Microscopy

Introduction

A biophysical technique which is used to study the objects and its areas which cannot be seen through naked eye. (for example: microorganisms like bacteria and yeast and cells like cancer cells & blood cells). A microscope consists of lens, or combination of lenses for making enlarged or magnified images of minute objects. the ability of microscope to detect, resolve and image the smallest items of evidence, often without any alteration or destruction. The aim of any microscope is to magnify images or photos of a small object and to see fine details. There are various types of microscopy. 
Some of them are bright field microscopy, dark field microscopy, phase contrast microscopy, fluorescent microscopy, electron microscopy, transmission electron microscopy and scanning electron microscopy

Light Microscope or Compound Microscope: 

Image Source: www.microscope.com

A compound microscope has two set lenses. One is objective lens and other one is an eye piece. The lens which is nearest to the specimen is objective lens which magnifies the image and other lens which is nearest to the eye called eye piece which further magnifies the image formed by the objective. There are also fine adjustment screws which are used to attain the proper image of the specimen under study. 

Parts of compound microscope are objective lenses, eye piece and condensers.

Objective lenses gather light rays coming from any point to the object and they unite the light in a point which is further to magnify the image. There are 3 types, achromatic – simplest and adequate for most purpose, fluorites – those in which the aberrations are largely eliminated by the use of achromatic ones. Aberration meaning- the failure of rays to converge at one focus because of a defect in a lens or mirror. Apochromatic- corrected with respect to aberrations.

Eye piece - the main use of the eye piece are they magnify the real image of the specimen under study formed by the objective lenses and with help of fine adjustment to correct the defects of the objective. Eye pieces are also called as Oculars. There are 3 types of eye pieces, huggenian, hyper-place and compensating eye pieces.

Condensers, series of lenses for illumination to the object on the stage of microscope by transmitted light. There are two methods, one is illumination by transmitted light and other one is dark field illumination.

Resolving power, it is defined as the power of an objective which is able to distinguish two adjacent points. R.P of a microscope is a function of wavelength of light used and the numerical aperture (NA) of the lens system). Larger the NA, Greater the resolving power.

Numerical Aperture, the sine value of the half aperture angle multiplied by the refractive index (n) of the medium gives the numerical aperture. With dry objectives the value of n is 1 because 1 is the refractive index of air. But when immersion oil is used as the medium, n =1.56.

Limit of resolution, smallest distance between two separate objects and distinguished as two separate objects. The greater resolution in light microscopy is obtained with the shortest wavelength of visible light and an objective with maximum NA. relation can be expressed as d = λ/2NA

Magnification is the increase in size of the object. Magnification beyond the resolving power has no value.

Bright-Field Microscopy

Image: Bright Field Microscopy I

The microscope field is bright and the microorganisms or specimen under study appear dark because they absorb some of the light. Staining the specimens or microorganisms greatly increases their absorbing ability and which produces fine and good image of the specimen. For Example: H&E Staining, which is Hematoxylin and Eosin Staining, in which Hematoxylin stains the nuclei of the cell and Eosin stains the cytoplasm. Generally, microscope of this type produces useful magnification of about X1000 to X2000. This type of microscopy is also called as transmitted light. It has transillumination light source, usually halogen lamp, and it has a condenser lens, which focuses light into the sample, an objective lens which collects light from the sample and magnifies the image. Eye piece or a camera to view the sample image. Bright field microscope is simple to set up and required for all the basic imaging which makes it basic equipment required. Limitations of this type of microscopy are that very low contrast of most biological samples. It has practical limit to the magnification. Beyond X2000, the image become fuzzy or appears blur. And samples that are colorless and transparent cannot be imaged properly. Although you can enhance brightness of the image by reducing or increasing the light source, using an oil-immersion objective lens for some specific specimens, using sample-staining methods for example Hematoxylin and eosin staining.

