Types of Microscope

Type of microscopes. Light and Electron Microscope differences. Microscopes principles and uses.

1. TYPES OF MICROSCOPE & THEIR USES MADE BY: AMAL MERAJ

2. HISTORY OF MICROSCOPE PRINCIPLES OF MICROSCOPY TYPES OF MICROSCOPE LIGHT MICROSCOPE vs ELECTRON MICROSCOPE SUMMARY OF TYPES OF MICROSCOPES 01 02 03 04 05 CONTENTS

3. MICROSCOPE A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscopic. Microscopic means being invisible to the eye unless aided by a microscope.

4. HISTORY OF MICROSCOPE Zacharias Jansen (1580–1638) of Holland invented a compound light microscope, one that used two lenses, with the second lens further magnifying the image produced by the first. Englishman Robert Hooke (1635–1703) further refined the compound microscope, adding such features as a stage to hold the specimen, an illuminator, and coarse and fine focus controls. Until 1800, compound microscopes designed by Hooke and others were limited to magnifications of 30x to 50x, and their images exhibited aberrations. Carl Zeiss (1816–1888) and Ernst Abbe (1840–1905) added the substage condenser and developed superior lenses that greatly reduced chromatic and spherical aberration, while permitting vastly improved resolution and higher magnification.

5. HISTORY OF MICROSCOPE Physicist Ernst Ruska and the electrical engineer Max Knoll (1931) developed the first prototype electron microscope which was capable of four-hundred-power magnification. Ernst Lubcke of Siemens & Halske (1932) built and obtained images from a prototype electron microscope, applying the concepts described in Rudenberg's patent. Ruska (1933) built the first electron microscope that exceeded the resolution attainable with an optical (light) microscope . Manfred von Ardenne (1937) pioneered the scanning electron microscope. Siemens (1939) produced a transmission electron microscope (TEM) in 1939.

6. HISTORY OF MICROSCOPE   Antony van Leeuwenhoek was a Dutch businessman and scientist in 1674, viewing a drop of rainwater , he observed things moving which he called "animalcules."  F irst to experimented with microbes, using single-lensed microscopes of his own design invented in 1670. Magnified up to 200x and achieved twice the resolution of the best compound microscopes of his day, mainly because he crafted better lenses. Father of Microbiology ( 1632–1723)

7. MICROSCOPE USED BY LEEUWENHOEK REPLICA OF LEEUWENHOEK’S MICROSCOPE Leeuwenhoek’s drawings of animalcules

8. MAGNIFICATION RESOLUTION NUMERICAL APERTURE ILLUMINATION PRINCIPLES OF MICROSCOPY ABERRATION 01 02 03 04 05

9. MAGNIFICATION Magnification is the factor by which an image appears to be enlarged. It is dependent upon the curvature and size of the lens. The image formed is enlarged to a particular degree called the “Power of Magnification”. Magnifying Glass Crystal Ball

10. RESOLUTION Resolution or Resolving P ower is the ability of a lens to show two adjacent objects as discrete entities. Degree to which detail in specimen is retained in a magnified image The minimum distance between two visible bodies at which they can be seen as separate is the “Limit of Resolution (LR)”. Resolution is best when LR is low. The shorter the wavelength of the illumination, the better the resolution. Resolving Power (R.P)  

11. NUMERICAL APERTURE The numerical aperture of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object. Each objective has a fixed numerical aperture reading Higher the NA the better will be the resolution. Greater the refractive index (n) the greater will be the NA.

12. ILLUMINATION Effective illumination is required for efficient magnification and resolving power. A rtificial light from a tungsten lamp is the most commonly used light source in microscopy . I ris diaphragm regulates the amount of light entering the condenser . Too much light can reduce contrast and burn out the image. Other ways to increase contrast in cell components are by using special lenses (phase-contrast microscope) and by dyes.

13. ABERRATION A berration is a property of optical systems such as lenses that causes light to be spread out over some region of space rather than focused to a point. Aberrations cause the image formed by a lens to be blurred or distorted. Aberrations associated with microscope are spherical aberration and chromatic aberration.

