light microscopy and its components and also errors in light microscopy

microscope and its parts
condensor substage objective lens eyepiece lens ray optics

1. LIGHT MICROSCOPYY Dr SRUTHY P 3 RD MDS

2. INTRODUCTION Microscope , instrument that produces enlarged images of small objects, allowing the observer an exceedingly close view of minute structures. The microscope must accomplish three tasks: produce a magnified image of the specimen separate the details in the image render the details visible to the human eye or camera

3. History……. 1590 - Hans & Zacharias Janssen of Middleburg, Holland manufactured the first compound microscopes While experimenting with several lenses in a tube, they discovered a combination of two lenses in a particular fashion. One lens was at the eyepiece of the microscope (ocular), and the second lens was near the sample to be studied (objective). This arrangement significantly enlarges the sample under observation.

4. Galelio In 1610, Galelio used a telescope at close range to magnify the parts of insects 1624 invented a compound microscope During this time, the word "microscope" was beginning to be accepted and regularly used by the members of the "Academia dei Lincei ," a scientific society that included Galileo.  Origin was from the Greek words μικρόν ( micron ) meaning "small," and σκο πεῖν ( skopein ) meaning "to look at."

5. Robert Hooke Robert Hooke (1635-1703), is an English scientist and mechanical genius, who was educated at the University of Oxford. Hooke was also a pioneer in microscopic research and published his observations, which included the discovery of plant cells. His Micrographia was an important milestone in proving the importance of microscopy, and has granted him the title of English father of microscopy Hooke also developed and modified the Jansen compound microscope into a more modern one, similar to the one we currently use.

6. Antoni van Leeuwenhoek A Dutchman, Anton van Leeuwenhoek , is considered the father of microscopes He worked as an apprentice in a dry goods store where magnifying lenses were used to count the threads in cloth. He made lenses which magnified up to 270x. This led to the first practical microscopes . In 1674, Anton was the first to see and describe bacteria, yeast, plants, and life in a drop of water.

7. LIGHT AND IT’S PROPERTIES Light or visible light, is electromagnetic radiation of a wavelength that is visible to the human eye (about 400–700 nm) Speed - approximate value of 3×10 8 meters per second Travels in straight line Illustrated as a sine wave

8. Wavelength – color , monochromatic or polychromatic Amplitude – brightness, energy content of light

9. Refraction Reflection Dispersion

10. Refraction Bending of the light ray when passing from one medium to another is called refraction

11. The extent of refraction depends on a) Angle of incidence b) Density of medium c) Curvature of interface between two medium As a rule When light enters a less optically dense medium it bends away from the perpendicular. When light enters a more optically dense medium it bends toward from the perpendicular.

12. The angle to which light rays are deviated called as angle of refraction . Ratio of sine values of angle of incidence ( i ) and refraction (r) called as refractive index.

13. It’s a dimensionless number that describes how fast light travels through the material. For example, the refractive index of water is 1.333, meaning that light travels 1.333 times slower in water than in a vacuum . Increasing the refractive index corresponds to decreasing the speed of light in the material.

14. REFLECTION When a ray of light approaches a smooth polished surface and the light ray bounces back, it is called the reflection of light . The incident light ray which lands upon the surface is said to be reflected off the surface. The ray that bounces back is called the reflected ray. If a perpendicular were to be drawn on a reflecting surface, it would be called normal. The figure below shows the reflection of an incident beam on a plane mirror.

