Microscopy

 The studies of living organisms of microscopic dimensions, its development depended entirely upon the refinement of the microscope. 

Type of microscopy 

1.Simple Microscope 

In the 17th century A.  van Leeuwenhoek was used single-lens microscopes of the simplest possible design.He could make lenses and using them to build magnifying glasses to provide a magnification of about 200.

2. Compound Microscope 

In the 18th century, Robert Hooke had used a compound microscope

3. Bright-field (Light) microscopy

 The light microscope is a two-lens system in which the lens nearest the object is known as the objective lens and that nearest the eye, is known as the ocular lens. 

Visible light rays are projected through a condenser that focuses them into a sharp cone, then sends them through the opening in the stage on which the slide rests.

 The light passes through the slide, bounces off the object, then passes through the objective lens to form a magnified image darker than the background around it. This image is a real image that can be projected onto a screen. 

The image now becomes an object for the ocular lens and the light rays are magnified a second time creating a virtual image in space visible only in the observer.

The dual-lens system is referred to as a compound microscope to distinguish it from a single-lens system such as used by van Leeuwenhoek. 

In addition laboratory microscope usually has three objective lenses called the low-power. high-power, and oil-immersion lenses. 

Generally, these magnify an object 10x, 40,x, and 10x diameters, respectively, and the magnification is represented by the multiplication symbol "X" 

Resolving power. In order for an object to be seen distinctly, the lens system must have good resolving power. This is the ability to transmit light without variation. 

If two objects are very close to each other, these must be distinguished clearly from each other as distinct, sharp objects. 

The resolving power (RP) of a lens system is a number (i.e. the ability, represented numerically) used to determine the size of the smallest object that can be clearly seen. It varies  for each objective, and calculated as follows:

                              

 RP= 𝝺/2 X N.A.        

 

 where 𝝺 =wave-length of light used

 (which is usually set at 550 nm, the halfway point between the limits of visible light)

The N.A.-numerical aperture of the lens (usually written on the objective lens 

Numerical aperture relates to the size of the cone of fight that will enter the objective, and to the medium in which the lens is suspended, usually air. 

For the low power objective, a common N.A. is 0.25, for the high-power objective, 0.65, and for the oil-immersion objective, 1.25. Thus the R.P. for the low-power objective may be calculated as follows a

R.P.= 550nm/2X0.25=1100nm=1.1µm

Since the R.P. for this lens system is 1.1µm, objects smaller than this figure can not be seen clearly, or the two objects placed close to each other at a distance smaller than this figure (1.1 um) may not be seen as two clear objects.

 Thus, with low power objective, certain bacteria cannot be seen clearly but larger objects, such as protozoa may be easily discerned

For the oil-immersion objective, the R.P. is calculated as follows:

R.P.=550nm/2X1.25=220nm=0.22µm

 

Thus most bacteria except chlamydiae may be seen clearly with oil-immersion objective. However, viruses, which are generally much smaller than 0.22 μm continue to escape the vision.

 Another factor of the compound microscope is, the working distance, or amount of clearance between the slide and the bottom of the lens. For low-power,

high power, and oil-immersion objective, this figure is commonly 6.8 mm, 0.73

mm and 0.12mm respectively. 

The oil used with oil-immersion objective increases the numerical aperture of the lens to 1.25 and therefore lowering the resolving power to 0.22µm

. 

3. Dark-field microscopy

In this scenario, only the object is lighted against a dark background.

A particular condenser scatters the light, causing it to strike the item from various angles.

The object is seen clearly because some light is reflected into the lens, but the surrounding area appears dark because there is no direct background fight:

 This microscopy is important in the diagnosis of certain diseases when small live organisms near the microscope's limit of resolution must be observed. 

Scrapings obtained from a skin lesion, for example, may reveal the syphilis spirochete Treponema pallidum. 

The organisms may be seen moving around in a rotating motion because no stain is used.

4. Phase-contrast microscopy 

 In 1953, the Dutch physicist Fritz Zernike  design this microscope. 