14. ABERRATION Creates a curved image rather than flat. SPHERICAL ABERRATION SPHERICAL ABERRATION Image formed by Spherical Aberration

15. ABERRATION Creates a blurry image like a rainbow Caused by the lens acting as a prism For correction- Achromatic objective and Apochromatic objectives. CHROMATIC ABERRATION Image formed by Chromatic Aberration

16. TYPES OF MICROSCOPE Bright-field Dark-field Phase-contrast Differential Interference Contrast (DIC) LIGHT MICROSCOPES 01 Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM) ELECTRON MICROSCOPES 03 Fluorescent Confocal Laser Scanning Inverted Comparison Polarized Stereoscopic MODIFIED MICROSCOPES 02 Digital Scanning Probe Acoustic USB Computer OTHER MICROSCOPES 04

17. LIGHT MICROSCOPE A light microscope (LM) is an instrument that uses visible light and magnifying lenses to examine small objects not visible to the naked eye, or in finer detail than the naked eye allows. Magnification, however, is not the most important issue in microscopy . The usefulness of any microscope is that it produces better resolution than the eye .

18. SIMPLE MICROSCOPE Simple microscope  is a magnifying glass that has a single lens with a short focal length. Old Simple Microscopes Present-Day Simple Microscopes

19. OPTICAL MICROSCOPE A simple microscope uses a lens or set of lenses to enlarge an object through angular magnification alone Gives the viewer an erect enlarged virtual image . The use of a single convex lens or groups of lenses are found in simple magnification devices such as the magnifying glass, loupes, and eyepieces for telescopes and microscopes . Principle of Optical Microscope Loupe Telescope

20. COMPOUND MICROSCOPE Hooke devised the compound microscope and illumination system (1665). It was one of the best such microscopes of his time. Robert Hooke’s Micrographia

21. MODERN COMPOUND MICROSCOPE

22. PRINCIPLES OF COMPOUND MICROSCOPE The magnification from compound microscope occurs in two series of lenses. ( i ) The objective lens close to the object to be observed. (ii) the ocular lens or eyepiece; the one closest to the eye. The objective lens produces an initial magnified ‘real image’ This image is again magnified by the ocular lens (eyepiece) to obtain a final ‘virtual image’. This image is received by the eye and converted to a retinal and visual image.

23. PRINCIPLES OF COMPOUND MICROSCOPE The total power of magnification of the final image is formed by the combined product of two separate powers of each lens. It can magnify up to x2000. Structures less than 0.2um cannot be resolved with the compound light microscope.

24. BRIGHT-FIELD MICROSCOPE Also known as compound light microscope. Produces a image when light is transmitted through the specimen. Through differential absorption and differential refraction it produces a contrasting image . Produces a dark image against a bright background. U sed for live, unstained material and preserved, stained material. Principle of Bright-field microscopy

25. BRIGHT-FIELD MICROSCOPE USES Vastly used in Biology, Cellular Biology, and Microbiological Laboratory studies. It can be used to identify basic bacteria cells and parasitic protozoans such as  Paramecium. Image Produced by Bright-field microscopy Bone marrow biopsies of cynomolgus monkeys experimentally infected with hepatitis E virus at 160 dpi. Histological analysis showing: (A-B) vacuolization in mononuclear cells (!); (C) lymphocyte proliferation and activation clusters; (D) megakaryocytosis (>5 megakaryocytes/field); (E) absence of megakaryocytosis (0-2 megakaryocytes/field); and (F) vacuolization in endosteal cells (!). Hematoxilin and Eosin stain.

26. DARK-FIELD MICROSCOPE Specialized type of bright field light microscope which has several similarities to the Phase-Contrast Microscope. To make a dark field Microscope place a dark-field stop to the condenser lens. When a hollow cone beam of light is transmitted to the specimen, deviated light ( unreflected / unrefracted ) rays do not pass through the objectives but the undeviated (reflected/refracted) light passes through the objectives to the specimen forming an image.  Resulting image is brightly illuminated surrounded by a dark (black) field. It is used to visualize living cells which cannot be stained or be distorted by drying. Principle of Dark-field microscopy

27. DARK-FIELD MICROSCOPE USES It is used to visualize the internal organs of larger cells such as the eukaryotic cells Identification of bacterial cells with distinctive shapes such as  Treponema pallidum , a causative agent of syphilis. Image Produced by Dark-field microscopy Treponema pallidum bacteria (syphilis) is shown