15. Dispersion Separation of light into its constituent wavelengths when entering a transparent medium - such as a prism di sp er si on Short wavelengths are “ bent ” more than long wavelengths

16. Lens : a piece of glass or other transparent material, usually circular, having the two surfaces ground and polished in a specific form in order that rays of light passing through it shall either converge or diverge Positive Negative

17. Parallel rays of light entering a simple lens are brought together by refraction to a single point, the principal focus or focal point , where a clear image will be formed of an object The distance between the optical center of the lens and the principal focus is the focal length Focal length o

18. Real image formation in eye

19. Eg:magnifying lenses

20.     a) Distant object                                                   Real Inverted Smaller than object At F b) Object at 2F                                                    Real Inverted Same size At 2F c) Object between 2F and F                                                     Real Inverted Larger than object Beyond 2F d) Object at F                                                     No image Refracted rays are parallel e) Object between F and lens                                                     Virtual Erect Larger than object Behind the object on the same side of the lens

21. ABERRATIONS IN LENS SYSTEMS

22. Chromatic aberration Chromatic aberration, is a common optical problem that occurs when a lens is unable to bring all wavelengths of color to the same focal plane . a “color fringing” or “purple fringing”,

23. Spherical aberration is caused when the light rays entering a curved lens at its periphery are refracted more than those rays entering the center of the lens and are thus not brought to a common focus.

24. an   achromatic doublet , composed of two individual lenses made from  glasses  with different amounts of  dispersion . Typically, one element is a negative ( concave ) element made out of  flint glass  such as F2, which has relatively high dispersion, and the other is a positive ( convex ) element made of  crown glass  such as BK7, which has lower dispersion. The lens elements are mounted next to each other, often cemented together, and shaped so that the chromatic aberration of one is counterbalanced by that of the other . achromat  is a  lens  that is designed to limit the effects of  chromatic  and  spherical aberration

25. Classification Of Light Microscopes Depending on lens system: Simple And Compound Depending on optical technique: Bright Field, Dark Field, Phase Contrast, Interference Depending on microscope frame type Upright Microscope, Inverted Microscope

26. One Light Path & One Lens = Simple Microscope One Light Path & Multiple Lenses = Compound Microscope

27. COMPONENTS OF A COMPOUND MICROSCOPE

28. MICROSCOPE PROPER : Incorporating the body tube with the objective at one end and the eyepiece at the other end. THE STAND: which include the supporting, adjusting and illuminating apparatus

29. Light source The source of illumination should be: Uniformly intense Should completely flood the back lens of the condenser with light when the lamp iris diaphragm is open Make the object appear as though it were self-luminous Day Light Oil Lamp Low Voltage Electric Lamps 6V, 9W tungsten halogen lamp

30. Afocal or Nonfocused Illumination Illumination systems that do not form an image of the light source at some point in the optical pathway are called afocal or nonfocused illumination. Before the invention of electric bulbs, microscopists were limited in their choice of suitable sources for microscope illumination. During daylight hours, they could point their microscopes (or substage reflector mirrors) towards the sky.

31. Mirror (or internal lamp) Combined with an out board light source the mirror serves to direct light into the condenser. In most cases there are two surfaces, a flat surface for directing a parallel light beam into the condenser, and a curved or concave surface for directly focusing light onto the specimen with the condenser removed.

32. CONDENSER It is a vital part of the illumination system Designed to collect, control and concentrate light from the lamp on to the specimen .

33. Basically a group of lenses. The substage condenser gathers light from the microscope light source and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire view field .

34. All condensers have an aperture diaphragm with which the diameter of cone of the light beam can be controlled. If the diaphragm is closed too much – too contrast and refractile If the diaphragm is left wide open – glare (due to extraneous light interference) In both cases – resolution - poor

35. Each time an objective is changed, a corresponding adjustment must be performed on the substage condenser aperture iris diaphragm to provide the proper light cone for the numerical aperture of the new objective .

