Light Microscope Principle and Applications or Uses

Light Microscope Principle & Uses

(By: Dr. Akalesh K Verma, Asst. Prof., Cotton University)

Fig 1: Components of Light Microscope.

  Parts of Light Microscope

a.      Illuminator: the light source in the base of the microscope;

b.      Abbe Condensor: a two lens system that collects and concentrates light from the illuminator and directs it to the iris diaphragm;

c.      Iris diaphragm: regulates the amount of light entering the lens system;

d.      Mechanical stage: a platform used to place the slide on which has a hole in the center to let light from the illuminator pass through. Often contains stage clips to hold the slide in place;

e.      Body tube: houses the lens system that magnifies the specimens;

f.       Upper end of body tube—oculars/eye pieces: what you view through;

g.      Lower end of body tube—nose-piece: revolves and contains the objectives.

h.      Essentially, a light microscope magnifies small objects and makes them visible. The science of microscopy is based on the following concepts and principles:

i.        Magnification is simply the enlargement of the specimen. In a compound lens system, each lens sequentially enlarges or magnifies the specimen;

j.        The objective lens magnifies the specimen, producing a real image that is then magnified by the ocular lens resulting in the final image;

k.      The total magnification can be calculated by multiplying the objective lens value by the ocular lens value.

KEY TERMS IN MICROSCOPY

Magnification

In most light microscopes, the objective lens, which is closest to the specimen, magnifies objects 100× (times), and the ocular lens, which is nearest the eye, magnifies 10×. Using these two lenses in combination, organisms in the specimen are magnified 1000× their actual size when viewed through the ocular lens. Objective lenses of lower magnification are available so that those of 10×, 20×, and 40× magnification power can provide total magnifications of 100×, 200×, and 400×, respectively. Magnification of 1000× allows for the visualization of fungi, most parasites, and most bacteria, but it is not sufficient for observing viruses, which require magnification of 100,000× or more (Electron Microscopy needed).

Total magnification:

The total magnification obtained in a compound microscope is the product of objective magnification and ocular magnification.

M_t = M_ob X M_oc

Where,

M_t = Total magnification,

M_ob = Objective magnification and

M_oc = Ocular magnification

Resolution

In microscopy, the term 'resolution' is used to describe the ability of a microscope to distinguish in details. In other words, this is the minimum distance at which two distinct points of a specimen can still be seen - either by the observer or the microscope camera - as separate entities.

                                  Fig 2: Difference between high and low resolution image of an object.

To optimize visualization, other factors besides magnification must be considered. Resolution, defined as the extent to which detail in the magnified object is maintained, is also essential. Without it everything would be magnified as an indistinguishable blur. Therefore, resolving power, which is the closest distance between two objects that when magnified still allows the two objects to be distinguished from each other, is extremely important. The resolving power of most light microscopes allows bacterial cells to be distinguished from one another but usually does not allow bacterial structures, internal or external, to be detected.

To achieve the level of resolution desired with 1000× magnification, oil immersion must be used in conjunction with light microscopy. Immersion oil has specific optical and viscosity characteristics designed for use in microscopy. Immersion oil is used to fill the space between the objective lens and the glass slide onto which the specimen has been affixed. When light passes from a material of one refractive index to a material with a different refractive index, as from glass to air, the light bends. Light of different wavelengths bend at different angles creating a less distinct distorted image. Placing immersion oil with the same refractive index as glass between the objective lens and the coverslip or slide decreases the number of refractive surfaces the light must pass through during microscopy. The oil enhances resolution by preventing light rays from dispersing and changing wavelength after passing through the specimen. A specific objective lens, the oil immersion lens, is designed for use with oil; this lens provides 100× magnification on most light microscopes.

Lower magnifications (i.e., 100× or 400×) may be used to locate specimen samples in certain areas on a microscope slide or to observe microorganisms such as some fungi and parasites. The 1000× magnification provided by the combination of ocular and oil immersion lenses usually is required for optimal detection and characterization of bacteria.

