BSc Biophysics_Microscopy Notes

MICROSCOPY

Most cells are exceedingly small in size, and all cell components are still smaller and almost transparent to visible light. So, unaided human eye cannot resolve them. Resolution is the ability to discriminate between two closely adjacent points or objects as separate units, rather than viewing them as a single unit. The lowest minimum distance between two objects, required for their resolution (discrimination), is known as limit of resolution. The normal resolving power of human eye is 100 microns. 

Microscopes (Gk. mikros = minute, skipein= to see) are the instruments used to produce magnified visual or photographic images of very minute objects. The ratio between the size of the microscopically magnified image and that of the image formed in the retina of an unaided eye is called the magnifying power of the microscope.

The resolution of a microscope depends directly on its magnifying and resolving powers. If the resolving power is very low, then the images of close by objects would overlap, making the vision blurred and indistinct. On the other hand, a very high resolving power would make the images of adjacent objects sharply distinct from each other.

Resolution of a microscope

The resolving power of a microscope depends upon the wavelength (λ) of the illuminating agent and the numerical aperture (NA- light-gathering power) of the objective lens. Shorter the wavelength, greater would be the numerical aperture and higher would be the resolving power. The resolving power of a micro­scope is inversely proportional to the limit of resolution of its objective lenses. So, the resolving power can be increased by decreasing the limit of resolu­tion i.e., by using illuminating agents of shorter wavelength.

The resolving power of a microscope can be determined by applying the formula
Numerical Aperture
The resolving power of a light microscope depends on the wavelength of light used and the numerical aperture (NA) of the objective lenses
The numerical aperture of a lens can be increased by 

• increasing the size of the lens opening and/or 
• increasing the refractive index of the material between the lens and the specimen.

The larger the numerical aperture the better the resolving power. It is important to illuminate the specimens properly to have higher resolution. The concave mirror in the microscope creates a narrow cone of light and has a small numerical aperture. However, the resolution can be improved with a sub stage condenser. A wide cone of light through the slide and into the objective lens increases the numerical aperture there by improves the resolution of the microscope.

Based on the source of illumination two major groups of microscopes can be recognized, namely light microscopes and electron microscopes.

Light microscopes

These are the microscopes in which the object is illuminated by visible light, and magnified by an optical lens system.

 Bright field microscopes

These are the microscopes in which the object is illuminated against a bright background. Bright field microscopes are of two kinds, simple and compound.

(a) Simple light microscope

Simple microscope consists of a single convex glass, which can produce only a slightly magnified visual image of the object.  In a simple microscope the image appears on the same side where the lens and the object are placed, and it cannot be projected on a screen. So the image is virtual. It is upright (erect), and not upside down (inverted). Simple hand lenses, dissection microscopes, etc. are simple microscopes

(b)        Compound light microscope

Compound microscopes are the microscopes in which there is a series of lenses, in place of the single magnifying glass in a simple microscope. The optical system of an ordinary light microscope has two magnifying lenses namely an objective lens, seen close to the object, and an ocular lens or eye piece, seen close to the observer's eye. After illuminating the object, the light rays pass through the objective lens. The objective lens produces a slightly magnified, real and inverted initial image of the object. This initial image serves as the object of the ocular lens, which produces a further magnified virtual and inverted image on the retina of the observer's eye.

Condenser lens system

Conventional light microscopes do not have devices to improve the quality of the light beam. In modern microscopes it is effected with the help of a condenser lens system, fixed just below the platform of the microscope. It consists of a condenser lens and a diaphragm, placed in between the light source and the object. Diaphragm has an aperture to control the incident light. The lens focuses the illuminating light on the object. High resolving power can be obtained by modifying the aperture of the condenser system.The resolving power of a compound microscope depends on visible light whose wavelength is much higher, rang­ing between 4,000 and 8,000 A⁰. So, compound light microscopes cannot resolve objects closer than half the wavelength of visible light.

Oil immersion: This is a microscopic technique that uses high powered ob­jective lenses, called oil immersion objectives. Oil immersion objective is a microscopic objective in which the front part of the lens gets immersed in a liquid, when lowered. In this case a thin film of immersion oil (such as cedar wood oil) is placed on the cover slip, in between the objective lens and the specimen. This oil has precisely the same refractive index as that of the glass lens, and it increases the numerical aperture by letting a wide angle of rays to enter the objective lens. This, in turn, enhances the resolving power and thereby gives the maximum magnification obtainable with a light microscope.