36. Type of condenser Abbe condenser simplest, least corrected, least expensive. 2 lens elements. Achromatic 4 lens elements Aplanatic 5 lens elements Aplanatic -achromatic corrected for both chromatic and spherical aberrations use in critical color photomicrography with white light; 8 internal lens

37. The two-lens Abbe condenser is in common use but is not very efficient, forming only an imperfect image of the light source.(NA = 0.25)

38. These are corrected chromatic aberration . NA – 0.95 Recommended for research microscopy and for color photomicrography

39. optically corrected for spherical aberration NA =1.40

40. the iris diaphragm iris diaphragm controls the angle of illuminating rays (and thus the aperture) which pass through the condenser, through the specimen and then into the objective. When the condenser aperture diaphragm is opened too wide, stray light generated by refraction of oblique light rays from the specimen can cause glare and lower the overall contrast. When the aperture is made too small, the illumination cone is insufficient to provide adequate resolution and the image is distorted due to refraction and diffraction from the specimen. Correct setting N .A of condenser is matched to the N.A of objective

41. When properly adjusted, light from the condenser will fill the back focal plane of the objective with image-forming light by projecting a cone of light to illuminate the field of view .

42. object stage Rigid platform with an aperture through which light pass. Allows movement of the object in two directions by means of two micrometer threads. 3 x 1 inch slide Moves over an area approximately 3½ x 1¼ inches Vernier scale 0-80, 80-110. Useful to locate slide at same position at a later date.

43. OBJECTIVE The most important lens of microscope collect maximum light possible from the object, unite it and form a high quality real image Its properties largely determine depth of focus, resolution, and contrast of the specimen.

44. switching from one lens to another requires only a little turn of the fine adjustment to bring the image into sharp focus. This arrangement of lenses is called parfocal system Parcentral : the image remains in the center of field of view when the lenses are changed

45. TYPES OF OBJECTIVES Achromatic objectives Fluorite objectives Apochromatic objectives Plan objectives

46. Achromatic Objectives Chromatic aberration can be corrected to within useful limits by using a two component lenses. A positive lens is combined with a negative lens made of glass producing a greater chromatic aberration . The negative lens corrects the chromatic aberration in the positive lens, and only partially neutralizes its magnifying power. This method will correct a thin positive lens for any two colors, leaving a small error in the intermediate colors. This type of lens is known as an achromatic lens.

47. Apochromatic Objectives They are highly corrected lens and always used for photomicrography. They are corrected chromatically for three colors (red, green, and blue), When fluorspar is incorporated in the glass of achromatic lens, three colors can be brought to the one focal point, and the amount of chromatic aberration visible in the image will be negligible.

48. Fluorite Objectives (CaF2) Fluorite or semi apochromatic objectives have fluorite incorporated into the lens system to give better color correction. . They produce a quality image mid way between that of the achromat and apochromat .

49. Levels of optical correction for aberration in commercial objectives. (a) Achromatic objectives, the lowest level of correction, contain two doublets and a single front lens; (b) Fluorites or semiapochromatic objectives, a medium level of correction, contain three doublets, a meniscus lens, and a single front lens; and (c) Apochromatic objectives, the highest level of correction, contain a triplet, two doublets, a meniscus lens, and a single hemispherical front lens.

50. Plan Objectives All three types of objectives suffer from pronounced field curvature and project images that are curved rather than flat. To overcome this inherent condition, lens designers have produced flat-field corrected objectives that yield flat images. Such lenses are called plan achromats, plan fluorites, or plan apochromats . In Plan Objectives, when imaging the field, parts of the specimen at the edge of the visual field are almost as well focused as those in the center . A Plan Objective lens will also have the same corrections for spherical and chromatic aberrations .

51. Polarizing objectives - strain-free objectives for use on the polarizing microscope Phase objectives contain a phase-plate for use in phase-contrast microscopy

52. Every objective has a fixed working distance focal length magnification and numerical aperture(NA).

53. Magnification Total magnification of microscope = magnification of objective x magnification of eye piece. With the eye piece of 10x, the total magnifications with the three objectives will be as follows

54. Total magnification is the product of magnification values of the objective and eyepiece , provided the system is standardized to an optical tube length of 160mm For variation M= optical tube length x M of eyepiece focal length of O With lower magnification , a large area on the slide can be seen, with higher magnification , though details can be seen, the area of view is restricted.

