1. Microscope and Microscopy

2. Microscopy: The Instruments
The simple microscope used by van Leeuwenhoek in the seventeenth century had only one lens and was similar to a magnifying glass. However, van Leeuwenhoek was the best lens grinder in the world in his day. His lenses were ground with such precision that a single lens could magnify a microbe 300X. His simple microscopes enabled him to be the first person to see bacteria.
Contemporaries of van Leeuwenhoek, such as Robert Hooke, built compound microscopes, which have multiple lenses. In fact, a Dutch spectacle maker, Zaccharias Janssen, is credited with making the first compound microscope around However, these early compound microscopes were of poor quality and could not be used to see bacteria. It was not until about 1830 that a significantly better microscope was developed by Joseph Jackson Lister (the father of Joseph Lister). Various improvements to Lister's microscope resulted in the development of the modern compound microscope, the kind used in microbiology laboratories today.
Microscopic studies of live specimens have revealed dramatic interactions between microbes.

4. Principles of the Compound Light Microscope
Magnification: Ocular and objective lenses of compound microscope
Resolution: The ability of the lenses to distinguish two points.
Contrast: Stains change refractive index  contrast between bacteria and surrounding medium

5. Refractive Index Measures light-bending ability of a medium
Light may bend in air so much that it misses the small high-magnification lens.
Immersion oil is used to keep light from bending.

6. Numerical aperture
The light-gathering ability of a microscope objective is quantitatively expressed in terms of the numerical aperture,
Higher values of numerical aperture allow increasingly oblique rays to enter the objective front lens, producing a more highly resolved image.

7. Numerical aperture
The aperture angle is the angle between the microscope optical axis and the direction of the most oblique light rays captured by the objective.

8. Numerical aperture
The sine value of the half-aperture angle (θ) multiplied by the refractive index n of the medium filling the space between the front lens and the cover slip gives the numerical aperture: NA= n sin θ
The value of n varies between 1.0 for air and 1.52 for a majority of immersion oils utilized in optical microscopy. 

9. Color code & number on objective lens
Objective lenses are color coded and have numbers written on them.  Let's look at 
The yellow band tells us that it is a 10X objective lens.  Red is 4X, Blue is 40X and White is 100X.  The first number "10" is the power (10X).  The 0.25 is the Numerical Aperture. 
The 160 is a standard DIN measurement in millimeters of the tube length of the microscope required for this lens to work properly. Finally, the 0.17 is the thickness in mm of the cover slip that you should use. 

10. Color code & number on objective lens

11. Limit of resolution
The smallest distance by which two objects can be separated and still be distinguishable as two separate objects. This can be expressed as follows:
d = λ/2 NA
where d = Limit of resolution
λ = Wavelength of light

12. Brightfield Microscopy
Simplest of all the optical microscopy illumination. techniques
Dark objects are visible against a bright background.

13. Brightfield Microscopy

14. Brightfield Microscopy

15. Brightfield Microscopy

16. Darkfield Microscopy
A darkfield microscope is used to examine live microorganisms that either are invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by staining that their characteristics then cannot be identified.
Instead of the normal condenser, a darkfield microscope uses a darkfield condenser that contains an opaque disk. The disk blocks light that would enter the objective lens directly. Only light that is reflected off (turned away from) the specimen enters the objective lens. Because there is no direct background light, the specimen appears light against a black background-the dark field.
This technique is frequently used to examine unstained microorganisms suspended in liquid.
One use for darkfield microscopy is the examination of very thin spirochetes, such as Treponema pallidum, the causative agent of syphilis.

18. Fluorescence Microscopy
Uses UV light.
Fluorescent substances absorb UV light and emit visible light.
Cells may be stained with fluorescent chemicals (fluorochromes).
Immunofluorescence

19. Fluorescence Microscopy
Fluorescence microscopy takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible).
Some organisms fluoresce naturally under ultraviolet light; if the specimen to be viewed does not naturally fluoresce, it is stained with one of a group of fluorescent dyes called fluorochromes.
When microorganisms stained with a fluorochrome are examined under a fluorescence microscope with an ultraviolet or near ultraviolet light source, they appear as luminescent, bright objects against a dark background.

20. Fluorescence Microscopy
Fluorochromes have special attractions for different microorganisms. For example, the fluorochrome auramine o, which glows yellow when exposed to ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis, the bacterium that causes tuberculosis.
Bacilllls anthracis, the causative agent of anthrax, appears apple green when stained with another fluorochrome, fluorescein isothiocyanate ( FITC).
The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent -antibody (FA) technique, or immunofluorescence.
Antibodies are natural defense molecules that are produced by humans and many animals in reaction to a foreign substance, or antigen.

