Biophysics lecture 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.

Centrifugation

Centrifugation

Centrifugation is the separation and sedimentation of sub- cellular frac­tions of the homogenate according to their size, mass, density and specific gravity. The instrument used for this is called centrifuge . It consists of a pivoted cylinder, called rotor, in which the samples are placed. The rotor rotates or spins around a central axis at very high speeds. This produces gravitational fields, which are higher than earth's gravity. The high rotational speed of the rotor produces a centrifugal force, directed away from the cen­tre. It may be as much as 100,000 times greater than gravitational force. The magnitude of the centrifugal force is expressed in terms of earth's gravita­tional force, namely 'g' (g = 980 cms/sec²). The relative centrifugal force (RCF) is directly proportional to the number of revolutions per minute (rpm-ω), radius of rotation (r) and the gravitational force

 Centrifugal force causes the sample particles to move away from the cen­tre and also to settle in different layers according their sedimentation ratio. Heavier particles move away first and get deposited on the outer wall of the tube. Lighter particles follow them in regular sequence in the order of de­creasing mass.

The intensity of the centrifugal force in relation to the sedimentation of particles can be represented by the equation.

The rate of sedimentation of particles in centrifugation is called sedimen­tation coefficient. The unit of its measurement is called Svedberg unit(S). The suspension, left above the sediment after centrifugations, is called superna­tant. It may be subjected to repeated centrifugation, with progressively in­creasing speed. As a result, different particles settle down differently at different times, at different rates and at different sites. This may be called differential centrifugation.

In classical fractionation and centrifugation cell components separate into four successive fractions in the order nuclear fraction, mitochondrial fraction, microsomal fraction and soluble fraction. For the separation of DNA and RNAs an improved method of centrifugation has been deviced. It is called density gradient centrifugation.

Kinds of centrifuges

Three major kinds of centrifuges can be recognized, namely low-speed centrifuges, high-speed centrifuges, and ultracentrifuges. Low-speed centri­fuges are the most ordinary types of centrifuges, used for the routine sedimentation of heavy particles. Their rotor has a maximum speed up to 4,000 or 5,000 rpm, with RCF values up to 3000g. Low-speed centrifuges are used at room temperature, and they have no means for temperature control of the samples. High-speed centrifuges are advanced types of centrifuges, with higher speed and temperature control of the rotor chamber. They are especially important in the centrifugation of temperature-sensitive biological samples. Their rotor revolves at an average speed of 20,000 rpm, with an RCF value up to 50000g.

Ultracentrifuges are the most sophisticated and refrigerated types of cen­trifuges. In them the rotor rotates at extremely high speeds (75,000 rpm), producing a gravitational pull of above 50,000g. Ultracentrifuges are used for the separation of viruses and extremely minute sub-cellular components and also for the determination of molecular weights. They are used for pre­parative work and analytical measurements. So, there are two kinds of them, namely preparative Ultracentrifuges and analytical Ultracentrifuges. Analyti­cal Ultracentrifuges are provided with window and optical system. So, sedi­mentation of particles can be observed and their sedimentation rates can be measured during the run.

Methods of centrifugation

There are two main kinds of centrifugation, namely differential centrifuga­tion and density -gradient centrifugation.

(i) Differential centrifugation

Differential centrifugation is the successive centrifugation of the homogenate at progressively increasing rotor speeds, leading to the succes­sive separation of cell components. It involves the sedimentation of particles in a medium of homogeneous density.

(ii) Density-gradient centrifugation

Density-gradient centrifugation is the technique used for the separation of multi component mixtures of macromolecules and also for the measure­ment of sedimentation coefficients (S values). In this case, the suspending liquid and the multi component sample exhibit density difference. Density will be highest at the bottom of the centrifuge tube, and lowest at the top. In between these two extremes there will be a gradient (graded series) of den­sity- different zones. This may be called density gradient. The multi compo­nent sample is layered at the top of the gradient. Ultracentrifugation at a constant speed results in the sedimentation of the sample components according to their density. When a fraction reaches a liquid zone, whose density is the same as that of the fraction, the fraction stays there without any further sedimentation. In this manner different fractions of the sample sepa­rate into distinct density- different zones or bands.

Density-gradient centrifugation is widely used in the separation and puri­fication of biological macromolecules and also for the determination of S values. 