55. Useful magnification range - At a selected numerical aperture, when the microscope provides a magnified image that has a magnitude equal to the resolution limit of the human eye and any additional magnification beyond this point does not result in the resolution of even smaller specimen detail Microscope resolution is limited by NA and wavelength Empty magnification: Increase in size of image which does not increase information. Magnification which does not contribute to improved resolution or resolving power and which is beyond what the system can deliver. 

56. Resolution The ability of a lens to define detail. ie the ability to tell two points apart as separate points. If the resolving power of your lens is 2um that means two points that are 2um apart can be seen as separate points. If they are closer together than that, they will blend together into one point . Resolution is restricted by two factors: (1) the numerical aperture of the lens; and (2) the wavelength of light employed. The relationship is as follows Resolution=1.2 lambda/2 NA Lambda –wavelength of light employed

57. Numerical aperture the numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light . Numerical aperture of objective lens is the ability of the objective lens to gather the cone of light from the specime . higher the NA the greater the resolution NA = n sin µ(n=the refractive index of the medium between the object and the lens) µ is half of the angular aperture(maximum cone of light) of the objective The apertures are measured by, the angle formed by the outer edges of the lens, and a point on the object .

58. Condenser Object Real image Eye lens Eye piece lens Objective Virtual image

59. Refractive Index Higher numerical apertures can be obtained by increasing the imaging medium refractive index ( n ) between the specimen and the objective front lens. Microscope objectives are now available that allow imaging in alternative media such as water (refractive index = 1.33), glycerin (refractive index = 1.47), and immersion oil (refractive index = 1.51).

60. Color-Coded Rings on Microscope Objectives Immersion color code Black - Oil immersion Orange - Glycerol immersion White - Water immersion Magnification color code Black - 1x, 1.25x Brown - 2x, 2.5x Red - 4x, 5x Yellow - 10x Green - 16x, 20x Turquoise blue - 25x, 32x Light blue - 40x, 50x Cobalt (dark) blue - 60x, 63x White (cream) - 100x

61. Nosepiece: Nose piece is fitted at the lower end of the body tube and a number of objectives (usually three) are fitted on it The nosepiece should bring each objective into its central position. Any lens can be rotated into position when desired, its correct position being indicated by a ‘click’ 18 mm

62. BODY TUBE Supports the eyepiece and objectives Standard 160 mm = 142 + 18 mm connects the eyepiece to the nosepiece Monocular Binocular A) Seidentopf (sigh-den-top-ff) binocular head B)Slider binocular head Combined photo binocular

63. Mechanical Tube Length is the distance between the nosepiece to the top edge of the observation tubes where the eyepieces (oculars) are inserted

64. Monocular microscopes Microscopes with one body tube © J.Paul Robinson

65. Binocular microscopes The light rays emerging from the objective in the binocular microscope are equally divided between the two eyepieces. The modern binocular microscope, achieves this by the use of four prisms thus eyes will receive two parallel beam of light. Advantages Long period may be spent viewing through with minimum amount of eye fatigue The eye pieces can be easily moved together or apart to adjust inter ocular distance to suit individual requirements.

66. Types A) Seidentopf (sigh-den-top-ff) binocular head A head design where the increasing or decreasing the distance between the two eyepieces (interpupillary distance) is done by twisting the eyepieces in an up and down arc motion similar to most binoculars. B)Slider binocular head Inter-pupillary distance is adjusted side-to-side, by sliding the eyepieces towards and away from each other.

67. Photo binocular It has a prism system allowing 100% of the light to go either to the observation eye piece, or to camera located on the vertical part, and some times has a beam splitting prism dividing the light

68. Two body tube styles are also available; Dual head: has one vertical eyepiece lens and a second eyepiece off the side at 45 degrees (so that two people can view the sample at one time, or one person and camera). Trinocular: a microscope with a vertical tube at the top and regular binocular eyepieces at 30 degrees. Vertical tube is often used for a digital camera or a second observer.