21. Fluorescence Microscopy
Fluorescent antibodies for a particular antigen are obtained as follows: an animal is injected with a specific antigen, such as a bacterium, and the animal then begins to produce antibodies against that antigen. After a sufficient time, the antibodies are removed from the serum of the animal. Next, a fluorochrome is chemically combined with the antibodies. These fluorescent antibodies are then added to a microscope slide containing an unknown bacterium. If this unknown bacterium is the same bacterium that was injected into the animal, the fluorescent antibodies bind to antigens on the surface of the bacterium, causing it to fluoresce.
This technique can detect bacteria or other pathogenic microorganisms, even within cells, tissues, or other clinical specimens. Of paramount importance, it can be used to identify a microbe in minutes. Immunofluorescence is especially useful in diagnosing syphilis and rabies.

23. The Phase-Contrast Microscope
The principle of phase-contrast microscopy is based on the wave nature of light rays and the fact that light rays can be in phase (their peaks and valleys match) or out of phase.
If the wave peak of light rays from one source coincides with the wave peak of light rays from another source, the rays interact to produce reinforcement ( relative brightness ).
However, if the wave peak from one light source coincides with the wave trough from another light source, the rays interact to produce interference (relative darkness).

24. The Phase-Contrast Microscope
In a phase-contrast microscope, one set of light rays comes directly from the light source. The other set comes from light that is reflected or diffracted from a particular structure in the specimen. (Diffraction is the scattering of light rays as they " touch" a specimen's edge).
The diffracted rays are bent away from the parallel light rays that pass farther from the specimen.) When the two sets of light rays- direct rays and reflected or diffracted rays- are brought together, they form an image of the specimen on the ocular lens, containing areas that are relatively light (in phase), through shades of gray, to black (out of phase ) .
In phase-contrast microscopy, the internal structures of a cell become more sharply defined.

25. The Phase-Contrast Microscope
enhances the contrast between intracellular structures having slight differences in refractive index
excellent way to observe living cells

26. The Phase-Contrast Microscope

27. Phase-Contrast Microscopy

28. Differential interference contrast (DIC)
Like phase-contrast microscopy, DIC microscope uses differences in refractive indexes. However, a DlC microscope uses two beams of light instead of one.
In addition, prisms split each light beam, adding contrasting colors to the specimen.

29. Differential interference contrast (DIC)
Therefore, the resolution of a DIC microscope is higher than that of a standard phase-contrast microscope.
Also, the image is brightly colored and appears nearly three-dimensional

31. Electron Microscopy Uses electrons instead of light.
The shorter wavelength of electrons gives greater resolution.
There are two types of electron microscopes:
Transmission electron microscope
Scanning electron microscope.

32. Transmission electron microscope

33. Scanning Electron Microscopy (SEM)

34. Transmission Electron Microscopy
A beam of electrons from an electron gun is focused on small area of a specially prepared, ultra thin section of the specimen by an electromagnetic condenser lens. The specimen is usually placed on a copper mesh grid.

35. Transmission Electron Microscopy
The beam of electrons passes through the specimen and then through an electromagnetic objective lens, which magnifies the image.
Finally, the electrons are focused by an electromagnetic projector lens onto a fluorescent screen or photographic plate.

36. Transmission Electron Microscopy
The final image called a transmission electron micrograph appears as many light and dark areas, depending on the number of electrons absorbed by different areas of the specimen. 
The transmission electron microscope can resolve objects as dose together as 2.5 nm, and objects are generally magnified 10,000 to 100,000 x.

37. Transmission Electron Microscopy
Contrast can be greatly enhanced by using a "stain" that absorbs electrons and produces a darker image in the stained region.
Salts of various heavy metals, such as lead, osmium, tungsten, and uranium, are commonly used as stains.

38. Transmission Electron Microscopy
These metals can be fixed onto the specimen (positive staining) or used to increase the electron opacity of the surrounding field (negative staining).
Negative staining is useful for the study of the very smallest specimens, such as virus particles, bacterial flagella, and protein molecules.

39. Transmission Electron Microscopy
A microbe can also be viewed by a technique called shadow casting which gives a three-dimensional effect to the specimen and provides a general idea of the size and shape of the specimen.

40. Transmission Electron Microscopy
Advantage:
Transmission electron microscopy has high resolution and is extremely valuable for examining different layers of specimens.

41. Transmission Electron Microscopy
Disadvantages:
Only a very thin section of a specimen (about 100 nm) can be studied effectively.
Specimen has no three-dimensional aspect.
Specimens require treatments which not only kill the specimen, but also cause some shrinkage and distortion.
Sometimes there may be additional structures called artifacts.

42. Scanning Electron Microscopy
An electron gun produces a finely focused beam of electrons called the primary electron beam. These electrons pass through electromagnetic lenses and are directed over the surface of the specimen.

43. Scanning Electron Microscopy
The primary electron beam knocks electrons out of the surface of the specimen, and the secondary electrons thus produced are transmitted to an electron collector, amplified, and used to produce an image on a viewing screen or photographic plate. The image is called a scanning electron micrograph.

44. Scanning Electron Microscopy
Advantages:
It overcomes the problem of sectioning associated with a TEM.
It provides three -dimensional views of specimens .

45. Scanning Electron Microscopy
Advantages:
This microscope is especially useful in studying the surface structures of intact cells and viruses.
It can resolve objects as close together as 10 nm, and objects are generally magnified 1000 to 10,000 x.