This enables organisms to be observed alive and undamaged. 

Bacterial granules, the fine structure of protozoa and fungus, and other interior characteristics that are difficult to view with bright-field microscopy can be investigated. 

A set of sophisticated filters and diaphragms in the phase-contrast microscope split the light beam and fling the rays slightly out of phase. Separated light then travels both around and through microscopic things.

 Small changes in item densities show up as varying degrees of brightness and contrast. 

5. Fluorescent microscopy

The process consists of coating a microbe with a fluorescent dye such as Tuorescein and illuminating it with ultraviolet light.

 As the electrons in the dye are excited, they move to high energy levels, then quickly drop back giving off the excess energy as visible light. The object appears to fluoresce. The most important application of this microscopy is in the fluorescent antibody technique.

6. Electron microscopy

Invented by Ernst Ruska , Max Knoll in 1931 at the Berlin Technische Hochschule or Berlin Technical University

The essential point here is the electron beams' unusually small wavelength, which acts as a light substitute. The resolving power of the device is increased to fractions of a nanometer at wavelengths of about 0.005 nm. It allows viruses and bigger molecules to be seen in detail.

Two types of electron microscopes are in use today

 1. Transmission electron microscopy (TEM). 

This is used to see the fine structure of cells. Ultra-thin sections of the object are prepared, by embedding freezing the specimen, and sectioning it with a diamond or glass knife. Sections are floated in the water and picked up on a wire grid. 

They are stained with a heavy metal (gold or palladium) to make certain parts dense and inserted in the vacuum chamber of the microscope. A 100,000-volt electron beam is focused on the section and manipulated by magnetic lenses.

 A photograph prepared from the image may be enlarged with enough resolution to achieve a total magnification of over 20 million times. Objects are small as 1.0 nm may be observed.

2. Scanning electron microscopy (SEM). 

This microscopy allows surfaces of objects to be seen in their natural state without staining. The specimen is put into the vacuum chamber and covered with a thin coating of gold to increase electrical conductivity and thus forms a less blurred image. The electron beam then sweeps across the object building an image line by line as in a TV Camera.  As electrons strike the object, they knock loose showers of electrons that are captured by a detector to form the image. Magnifications with this microscopy are limited to about 75,000-100,000 diameters

The degree of magnification needed to observe a microbe depends upon its size. Bacteria, fungi, algae, protozoa can be viewed with a light microscope, Smaller microorganisms, like viruses, as well as the internal structures of cells, require the use of an electron microscope. The choice of a particular microscope depends upon the size of the object, the degree of detail that must be viewed, the nature of the specimen, and the overall purpose of the microscopic observations 

Table1.   shows features of different types of microscopes including their strength, limitation, and applications.

Table 1. A comparison of various types of microscopes.

Type of Microscope          Maximum magnification            Resolution                       Remarks

Bright-field (Light)                     1,500x                               100-200nm             Extensively used in microbiology

                 

                                                                                                                          usually necessary to stain specimens

                                                                                                                          for observation

Dark-field                                  1,500x                           100-200nm              Used for live microbes, particularly 

                                                                                                                        those with characteristics morphology, 

                                                                                                                       staining not required

Ultraviolet                                 2,500X                       100nm                 Improved resolution over the light microscope                                                        

                                                                                                                   largely replaced by electron microscope

 Fluorescence                        1,500X                      100-200nm                 Fluorescent staining used useful in 

                                                                                                                   several diagnostic procedures for identifying                                                                                                          

                                                                                                                  microbes Used to examine the structure of live microbes;

Phase-contrast                        1,500X                    100-200nm      produce a sharp multicolored three-dimension  image 

               -    

                                                                          

TEM                                       500,000-                1 nm               used  to view the ultrastructure of microbes including 

                                               1,000,000X                                 virus  much greater resolving power and magnification

                                                                                                      then the light microscope

SEM                                     10,000-

1,000,000X   1-10nm            Used for detailed surface structure 

                                                                                                      of microbes three dimensional