AUTORADIOGRAPHY

This is the technique used to observe the distribution of a particular chemical substance in living cells and tissues, and to track the routes and conversions of macromolecules in different biochemical reactions. In autoradiography, radioisotopes (isotopes which can emit ionizing radiations) of some elements (e.g. ^I4C, ³H, ³²P, ³⁵S, etc.) are introduced to the cell. Each of them gets incor­porated with a specific substance and makes it radioactive. This is called la­belling of the substance with radioisotopes, or simply radioactive labelling. Then, the movement and distribution of the labelled substance is observed. This helps to detect the presence of the substance and also to track the syn­thesis of a compound out of it. The radioisotopes can emit one or more of the three radiations, namely alpha and beta particles and gamma rays. Most of the isotopes used in autoradiography are beta-emitters.The cells or tissues, containing radioactively labelled molecules, are placed in contact with a photographic emulsion for a certain period. The ionizing radiations, emitted by the radioisotopes, blacken the emulsion and produces an image, known as autoradiograph. A comparison of this image with the normal cells observed under microscope will enable to detect the location of the radioactive isotope and also to track the route of the radioactively la­belled molecule. Autoradiographs are usually observed with the help of phase contrast microscopes

ELECTROPHORESIS

Electrophoresis  is the analytical technique applied for the separation of charged molecules, based on their movement in an electric field ; positively charged particles move to the cathode, and negatively charged ones to the anode. This makes their separation easy. It is a very convenient technique for analysing and purifying several kinds of biomolecules, especially peptides, proteins, nucleotides and nucleic acids. Separation of the constituents of mixtures or solutions occurs at pH values .above or below the isoelectric point.

Electrophoresis relies on the principle that biological molecules in solu­tion carry a net electric charge (except at the isoelectric point). If two elec­trodes are placed in such a solution, and an electric field is applied, the charged particles move towards oppositely charged poles at different rates and get localized in different zones. Their movement is greatly influenced by their shape, size, molecular weight and electric charge, and also by the matrix of the gel support and the applied electric field. The greater the charge and the lesser the molecular weight, the greater would the electrophoretic mobility of the particles. The charge, in turn, depends on the pH of the buffer solution. A molecule with a double charge will move at twice the speed of a molecule with a single charge. This difference is the basis for the separation of the differ­ent molecules of mixtures or solutions. It is very unlikely that two different kinds of molecules of a mixture will have exactly the same size, molecular weight and charge level.

Modern elec­trophoretic techniques use wetted filter paper or a polymerized gel - like matrix (acrylamide gel, starch gel, cellulose acetate gel, agarose gel, silica gel, etc.) as an inert support medium. The sample to be analysed is applied to this medium as a spot or as a thin band.

The movement of charged molecules in an applied electric field is repre­sented by the equation

Thus , charged particles move at a velocity which depends directly on the applied electric field or voltage (E) and charge (q) , but inversely on a counteracting force generated by the viscous drag (f)

Types of electrophoresis

All electrophoretic methods are based on the same principle. So, they differ from each other only in the nature of the support medium. Some common types of electrophoretic methods are paper electrophoresis, polyacrylamide gel electrophoresis (PAGE - significant in the separation of pro­teins ), agarose gel electrophoresis (AGE - important in the separation of nucleic acids), sodium dodecyl sulphate - polyacrylamide gel electrophore­sis (SDS- PAGE), significant in the measurement of the molecular weight of biomolecules), pulsed field gel electrophoresis (PFGE), significant in the sepa­ration of large chromosomal DNA, capillary electrophoresis (CE- combina­tion of the resolving power of electrophoresis with the speed of high perfor­mance liquid chromatography to analyse very small samples ), etc.

Paper electrophoresis

This is the electrophoretic  method in which a filter paper wetted with a buffer solution forms the support medium. The pa­per spans between two reservoirs of the buffer solu­tion, with its ends immersed in the solution. A small amount of the solu­tion or mixture to be analysed is placed somewhere near the centre of the paper. Now, electrodes are placed in the reser­voirs of the buffer and a voltage is applied. The charged constituents of the mixture or solution move to their respective poles at different velocity. This brings about their separation.

 Polyacrylamide gel electrophoresis (PAGE)

PAGE is the electrophoretic method in which polyacrylamide gel is used as the support medium. Polyacrylamide gel is a synthetic polymer (prepared by the free radical polymerisation of acrylamide and the cross linking agent methylene-bis-acrylamide). Polyacrylamide gels are produced either as col­umns or as slabs. Slab gels are now more widely used than column gels. A slab gel is more convenient, because several samples can be analysed on it; only a single sample can be analysed on a tube (column) gel.

Acrylamide gel is something like a spongy network. Macromolecules can­not freely pass though it, but can only squeeze through its narrow channels. The rate of this movement depends largely on the size and molecular weight of the migrating molecules. Smaller the molecules and lower their molecular weight, faster would be their movement.

In PAGE gel column or gel slab is inserted vertically between two buffer reservoirs. The upper reservoir usually contains the anode, and the lower one contains the cathode. The sample to be analysed is placed on the gel slab or gel tube and a voltage is applied. The charged molecules of the sample move to their respective poles.PAGE is significant in that in it separation of the sample components involves both molecular sieving and electrophoretic movement So, it results  in enhanced resolution of sample components The order of molecular movement in gel filtration and PAGE is different. In gel filtration large mol­ecules move through the matrix faster than small molecules, but the reverse is the order in PAGE. Usually, PAGE is used for the separation of large mol­ecules, such as proteins.