69. Light pathway in binocular The light rays emerging from the objective in the binocular microscope are equally divided between the two eyepieces. It is not sufficient simply to insert a single prism and divert one half of the rays, since this would cause eyestrain due to both the observer's eyes being focused on a single point a short distance away, and the advantage of a binocular microscope is that long periods may be spent viewing through it with the minimum amount of eye fatigue.

70. The modern binocular microscope achieves this by the use of four prisms. from Figure the eyes are receiving two parallel beams of light . The lower central prism consists of two prisms cemented together, at the interface of which there is a semi-silvered surface : this silvering is a very special process, fine grains of silver being deposited so that alternate light rays are differentially treated, one being reflected to the right (Figure ) and the other passing into the upper prism.

71. The light rays passing through the semi-silvered surface to the upper prism travel through a greater thickness of glass than those that are reflected — having the effect of retarding them — and this is compensated for by making the right-hand prism with an extra thickness of glass as will be seen by comparing the two outside prisms in Figure 30.15.

72. Finite and Infinite body tubes Over the past 10 years, the major microscope manufacturers have largely all migrated to the utilization of infinity-corrected optical systems in both research-grade biomedical and industrial microscopes. In these systems, the image distance is set to infinity, and a tube (or telan ) lens is strategically placed within the body tube between the objective and the eyepieces (oculars) Here objective produces a flux of parallel light wave trains imaged at infinity (often referred to as infinity space )which are brought into focus at the intermediate image plane by the tube lens

73. EYE PIECE The final stage in the optical path of the microscope function is to magnify the image formed by the objective with in the body tube, and present the eye with a virtual image. 5x, 8x, 10x eyepieces Two lenses-one at the top-the eye lens and the other ‘field lens ’ fitted at the bottom .

74. Approximately mid-way between these lenses there is a fixed circular opening or internal diaphragm which, by its size, defines the circular field of view that is observed in looking into the microscope. Eyepieces can be adapted for measurement purposes by adding a small circular disk-shaped glass reticle (graticule or reticule) at the plane of the field diaphragm of the eyepiece. Reticles usually have markings, such as a measuring rule or grid, etched onto the surface. Because the reticle lies in the same plane as the field diaphragm, it appears in sharp focus superimposed over the image of the specimen.

75. Negative eyepieces In Negative eyepiece the focus is within (between) the lenses of the eyepiece. The lower or field lens collects the image that would have been formed by the objective and cones it down to a slightly smaller image at the level of the field stop with in the eyepiece, the upper lens then, produces an enlarged virtual image which is seen by the microscopist . In their simplest form, both lenses are plano-convex, with convex sides facing the specimen.

76. Positive eyepieces In Positive eyepieces the focus is outside the eyepiece lens system. The field stop (or diaphragm) is outside the eye piece, from which the virtual image is focused and magnified by the entire eyepiece This eyepiece has an eye lens and field lens that are also plano-convex, but the field lens is mounted with the curved surface facing towards the eye lens. The front focal plane of this eyepiece lies just below the field lens, at the level of the eyepiece fixed diaphragm

77. Huygenian & Ramsden Eyepiece Negative oculars Originally designed for telescopes Most commonly used in microscope Positive oculars Compensated eye pieces are mostly of the Ramsden type Huygenian Eyepiece Ramdsen Eyepiece

78. Kellner or Achromat Eyepieces Also called an " achromatized Ramsden ". Kellner eyepieces are a 3-lens design. An achromatic doublet is used in place of the eye lens in the Ramsden design to correct the residual chromatic aberration. They are inexpensive and have fairly good image from low to medium power and are far superior to Huygenian or Ramsden design.

