses The Objective Lenses increase magnification (usually from 10x to 100x) Diagram

15 Second lens system of a microscope Mounted on nosepiece and can be rotated into the place There are usually three objective lenses on a microscope. Objective lenses are generally equipped with microscope having low power, high power and oil immersion lens and magnification of 10x, 45x and 100x respectively

16 Function: to make real image Working distance: it is the distance between objective and object under observation

17 Stage Clips These 2 clips hold the slide/specimen in place on the stage. Diagram

18 Iris Diaphragm The Diaphragm controls the amount of light on the slide/specimen Turn to let more light in or to make dimmer. Diagram

19 It is equipped with condenser Control intensity of light condenser and therefore controls the amount of light intensity. Lever is equipped with it to adjust the light intensity. Blue colour filter is also equipped below the condenser

20 Condenser It is third lens system of microscope It is located below the stage It is responsible for focusing the light on the specimen There are several different types of condensers depending upon the type of microscope to be employed Abbe s condenser is most commonly used

21 Light Source (Illuminator) Projects light upwards through the diaphragm, the specimen and the lenses Some have lights, others have mirrors where you must move the mirror to reflect light Diagram

22 illumination Abbe and Nelson In this system light source, such as sun light through a window, or an open lamp flame is placed before the microscope mirror. Any structure or irregularity of the source is seen directly in the field of view. It creates a problem to some extent during examination of the specimen

23 Koelhler s type: It is second,method of illumination Prepared by Dr. August Koehler. It eliminated field of view The koehler form of illumination is mostly used today Here parallel rays of light generated usually by a tungsten filament lamp are used to illuminated the specimen

24 Ocular Lens/Eyepiece Magnifies the specimen image Diagram

25 It is the first lens system of microscope. It is present at top of the microscope In the microscope, the occular is capable of 10x magnifications Eyepieces of 5x, 15x and 20x magnification potential are also available. One eyepiece can be replaced by another

26 Function: to make virtual image of specimen

27 Arm Used to support the microscope when carried. Holds the body tube, nose piece and objective lenses Diagram

28 Stage Supports the slide/specimen Diagram

29 It is the platform on which the specimen to be viewed is placed. Some stages have clips to hold the glass slide in place. Others have a mechanical stage, which make it possible to move the slide across the stage in both horizontal and vertical directions

30 Coarse Adjustment Knob Moves the stage up and down (quickly) for focusing your image Diagram

31 They are used to move the body tube/stage relative to the objectives and occular, making it possible to focus the image

32 Fine Adjustment Knob This knob moves the stage SLIGHTLY to sharpen the image Diagram

33 Base Supports and stabilizes the microscope Diagram

34 Types of Microscopes: 1. Compound Light Microscope (what we use most often) 2. Stereoscopes also known as dissecting scopes 3. Electron Microscopes

35 Parts of the Microscope Arm

36 Parts of the Microscope Diaphragm Light Source

37 Parts of the Microscope Stage Stage Clips

38 Parts of the Microscope Revolving Nosepiece Objective Lenses

39 Ocular Lens Parts of the Microscope

40 Parts of the Microscope Coarse adjustment knob Used only when low power objective is used!!

41 Fine adjustment knob Parts of the Microscope

42 Carrying a Microscope

43 Steps to Use: 1. Rotate the low power objective into place and make sure the stage is all the way down. 2. Place slide on stage making sure object to be viewed is centered over the hole in the stage. Use the stage clips to hold the slide in place. 3. Turn light on. 4. Focus first with the coarse adjustment knob. Once in focus on low power, turn the nosepiece until the next higher lens is in place. 5. Use FINE adjustment knob ONLY and focus the object.

44 Remember: 1. If you are seeing perfectly round, clear circles then you just may be looking at air bubbles. Check your slide and try again. 2. Microscopes must always be properly put away. 3. Slides and cover-slips should be washed, dried, and returned to their proper place.

45 Important Vocabulary : magnification \mag-ne-fe-'ka-shen\ n 1. apparent enlargement of an object 2. the ratio of image size to actual size A magnification of "100x" means that the image is 100 times bigger than the actual object. resolution \rez-e-loo-shen\ n 1. clarity, sharpness 2. the ability of a microscope to show two very close points separately

46 Highest Typical Resolution Optical Microscope ~200 nm Electron Microscope ~0.1 nm

47 Factor affecting magnification Optical tube length Focal length of objective Magnifying power of eye piece and objective

48 Formula to determine magnification Magnification of microscope = Objective Magnification x eye piece magnification

49 Magnification ocular power = 10x low power objective = 20x high power objective = 50x a) What is the highest magnification you could get using this microscope? 500x Ocular x high power = 10 x 50 = 500. (We can only use 2 lenses at a time, not all three.) b) If the diameter of the low power field is 2 mm, what is the diameter of the high power field of view in mm?