Darkfield microscopes

Darkfield microscopes are the light micro­scopes in which the object is illuminated against a dark background by scat­tering light rays. They are used for observing transparent and semitransparent objects, that are not readily visible in a bright background. Visibility natu­rally depends upon a contrast between the object and its background. In the case of transparent objects this contrast can be increased by using a dark background. The ordinary light microscope can be equipped for darkfield illumination either by replacing the ordinary condenser by a darkfield condenser  or by placing an opaque central disc in the centre of the ordinary condenser. This causes a cone of light to illuminate the object. The darkfield condenser illuminates the object only obliqoely. It prevents the central rays from the mirror from passing through it to illuminate the object. At the same time, it deflects the periph­eral rays obliquely on to the transparent object from where they get scattered. Thus, the field of vision is dark, and not bright. The scattered rays from the object pass through the ob­jective lens with the result that the object becomes visible as a bright speck in a dark background. It appears that in darkfield microscopes no direct light enters the objective lens, but only the scattered light. Darkfield microscopes are useful in detecting very small and transparent objects, and also objects that are poorly stained.

C. Fluorescence microscopes

These are the microscopes whose functioning is based on the phenomenon of fluorescence (the power to emit visible light). In them ultraviolet rays are the illuminating agents. When they illuminate the object, fluores­cence occurs in the object. This fluorescence is of two kinds, autofluorescence and secondary fluorescence. In autofluorescence some chemical compounds get excited at the molecular level, when ultraviolet rays illuminate them. Consequently, they become luminous and emit visible rays. In other words, they absorb ultraviolet rays, and in return, emit visible light rays. This is the case with chlorophyll, porphyrin, ribofiavin, etc. In secondary fluorescence luminosity is induced in certain substances by staining them with fluorescent dyes, known as fluorochromes. Consequently, they glow against a dark back­ground, making their detection very easy. These dyes combine with non-fluorescent compounds, such as proteins and carbohydrates, and make them fluorescent. Fluorescent dyes absorb light rays of a particular wavelength and emit them as light rays of another wave length, Fluorescein (emits red light when excited with blue light), rhodamine (emits red-green light when excited with green-yellow light), etc are fluorescent dyes

Both in auto and secondary fluorescence the fluorescent rays are detected by the microscope and the chemical nature of the object is determined. Thus. fluorescence microscopes are highly useful in testing the chemical nature of the objects and also in locating cellular components based on their chemical nature. An ordinary microscope can be converted to a fluorescence microscope by introducing a special filter between the source of illumina­tion and the object. There are two kinds of filters, namely ex­citation filters and barrier filters. Excita­tion filters transmit only the excitation radiations to the ob­ject; all other radiations would be "filtered" out or prevented from passing to the object. Barrier Filters prevent the excessive flow of excitation radiations to protect the eye of the observer especially from ultraviolet rays).

 Electron microscopes (EM)

       These are the optical instruments used for the direct study of the ultra structure of biological systems. The first functional EM was made by Knoll and Ruska in 1928. (Ruska shared the 1986 Nobel Prize in Physics with Binning and Rohrer).

Electron microscopes have exceptionally high magnifying and resolving powers. Their magnification is up to 500,000 times, and resolution up to 2-5A.  This high resolution is due to the very short wavelength of the elections, that are used as illuminating agents.

EM consists of an evacuated tube in which a beam of accelerated electrons interacts with the object. The electrons are accelerated through a potential of 500-1500 kV in an electron gun. These electron beams can penetrate relatively thick, hydrated and living specimens. They are deflected and focused on to a fluorescent screen by electrostatic or electro­magnetic fields, produced by electromagnetic lenses, much in the same-way that a beam of light is refracted by a lens in a compound microscope. An image is formed either directly or indirectly on the fluorescent screen. In the former, primary electrons, passing through the object, are directly focused on to the screen. But in the latter, X-rays or secondary electrons, emitted during the interaction of primary electrons with the object, are focused on to the screen.

EM is of two kinds, transmission EM (TEM) and scanning EM (SEM).In TEM electrons are transmitted right through the object to study the interior of the cell. But, in SEM electrons are scattered or reflected back from the object to study the cell surface.

(a)    Transmission electron microscope

The basic plan of the illumination source and the lens systems of TEM closely corresponds to that of a compound light microscope. Instead of visible light, TEM makes use of high speed electrons of very short wavelength as the illuminating agent. The source of these electrons is a cathode, which acts as a thermoionic gun, called the electron gun. It is, in fact, a metal filament placed in a vacuum tube. When it is heated, it emits streams of electrons, which are accelerated by high voltage electrical potential This high voltage considerably reduces their wavelength (to approximately 0.005 nm – for visible light it is 550 nm).The accelerated electrons tend to follow a straight path with properties similar to those of light. From the cathode the accelerated electrons travel through a cathode ray tube and reach the condenser lens system. The condenser system, in turn, consists of an electromagnetic lens, which is an electromagnetic coil, enclosed in a soft iron case. The electromagnetic condenser focuses the electrons on to the object. Thus, the object gets evenly illumi­nated by a broad beam of electrons at 40 - 100 kV. Most of these electrons pass right through the object. But, some get absorbed or scattered. The unscattered electrons travel straight and reach an elec­tromagnetic objective lens, it deflects the electron beam and produces a direct, magnified and real image of the object (nearly 2000 times magni­fied). This image is further magnified by a third electromagnetic lens, often called the projector lens It projects the final image on a fluorescent screen for direct viewing through an air tight "window". Often, the screen is re­placed by a photographic film with a camera attachment for recording the image. In that case a photograph of the object is registered on the film called   electron micrograph. The final image can be magnified up to 50000 times. The camera system can further magnify it 4 - 10 times. Thus, electron micrographs may be magnified up to 5 million times the size of the primary object. Adjustments in focusing, and alterations in magnification, can be made by changes in the currents of electromagnetic coils.