28. PHASE-CONTRAST MICROSCOPE This is a type of optical microscope. The phase-contrast microscope produces high contrast images when using a transparent specimen. The shifts that occur during light penetration, become converted to changes in amplitude which causes the image contrast . used to view unstained cells also known as the phase objects, which means that the morphology of the cell is maintained and the cells can be observed in their natural state, in high contrast and efficient clarity . Reveals internal details of cells. Useful in observing intracellular structures. Principle of Phase-contrast microscopy

29. PHASE-CONTRAST MICROSCOPE USES Determine morphologies of living cells such as plant and animal cells Studying microbial motility and structures of locomotion To detect certain microbial elements such as the bacterial endospores Image Produced by Phase-Contrast microscopy Cheek cells are shown

30. DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPE Also known as Nomarski interference contrast (NIC) or Nomarski microscopy. It is an optical microscopy technique. It has two prisms refinements which add contrasting colours to the images. Two beams of light instead of one. Produces well-defined images with detailed view. Creates a vividly coloured , three-dimensional image. USES Visualizes live and unstained biological samples, such as a smear from a tissue culture or individual water borne single-celled organisms.

31. DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPE Image produced by DIC is shown Principle of DIC microscopy

32. FLUORESCENCE MICROSCOPE Specially modified compound microscope. Uses a mercury arch lamp as a source of UV light. The microscope will also comprise excitation filter, dichromatic mirror and an emission filter.  Filter protects the viewer’s eye from UV rays. The specimen to be viewed is coated with dyes ( acridine , fluroscein ) or minerals which show fluorescence. Subsquent illumination by ultraviolet radiation causes the specimen to give off light that forms its own image; usually an intense yellow, orange or red against a black field. An advantage of fluourescence microscopy is that it can be used to detect and visualise multiple fluorescent molecules e.g. cells glowing as they are doing their work Principle of Fluorescence microscopy

33. FLUORESCENCE MICROSCOPE USES V isualization of bacterial agents such as  Mycobacterium tuberculosis. Identify specific antibodies produced against bacterial antigens/pathogens in immunofluorescence techniques by labeling the antibodies with fluorochromes . Used in ecological studies to identify and observe microorganisms labeled by the fluorochromes . D ifferentiate between dead and live bacteria by the color they emit when treated with special stains. Image produced by fluorescence microscopy of mycobacterium tuberculosis .

34. CONFOCAL LASER SCANNING MICROSCOPE (CLSM) Laser beam used to illuminate spots on specimen. Laser beam focused and scanned over the sample produces 3D and 2D images in the computer screen. Laser beam scans a single plane of 1 um thickness. Can scan many thin sections through the sample . Used on specimens which are too thick for a light microscope. Principle of CLSM

35. CONFOCAL LASER SCANNING MICROSCOPE (CLSM) USES Observing cellular morphology in multilayered specimen. Used in diagnosing CA cervix. Evaluation and diagnosis of basal cell or carcinoma of skin. Confocal Laser Scanning Microscopy image of dendritic cells Confocal Laser Scanning Microscopy image of Astrocyte Brain Cells

36. POLARIZED MICROSCOPE A polarizing microscope is an optical microscope composed of a detector, lenses and polarizing filters . It uses two polarizers. The polarizer is positioned at the light path somewhere between the specimen and an analyzer (second polarizer). Analyzer is placed in the optical pathway between the objective rear aperture and the observation tubes or camera port. Polarizers transmit one polarization angle of light. Crossed polarizers transmit no light Principle of Polarized Microscope

37. POLARIZED MICROSCOPE USES Study of birefringent materials; materials that split a beam of light into two. Used in crystallography, urine examination. Apple Green Birefringence in AMYLOIDOSIS Image of calcite crystal from polarized microscope

38. INVERTED MICROSCOPE Used in metallurgy. Double microscope. Produces 2D and 3D images. USES Examination of cultures in flat bottom dishes. Micro dissection. Examination of parasites. Observation of agglutination in serology. I nverted microscopic images of RBCs in human blood showing hemolysis after treatment with G7 amine terminated PAMAM dendrimers at dose of 10 mg/kg.

