1. Microscopy Microbial Biotechnology

2. Microscope an instrument for viewing objects that are too small to be seen by the naked eyes. First true microscope was made around 1595 in Netherlands by Hans Lippershey Zacharias Janssen and his son Giovanni Faber coined the name "microscope" Antony Van Leeuwenhoek (Father of microscope and microscopy) whose microscope allowed people to see things no human had ever seen before like bacteria, yeast, plant cell

3. Parts of Microscope Eye piece Tube Arm Base Illuminator Stage Resolving nose piece or turret Objective lenses Condenser Lens Diaphragm or Iris

6. Types Of Microscopes Optical microscopes (e.g. Light microscope) Electron microscopes (e.g.,TEM), Scanning probe microscopes (SPM). (e.g. SEM, STM) Types of Microscope based on application 1)Slide microscope Magnification: Uses lenses arranged in series, made of optical material that bends light. All magnification takes place via lenses. Uses diascopic illumination (that is, light is transmitted through the specimen). Magnification up to about 1000x. Depth of field: Small. Uses: Pathology, microbiology, forensics, education Advantages: Color viewing. Require no electricity except for the light source. Can be used on living samples. Simple models are relatively common and inexpensive. Disadvantages: Usually requires prepared slides and cannot examine objects well in three dimensions.

7. 2) Stereo microscope, also known as a dissecting microscope Magnification: As in a slide microscope, but arranged in two optical paths at slightly different angles to allow the user to view objects in three dimensions. Uses episcopic illumination (light is reflected from the specimen's surface). Maximum useful magnification is about 100x. Depth of field: Large. Uses: Microsurgery, fine repair, dissection, paleontology, quality control, sorting, forensics. Advantages: Color viewing. Can be used on living samples. Requires little or no preparation of the sample. Disadvantages: Low magnification.

8. 3) Digital microscope Magnification: Uses both optical lenses and CCD or CMOS sensors; up to at least 1000x magnification. Depth of field: Like a regular slide microscope. Uses: All the uses of slide microscopes. Especially useful for applications requiring digital information exchange such as telepathology and continuing medical education. Advantages: All the advantages of slide microscopes, plus the ability to create "virtual slides" and digital information sharing. Disadvantages: Requires a power source to operate. Most also require a computer, although some include their own viewscreens.

9. 4) 3D digital microscope Magnification: Works like a regular digital microscope to achieve up to 1000x magnification. Depth of field: Large, up to 20 times greater than that of a slide microscope. Uses: All the uses of other digital microscopes, as well as parts machining, materials science, and additional applications. Advantages: Object can be viewed from almost any angle and its three-dimensional features can be examined. Disadvantages: As with regular digital microscopes. Can be expensive. 5)TEM 6)SEM

10. What is microscopy? Use of microscope to visualize small objects. Types of microscopy Brightfield Microscopy Darkfied Microscopy Fluorescence Microscopy Confocal Microscopy TEM (Transmission Electron Microscope) SEM (Scanning Electron Microscope)

11. Properties of Light Distortion of Lenses Magnification Resolution Numerical Aperture Contrast

12. Properties of Light Physical properties of light (Wavelength) Interaction between light and object Transmission Reflection Absorption Fluorescence Refraction Distortions of Lenses Inherent problem in lenses distort magnified image Spherical aberration - based on the shape of lens Chromatic aberration – based on the color of light Achromatic lens (improved lens)lenses of different Apochromatic lens (highly improved)shapes and glass composition

13. Magnification Enlargement of object Two convex lenses ocular objective Contrast Necessary to discern an object from its background. Contrast can be increased by applying stains.

14. Resolution The degree to which the detail in the specimen is retained in the magnified image. Resolving power (R): The closest spacing between two points at which the points can still be seen clearly separate entities. OR it is the distance between two structure entities of a specimen at which the entities still can be seen as individual structure in magnified image. Depends on two properties –Wavelength of light –Numerical aperture Formula R = 0.5λ/NA R smaller – Better resolving power

15. Numerical Aperture A property of lens that describes the amount of light that can enter it. Depend on The refractive index (v) of the filling medium between the objective and lens. Angle (α) at which rays of light can enter in the lens Formula NA = v * sin α

17. Brightfield Microscopy Simple Most widely used Require a little sample preparation Easy to perform “Parfocal”

18. Darkfield Microscopy Simple No sample preparation required Apply to study to morphology of those objects which can not be stained

19. Fluorescence Microscopy Stain the specimen with fluorescent dye Widely used for immunological study Study location of structure in bacterial by using special stain nuclei - bis-benzimidazole mitochondria and actin cytoskeleton - MitoTracker Red CMXRos Epifluorescence – on specimen Transmitted fluorescence – below specimen

20. Phase Contrast Microscopy Frits Zernike No need to stain the material. Living objects can be seen.

22. Differential interference contrast Microscopy (Nomarski) Adv. Living objects can be seen. No need to stain the material. Dis. Material Should be transparent Unsuitable for thick sample Non biological can’t be visualized. Components A polarizing filter Interference contrast condenser A prism analyzer plate

25. Marvin Minsky CONFOCAL MICROSCOPY

26. ELECTRON MICROSCOPY Beam of electron Greater resolution & magnification 100,000X magnification than routine microscope Viruses and internal structure could be visualized

27. PREPARATION OF SPECIMENS dehydrated and fixed –Formaldehyde, glutraldehyde Staining Heavy metal (phosphotungstic acid) –Positive –Negative Thin sectioning Internal structure –Ultramicrotome – diamond knife

28. Freeze Etching

29. TRANSMISSION ELECTRON MICROSCOPE Max Knoll and Ernst Ruska in 1931

30. Grid copper, molybdenum, gold or platinum

31. Max Knoll SCANNING ELECTRON MICROSCOPE

32. Types of signals secondary electrons back-scattered electrons (BSE), characteristic X-rays Light cathodoluminescence Magnification Not a function of the power of objective lens.

33. Detection of secondary electrons The electrons are detected by an Everhart-Thornley detector, a type of scintillator-photomultiplier system. The secondary electrons are first collected by attraction by an electrically-biased grid at about +400 V, and then further accelerated towards a phosphor or scintillator positively biased to about +2,000 V. The accelerated secondary electrons are now sufficiently energetic to cause the scintillator to emit flashes of light (cathodoluminescence) which are conducted to a photomultiplier outside the SEM column via a light pipe and a window in the wall of the specimen chamber.

34. References Principles of Microbiology – R.M.Atlas Microbiology – Prescott http://www.microscopyu.com/tutorials