50 .8 mm The ratio of low to high power is 20/50. So at high power you will see 2/5 of the low power field of view (2 mm). 2/5 x 2 = 4/5 =.8 mm c) in micrometers? 800 micrometers To convert mm to micrometers, move the decimal 3 places to the right (multiply by 1000)..8 mm x 1000 = 800 micrometers d) If 10 cells can fit end to end in the low power field of view, how many of those cells would you see under high power? 4 cells. We can answer this question the same way we go about "b" above. At high power we would see 2/5 of the low field. 2/5 x 10 cells = 4 cells would be seen under high power.

51 Numerical Aperture It is a ratio of diameter of lens to its focal length Formula: Numerical aperture(na) = ηsinθ η = Refractive Index θ = Half angel of aperture

52 Numerical aperture θ θ BAC is cone of light θ is the half the angle of cone light formed at objective aperture Theoretical limit of BAC is 180 (2θ = 180 ) So θ = 90 NA of dry lens cannot be greater that 1, since the refractive index of air is 1 and value of sin θ = 1

53 Resolution (Resolving power) It is the abililty to reveal closely adjacent points as separate and distinct Formula: Where d = 0.5 x λ NA λ = Wavelength of light NA = Numerical Aperture

54 d= become small as resolution increase Hence resolution is inversely proportional to the wavelength and directly proportional to the numerical aperture. Maximum resolution = lowest wavelength light Compound microscope with blue filter below condenser help to resolve the image.

55 Example Green light wavelength =550nm Objective with NA = 1.4 then what will be the resolution? d = 0.5 x λ NA d = 0.5 x d = 196 nm Microscope can reveal two closely associated points by 196 nm.

56 Immersion oil and its use in compound Microscope Observation by compound microscope, using the 100x objective needs special oil. It is also known as immersion oil in microscopy Immersion oil is cedar wood oil obtained from gymnospermic juniperous vergiana It is colourless liquid and has refractive index 1.55 (same as glass)

57 Advantage Use of oil avoids diffraction of rays. If air is present between specimen and objective, some light is lost due to diffraction of ray. Thus the image observed is fuzzy and the finer detail may lost. Thus oil help to get sharper image.

58 For Maximum resolution NA value must be high. The value of θ cannot exceed 90. Hence by increasing refractive index NA value can be increased Refractive index is function of the bending of light from air though glass and back again. It can be made possible by filling medium which has refractive index larger than refractive index of air Refractive index of oil is larger than air

59 OPTICAL MICROSCOPES Image construction for a simple biconvex lens

60 Rayleigh criterion for resolution Numerical Aperature Resolution Rayleigh Criterion ; See more interactive tutorials at

61 Field Full aperture is illuminated Comparison Bright- Dark- Field A central obstruction blocks the central cone.

62 Dark-Field Optical Microscopy A central obstruction blocks the central cone. The sample is only illuminated by the marginal rays. These marginal rays must be at angles too large for the objective lens to collect. Only light scattered by the object is collected by the lens.

63 Dark-Field Optical Microscopy

64 THE ELECTRON MICROSCOPE The wavelength of the electron can be tuned by changing the accelerating voltage. de Broglie : λ = h/mv λ: wavelength associated with the particle h: Plank s constant Js; mv: momentum of the particle m e = kg; e = coulomb P.E ev = ½mv 2 λ = h/ (2meV) = 12.3/ V (for V in KV, λ in Å) V of 60 kv, λ = 0.05 Å Δx ~ 2.5 Å Microscopes using electrons as illuminating radiation TEM & SEM

65

66 Components of the TEM 1. Electron Gun: Filament, Anode/Cathode 2. Condenser lens system and its apertures 3. Specimen chamber 4. Objective lens and apertures 5. Projective lens system and apertures 6. Correctional facilities (Chromatic, Spherical, Astigmatism) 7. Desk consol with CRTs and camera Transformers: kv; Vacuum pumps: Torr

67 Schematic of E Gun & EM lens Magnification: 10, ,000; Resolution: nm

68 TEM IMAGES ; com ;

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