TEM has the advantage of much finer resolution than light microscope But, it is not usually suitable for living specimens, because within the specimens some electrons are scattered and some amount of energy is lost from them. Energy absorption by the specimen may damage it, especially at high magnification.

(b) Scanning electron microscope

SEM provides a clear surface view of the object. In it a very fine beam of electrons at 3 - 30 kV is made to scan a specific surface area of the specimen, just as a screen is scanned in a television tube. The primary electrons, emitted from it move back and forth across the specimen. During this secondary electrons are emitted either from the surface or from near surface of the specimen. They are collected by a photo multiplier tube or a positively charged grid. From there the signal is trans­ferred to a television tube with the result that an image is displayed on the screen.

The image produced by SEM resembles those seen in a hand lens. But, it has much finer resolution  and can be further magnified nearly 100,000 times.

Phase contrast microscopes

These are the microscopes used for examining the components of un­stained living cells. They are designed on the principle that small phase changes or phase differences in the light rays passing through an object can be trans­formed to differences in brightness or light intensity. The phase contrast prin­ciple was first worked out by Zernike in 1940.

Living cells are not usually coloured, but almost transparent to visible light. This means that they are pure phase objects and the light passing through them practically suffers no loss of intensity due to absorption. However, in reality, the light passing through a living cell does encounter regions of different refractive index and thickness, which al­ter its velocity and direction. This difference in refractive index produces a differ­ence in the optical path of the light transmitted by the different regions. As a result, the light trans­mitted by the region of higher thickness and greater refractive index gets retarded in velocity and emerges out of phase in relation to the light transmitted by the region of lower thickness and low refractive index. When the difference in refractive index is small, the magnitude of the phase change would also be small, and is measured in wavelength (     ). Phase contrast microscope can transform such phase changes into corresponding variations of brightness or intensity. This, in turn, enhances the contrast between the cell, its content and its surroundings, enabling the study of the cell in the living state.

The optical system of a phase contrast microscope differs from that of an ordinary light microscope only in the addition of (i) a sub stage annular dia­phragm to illuminate the object with a narrow cone of light, and (ii) a diffrac­tion plate (phase retardation plate or phase plate) in the objective. For enhancing the phase difference between direct and diffracted rays phase contrast microscope has special optical devices. To change the relative phase the diffracted waves have to be first separated from direct waves. This is achieved by inserting a sub stage annular diaphragm in the condenser system. This diaphragm permits the light to pass through the condenser in the form of a hollow cone of rays, the "central rays" being absorbed. This light cone illuminates the object. The objective lens will focus the annulus on its upper (back) focal plane. The direct rays passing through the thinner regions of the object will form the image of the annulus, while the diffracted rays passing through its denser region will spread over the entire focal plain  producing a number of overlapping images of the diaphragm. Thus, direct and diffracted rays are spatially separated from one another. By inserting phase retardation plate at the back focal plane of the objective lens the phase change of the diffracted rays can be further increased.The phase retardation plate is a channeled or grooved and transparent glass disc. Its channel is coated with a material that can absorb light, but cannot retard it. The remainder of the plate is coated with a thin film of light retarding material (such as magnesium fluoride). The channel is so designed that it exactly matches the image of the condenser annulus. So, the light passing directly through the specimen and the channel will not be further retarded. On the other hand, the diffracted light passing through the remainder of the plate gets retarded again. Thus, the phase contrast between direct and deviated rays gets exaggerated.The magnification by a phase contrast microscope is not much higher than that of  a compound microscope. Still,   it is much significant in the following respects :

1.    It enables to distinguish living cells from the surrounding medium

2.    It is useful to discriminate between various cell components and also to observe the effects of chemical and physical agents on living cells.

3.    All kinds of cellular movements, including the chromosomal movements during cell division, can be detected by phase contrast microscopy

4.        The changes undergone   by cytoskeleton during cellular movements can be observed with the help of phase contrast microscopy.

5.     The Phase contrast microscopy is particularly useful in observing living cells and tissues in tissue culture.

Phase contrast microscope has two major disadvantages also: 1) It is ideal only for observing individual cells or thin cell layers 2) It often results in halo formation, since the separation of direct and deviated rays is only partial or incomplete.