Dark Field Microscope

Dark filed microscopy image (fibers)

In dark field microscopy, the microscopic field is dark that means a dark background is produced against which objects are brilliantly illuminated. Used to enhance the contrast in the unstained samples For example, in this image you can see fibers appear bright and background for that appears dark. (The light microscope is equipped with a special kind of condenser that transmits a hollow core of light from the source of illumination. . And Illuminating an object where the object appears self-illuminous against a dark field is called dark-field illustration.) There are three types of condensers which are used in the dark field microscopy, Abbe Condenser, paraboloid condenser and cardoid condenser. This type of microscopy is particularly valuable for unstained samples and this is also used in hanging drop operations.

Phase Contrast Microscopy

Light passing from one object into another object of a slightly different refractive index or thickness undergoes a change in phase. According to principle of this type of microscopy, light waves have variable character for frequency and amplitude. When two light rays have similar amplitude and frequency but different phases then this type of microscopy is used to image the specimen. It is used to visualize unstained living cells. Most of the stains or staining procedures will kill the cells. Phase contrast microscopy enables the visualization of living cells. (This type of microscopy is based on the principle that small phase changes in the light rays, induced by differences in the thickness and refractive index of the different parts of an object, can be transformed into differences in brightness or light intensity). In a phase-contrast microscope, this difference in phase is translated into variation in brightness of the image and hence is detectable by eye. With a phase-contrast microscope, the differences among various cells with different refractive indices or thickness can be seen in unstained condition. A phase-contrast microscope is a compound microscope fitted with a phase-contrast condenser and a phase-contrast objective An annular aperture in the diaphragm placed in the focal plane of the sub-stage condenser controls the illumination of the object. The image of the aperture is formed at the rear focal plane of the objective. In this plane, there is a phase-shifting element or phase- plate. The phase plate also has an annular ring of phase altering pattern, which can increase the wavelength of light passing through it. Light coming through the annular aperture of condenser passes through the object. Those rays, which are not deviated by the object (solid lines in figure), pass through the phase-altering pattern of the phase plate and acquire longer wavelength. Those rays, which are deviated by the object structures due to different refractive index, pass through the phase-plate not covered by the phase altering pattern. Thus, their wavelength remains unchanged. The difference in phase (wavelength) gives the contrast for clear visibility of the object. 

Fluorescence Microscopy

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. To fluorescence to occur, the substance must absorb light at a shorter wavelength and emit the light at longer wavelength. I mean, when the substance is at ground state is excited by a light source for example laser, it will absorb the light at shorter wavelength and emit the light at a different or longer wavelength. A fluorescent microscope uses fluorescence or phosphorescence. Any microscope that uses fluorescence to generate an image is fluorescence microscope. Simplest ones are epifluorescence microscope. Simple to set up and simple to use. There are more complicated fluorescence microscopes are available for example confocal microscope which uses optical sectioning to get better resolutions of the fluorescent image by reducing the background and all.

GFP, YFP fluorescence merged image

Components of a typical epifluorescence microscope are Light source (Xenon Lamp or Mercury-vapor lamp), Excitation filter, dichroic mirror and emission filters. Example: Light source will emit white light and excitation filter will only the pass the required wavelength in this case here Blue light which will hit the dichroic mirror placed in front of the excitation filter and the blue light will travels through the objective lens to the specimen. Specimen will absorb the blue light and emits the green light. This green light will travels through the specific emission filter to the oculars and detectors will capture this green light. The camera which is placed near the detector will capture this green light emitted and forms the image. Immunofluorescence, where specific antibodies are tagged with a fluorophore molecule which will emit fluorescence when bound specific antigens. Fluorescent Proteins. Major limitation of fluorescence microscopy is that photobleaching because fluorophores lose their ability to fluoresce as they are illuminated in a process. That is why if you want to perform fluorescent imaging, then you should perform the imaging faster. But, it doesn’t apply to all the fluorescent molecules. There are some fluorophores which are stable and stay fluoresce for a quite some time.

But, remember, fluorescent microscopy is the most used imaging technique in biology research as it allows you to study the fine details of the cells for example, proteins, cell structures, nucleus, and other components of the cells.

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