39. COMPARISON MICROSCOPE Observe two different objects at the same time. Used to compare objects.. Composes of two independent objective lenses joined together by an optical bridge to a common eyepiece lens. Objects are observed side-by-side in a circular field that is equally divided into two parts. USES Examination of firearms. Used greatly in forensics applications for comparing different traces. Firearm comparison by Comparison Microscope

40. STEREOSCOPIC MICROSCOPE Double microscope It is an optical microscope variant designed observation of a sample. Low magnification and low resolution. Reflected illumination rather than transmitted one It uses two separate optical paths with two objectives and eyepieces. Provides different angles for each eye to view. Gives a three-dimensional view of objects. USES Dissections Forensic Applications for Questioned D ocument Examinations such as comparison of texture and condition of paper surfaces, ribbons, pen and pencil points, etc. Image produced by Stereoscopic Microscope

41. COMPARISONS OF LIGHT MICROSCOPES Fluorescene Stereo

42. ELECTRON MICROSCOPY Electron microscopy (EM) is a technique for obtaining high resolution images of biological and non-biological specimens. Uses a beam of accelerated  electrons  as a source of illumination . The wave of electrons are 100,000 times shorter than waves of visible light. EM images provide key information on the structural basis of cell function and of cell disease . This microscopy can yield information about topography, morphology, composition, and, crystallographic structure.  

43. SCANNING ELECTRON (SEM) MICROSCOPE SEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning ). Doesn’t transmit electrons. Shower of electron deflected from the surface is picked up by a sophisticated detector. The electron pattern displayed as an image on television screen. Produces an striking three-dimensional realistic images. Magnifies external surface of specimen.

44. SCANNING ELECTRON (SEM) MICROSCOPE

45. SCANNING ELECTRON (SEM) MICROSCOPE USES SEMs can be used in a variety of industrial, commercial, and research applications. SEMs are used in materials science for research, quality control and failure analysis. J ust about any material science industry, from aerospace and chemistry to electronics and energy usage, have only been made possible with the help of SEMs. Criminal and other forensic investigations utilize SEMs to  uncover evidence and gain further insight. In biological sciences, SEMs can be used on anything from insects and animal tissue to bacteria and viruses. Geological sampling using a scanning electron microscope can determine weathering processes and morphology of the samples.

46. SCANNING ELECTRON (SEM) MICROSCOPE Images produced by SEM

47. TRANSMISSION ELECTRON (TEM) MICROSCOPE Uses a high voltage electron beam to illuminate the specimen and create an image. Transmits electron through the specimen. The image-recording system usually consists of a fluorescent screen for viewing and focusing the image and a digital camera for permanent records. Electrons cannot readily penetrate thick preparations. The specimen must be sectioned into extremely thin slices (20-100nm thick) and stained or coated with metals to increase image contrast. Creates a two-dimensional image. Sections of specimen are viewed under very high magnification

48. TRANSMISSION ELECTRON (TEM) MICROSCOPE

49. TRANSMISSION ELECTRON (TEM) MICROSCOPE USES TEMs are ideal for a number of different fields such as life sciences, nanotechnology, medical, biological and material research, forensic analysis, gemology and metallurgy, and industry and education. TEMs provide topographical, morphological, compositional and crystalline information. TEMs can be used in semiconductor analysis and production and the manufacturing of computer and silicon chips. Colleges and universities can utilize TEMs for research and studies.

50. TRANSMISSION ELECTRON (TEM) MICROSCOPE Images produced by TEM

51. OTHER MICROSCOPES DIGITAL MICROSCOPE:  Variation of a traditional optical microscope that uses optics and a digital camera to output an image to a monitor sometimes by means of software running on a computer. Efficient tool to inspect and analyze various objects from micro-fabricated parts to large electronic devices.  Used in  a wide range of industries, such as education, research, medicine, forensics, and industrial manufacturing. SCANNING PROBE MICROSCOPE (SPM): Produces an image that represents the structure of surface rather than direct view of surface. Used to make images of nanoscale surfaces and structures, including atoms.

52. OTHER MICROSCOPES ACOUSTIC MICROSCOPE: Employs very high or ultra high frequency ultrasound. Sound waves produce an enlarged image of a small object. Operate non-destructively and penetrate most solid materials Used to visualize images of internal features, including defects such as cracks,  delamination and voids. USB COMPUTER MICROSCOPE (SPM):  L ow-powered  digital microscope which connects to a computer, normally via a  USB  port. Used in e xamination of flat objects like coins, circuit boards, banknotes. Examining irregular surfaces such as fibers with a high depth of field. Examining large items in situ when conventional microscopes can not be  used.

53. LIGHT MICROSCOPE VS ELECTRON MICROSCOPE

54. SUMMARY OF TYPES OF MICROSCOPE

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