79. High-eye point oculars With normal eyepieces the distance between the top of the eyepieces and the ‘exit pupil’ (eye point) is so small as to prevent the wearing of glasses Usually eyes are placed 8-10 mm above the eye lens. High point oculars have viewing distances approaching 20- 25 mm above the surface of the eye lens. Today, high eyepoint eyepieces are very popular, even with people who do not wear eyeglasses, because the large eye clearance reduces fatigue and makes viewing images through the microscope much more comfortable.

80. Compensating eyepieces Simple eyepieces such as the Huygenian and Ramsden will not correct for residual chromatic difference of magnification in the intermediate image To remedy this in finite microscopy systems, manufacturers produce compensating eyepieces that introduce an equal, but opposite, chromatic error in the objective lens elements. Compensating eyepieces may be either of the positive or negative type, and can be used at all magnifications with fluorite, apochromatic and all variations of plan objectives

81. SUPPORT SYSTEM FOOT LIMB FOCUSSING SYSTEM

82. Foot (base) It rests on the bench top and supports the stage and body of the microscope, and in many cases also houses the lamp

83. LIMB (ARM) The arm is attached to the foot and supports the body tube

84. FocusSing system Consists of coarse and fine adjustment screw-heads. It is employed for raising or lowering the optical system with reference to the slide under study till it comes into focus Coarse- rack and pinion Fine adjustment- micrometer screws

85. Operation of microscopes

86. Condenser Object Real image Eye lens Eye piece lens Objective Virtual image

87. There have been varying approaches to maximize the illumination within the microscope. Critical illumination is used in simple equipment where the light source is focused by the substage condenser in the same plane as the object, but this produces uneven illumination . Köhler illumination is used for photography and more specialized microscopes where an image of the light source is focused by the lamp collector or field lens in the focal plane of the stage condenser on the aperture diaphragm. The image of the field or lamp diaphragm is focused in the object plane and the illumination is even (Fig. 3.13). The illumination must be centered with respect to the optical axis of the microscope to prevent poor resolution CRITICAL ILLUMINATION(NELSONS) ILLUMINATION AND KOHLERS ILLUMINATION

88. Illuminating apparatus Light emitted from the tungsten-halogen lamp filament first passes through the collector lens located close to the lamp housing, and then through a second lens that is closer to the field diaphragm. The second lens in the light path is called the field lens, which is responsible for bringing the image of the filament into focus at the plane of the aperture diaphragm.

89. Focused light leaving the field lens is reflected by a mirror (positioned at a 45-degree angle to the light path) through the field diaphragm and into the substage condenser. The field diaphragm serves as a virtual source of light for the microscope and its image is focused by the condenser onto the specimen plane

90. The field diaphragm in the base of the microscope controls only the width of the bundle of light rays reaching the condenser It does not affect the optical resolution, numerical aperture, or the intensity of illumination. Proper adjustment of the field diaphragm is important for preventing glare than can reduce contrast in the observed image. When the field diaphragm is opened too far, scattered light originating from the specimen and light reflected at oblique angles from optical surfaces can act to degrade image quality.

91. Closing or opening the condenser diaphragm controls the angle of the light rays emerging from the condenser and reaching the specimen from all azimuths. Because the light source is not focused at the level of the specimen, the light at specimen level is essentially grainless and extended and does not suffer deterioration from dust and imperfections on the glass surfaces of the condenser. Opening and closing of the condenser aperture diaphragm controls the  angle  of the light cone reaching the specimen. .

92. Parallel light rays that pass through and illuminate the specimen are brought to focus at the back focal plane of the objective, where the image of the variable condenser aperture diaphragm and the image of the light source will be seen in focus.