Contrast

The another key component to light microscopy is contrast, which is needed to make objects stand out from the background. Because microorganisms are essentially transparent, owing to their microscopic dimensions and high water content, they cannot be easily detected among the background materials and debris in patient specimens. Lack of contrast is also a problem for the microscopic examination of microorganisms grown in culture. Contrast is most commonly achieved by staining techniques that highlight organisms and allow them to be differentiated from one another and from background material and debris. In the absence of staining, the simplest way to improve contrast is to reduce the diameter of the microscope aperture diaphragm increasing contrast at the expense of the resolution. Setting the controls for bright field microscopy requires a procedure referred to as setting the Kohler illumination.

Fig 3: High and low contrast images.

The resolving power

The resolving power is the inverse of the distance between two objects that can be just resolved. On the other hand it is the ability of an optical instrument or type of film to separate or distinguish small or closely adjacent images.

It is given by Abbe's criterion

Resolving power =  ​ 1/delta 'd' = 2a / 'λ'

 where a is the numerical aperture

d is distance

λ is the wavelength of the objective lens used

(a) When wavelength of incident light is decreased, the resolving power increases.

(b) When aperture of the objective lens is decreased, the resolving power decreases.

Numerical Aperture

The numerical aperture (NA) of an optical system (e.g. an imaging system) is a measure for its angular acceptance for incoming light. It is defined based on geometrical considerations and is thus a theoretical parameter which is calculated from the optical design. It cannot be directly measured, except in limiting cases with rather large apertures and negligible diffraction effects.

Numerical Aperture of an Optical System

Numerical aperture is a numerical value concerned with the diameter of the objective lens in relation to its focal length. Thus, it is related to the size of the lower aperture of the objective, through which light enters into it. In a microscope, light is focused on the object as a narrow pencil of light, from where it enters into the objective as a diverging pencil.

Numerical aperture (n.a.) = n sin θ

Where,

n = Refractive index of the medium between the object and the objective and

θ = Half aperture angle

NA of a Lens

A simple case is that of a collimating lens:

Fig 4: A collimating lens can theoretically accept light from a cone, the opening angle of which is limited by its size.

PRINCIPLES

The light microscope is an important tool in the study of microorganisms, particularly for identification purposes. The light microscope is an instrument for visualizing fine detail of an object. It does this by creating a magnified image through the use of a series of glass lenses, which first focus a beam of light onto or through an object, and convex objective lenses to enlarge the image formed. In the majority of light microscopes, the image is viewed directly through binocular eyepieces that act as a secondary lens in the form of a magnifying glass to observe the projected image. Such instruments are termed ‘compound microscopes,’ and the total magnification is the sum of the objective magnification and the eyepiece magnification. The magnification range extends from ×10 to ×1000, with a resolving power of the order of 0.2 μm, depending on the type and numerical aperture (area available for passage of light) of the objective lenses.

                                                Fig 5: Path of light in Light microscope.

Fig 6: Principle of Light Microscope

APPLICATIONS OF LIGHT MICROSCOPE

1.      For details study of cells or tissues section under high magnification and contrast.

2.      For histopathological study to understand toxic effect of drugs/chemicals.

3.      For studying anatomical structures of bones, tissues (Liver, kidney, spleen etc).

4.      For counting WBC, RBC and platelets Counts in patients sample.

5.      Rapid final identification of certain organisms by direct visualization in patient specimens

6.      Detection of different organisms or contaminations present in the specimen.

7.      Detection of organisms not easily cultivated in the laboratory.

8.      Evaluation of patient specimens for the presence of cells indicative of inflammation (i.e., phagocytes) or contamination (i.e., squamous epithelial cells).

9.      Determination of an organism’s clinical significance; bacterial contaminants usually are not present in patient specimens at sufficiently high numbers (×10⁵ cells/mL) to be seen by light microscopy.

10.  Provide preculture information about which organisms might be expected to grow so that appropriate cultivation techniques are used.

11.  Determine which tests and methods should be used for identification and characterization of cultivated organisms.

12.  Provide a method for investigating unusual or unexpected laboratory test results.