93. Field of view The diameter of the view field in an optical microscope is termed the field number and represents the diameter of the field measured in millimeters at the intermediate image plane From this can be calculated the actual diameter of the specimen being viewed Field Size = Field Number ( fn ) ÷ Objective Magnification (M o )

94. HOW TO OBSERVE A SLIDE ? 113

95. CAUSES OF ERROR IN FOCUSING Revolving Nose Piece is off centre Preparation is upside down Thick cover slip Dirt or Dried oil over Lens Air bubble in immersion oil Poor illumination – Condenser not fully racked up

96. MICROMETRY The standard unit of measurement in microscopy is a micrometer (  m), which is 0.001mm. The stage micrometer - 3 x 1 inch slide on which a millimeter scale is engraved in 1/10 and 1/100 graduations. Eyepiece micrometer- disc with an arbitrary scale on it. huygenian – placed on field stop kellner - disc permanently in place

97. Protocol for the use of microscope Step1 : Examine the slide with naked eye Step 2 : Slide is viewed under low magnification to get a general view all over. Lower the stage about 7-8 cm below the body tube. Put slide on the stage and bring the specimen over the central aperture. Looking from the side, and using the coarse adjustment, bring the stage up, so that lens is about 1 cm above the slide. Now look into the eyepiece and adjust till the specimen comes into focus. Scan the entire field, racking the fine adjustment all the time.

98. Step 3 : Choose an area of interest for viewing under high magnification and focus under high power objective.

99. Focusing under oil-immersion objective most frequently used in hematology because of its high magnification and resolution. Two important feature of this lens are its very small aperture and deep focusing position , which is very close to the slide because of its short focal length of about 2mm. cedar wood oil or Medias like glycerin or liquid paraffin during use.

100. Raise the body tube so that the lens is about 8-10 cm above the slide. Place a drop of cedar wood oil on the slide, and looking from the side, slowly bring the objective down till it just enters the oil drop. The oil will spread out in the capillary space between the slide and the lens. While looking into the eyepiece raise the objective with coarse adjustment till the cells come and into view. After use, clean the objective with xylene and then with alcohol.

101. Care of the microscope Apart from cleaning the outer surface of their lenses, objectives are best left alone. Prisms should never be touched, and cleaning should be confined to blowing of the dust Lenses should be wiped only with fresh lens tissue or cotton wool, otherwise they may be scratched. Immersion oil should be removed with lens tissue or cotton wool damped with xylene, or, preferably ether

102. Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for example Hold the plug (not the cable) when unplugging the illuminator Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done

103. Cleaning and maintenance Daily cleaning routine: The microscope should be dusted daily outer surface of the lenses of objectives polished with lens tissue or cotton wool. The top lens of the eyepiece should be polished to remove dust or fingermarks the microscope is set up for correct illumination. The substage condenser and the mirror should be cleaned.

104. Weekly cleaning routine The slides of the coarse adjustment, the mechanical stage and the substage condenser should be wiped with a cloth dampened with xylene to remove dust which would otherwise damage the slides. A little oil (as supplied for lubricating microscopes) is applied and the slides replaced: later models do not require this treatment. The lens system should be checked and cleaned

105. Limitations of standard optical microscopy (bright field microscopy) The technique can only image dark or strongly refracting objects effectively. Diffraction limits resolution to approximately 0.2 micrometre. Out of focus light from points outside the focal plane reduces image clarity. Live cells and internal structures of the cell could not be studied effectively. Live cells lack sufficient contrast to be studied successfully, internal structures of the cell are colourless and transparent. So to increase contrast special stains and dyes were used but this caused killing and fixing the cells. Staining may also produce artefacts.

106. CONCLUSION The light microscope, often the symbol of research and scientific discovery, has evolved over the last 350 years from Antonie van Leeuwenhoek’s simple magnifier to the more sophisticated instruments of today. Studies of biological structures and processes on both fixed and live specimens have advanced light microscopy into an indispensable tool for cell and molecular biologists.

107. references Theory and practical of histological techniques: John D Bancroft Cellular pathology technique: 4 th edition by C.F.A Culling, R.T.Allison & W.T Barr Basics in Light Microscopy- Dr. Arne Seitz, PT-BIOP Course, 2010, EPFL