Introduction: - It is an instrument which helps to visualise small structure which is otherwise not visible by naked eyes. It helps to produce images with higher magnification and clarity. It is used for the study of structures of the cell and subcellular components. The microscope is basically an instrument which is a combination of multi-lens which show the image with the help of light source (with the help of multi-lens combination). The higher resolution and magnification up to 2000 time can be achieved. In the electron microscope, the resolution is up to 100000 times.
The naked human eye cannot see any tiny object of 0.1 mm size. To see microorganism, much smaller than 0.1 mm, a system is required which have convex lenses.
The focal length of a lens is a point where Parallel Rays Converse to meet after passing through a convex lens. The focus is the point on the axis of a lens where parallel light converse and then appear to diverge after refraction or reflection. Focal length is the distance between the centre of the convex lens and the focus point.
Less focal length gives higher magnification.
Overall magnification is given as the product of lens magnification and distance over which the image is projected.
The magnification of the microscope is defined as the ratio of image size and object size. Magnification is the enlargement of the appearance of an object as compared to the appearance of an object as observed by the human eye.
For Example:- The left image of the figure represent the ts object seen by the uncorrected eye and has a magnification of 1 X. In the right image, one-fifth of the breadth and one-fifth of the height of an original image is projected on to the eye. This magnification is 5x and the field of reduced by 1 / m2 or 1/25 of the original view.
In an older microscope, the primary length of the microscope (objective lens) adds a back focal distance from the focus plane at a finite distance. In the modern microscope, the objective lens is said to be infinitely corrected because the objective lens focus as an infinite distance whereas the second lens (the tube lens) focuses the light from the objective lens.
The net effective distance from the focus to the tube lens (F1) divide by the distance from the objective lens to object (F0) is the magnification of the system (magnification is equal to F0/F1). The area of the scene is proportional to magnification squared (m2). If a smaller lens is projected onto the same detector (eg. : the eye) then the density of light also decrease by m2.
Resolution : -
Our naked eye is unable the resolve to objects that are separated less than 70 micrometres. The retina contains approximately 120 million rod and 8 million cone cells into a single layer. In the region of highest resolution known as fovea, the cones are packed so tightly that they are only 2 micrometres apart. This distance between the cones limits the resolving power of our eyes.
Resolution is a term that means distinguishing discrete objects even if two small objects brought closer and closer together until they no longer appear to be two separate objects but rather appear as one. The limit of resolution is the closest distance between two points at which each point still can be distinguished as separate images.
The specification should be coupled with a good resolution to visualise small microorganism as magnification alone will produce an unclear or blurred image.
The resolution of the microscope is determined by both wavelength of light (lambda) and maximum angle of flight which can be collected by the objective lens. The wavelength of light describes its energy. Both are inversely proportional. For eg. : Light that appears blue is shorter wavelength and contains more energy than red light. For a given wavelength of light, the resolution is determined by the angle of light that can be collected by the optical system (the angular aperture). Greater the angular aperture of light, more detail image is possible to be collected.
If the observation angle is fixed, the object appears to extend to infinity.
Numerical aperture :
The practical angle of the light that can be collected by the lens is described by the numerical aperture (N.A.) which is defined as the product of lowest refractive index between the sample and the front lens of objective lens (n) and the sine of half angle () that describes the cone of Light Between of focal panel and the front lens of objective lens
Numerical aperture (N.A) = n sin (theta)
The limit of resolution power depends upon three factors
* The wavelength of light used to illuminate the specimen.
* The angular aperture
* The refractive index
The effect of these three variables are limit of resolution is described by quantitatively by the following equation known as abbe equation
Where nsin is called a numerical aperture so the Limit of resolution = 0.61/N.A.
Contrast refers to the ability to recognize an object against the background. Depending on the specimen and the optical condition, the object is limited by either noise or background. Noise is the uncertainty in the measured signal due to which the desired response is not obtained properly. Some sources of noise are a statistic in nature while others are instrumental. The capturing and recording of the individual event (such as the arrival of photons at a camera) is the reason for the uncertainty. This uncertainty is commonly referred to as shot noise or Poisson noise.
Poisson noise = √ events
Shot noise is the result of uncertainty in measurement and is a statistic consequence of the number of measurement that is made. As the number of event increase, the absolute Poisson noise is Greater even though the relative Poisson noise (defined as the Poisson noise divided by the number of events) is reduced.
Image formation: Image formation by microscopy is based on illumination. The two type of microscope illumination i.e. Köhler and Critical.
In practice, köhler illumination is used in the most microscope. Whereas a specialised form of critical illumination is used in a confocal microscope.
Köhler illumination :
Köhler devised a method in which an image of the source is formed by a converging lens known as the collector lens, at the front focal plane of the substage condenser, while the image of field diaphragm is formed in the plane of the specimen by substage condenser. Substage condenser produced collimated light beams, each of which originates from a point on the source. Each point on source forms a collimated beam of light that illuminates the entire field of view. The point on the centre of source form collimated beam that is parallel to the optical axis. The points farther and farther away from the optical axis make collimated beams that strike the object at greater and greater angles. Thus, the specimen illuminated with a cone of light composed of both parallel and oblique illumination.
Critical illumination :
In critical illumination, an image of the light source is focused in the plane of the specimen. The illumination is intense but it is uneven unless a ribbon filament is used. Critical illumination does not require a substage condenser. In critical illumination, each point in the object acts as a point source of light. If the light radiating from two nearby points is truly incoherent, it will form two overlapping images of Airy discs, the intensity of which will be the sum of the two intensities. Since light from two nearby points will be somewhat coherent and will interfere with the intensity of each point. So, it will not be exactly the sum of the two intensities, but will in part be described by the square of the sum of the amplitudes of the light radiating from both points.
Bright field microscope: -
It is the original and most commonly used form of the microscope in which the specimen is viewed by transmitted light from a condenser lens. Light is emitted from a condenser towards the specimen and after specimen, light passes through the objective lens and a second magnifying lens i.e. the ocular or eyepiece. The specimen is visible in the light path because they are thick enough to absorb a significant amount of light despite being colourless.
Light microscope made up of more than one glass lens in combination. The major components are the condenser lens and the eyepiece lens and such instrument are therefore called a compound microscope. These components, in turn, are made up of the combination of lenses which are necessary to produce the magnified image with reduced aberrations. For example, chromatic aberration occurs when a different wavelength of light was separated and passed through a lens at different angles. This results in multiple colours around the edges of the object in the image.
The main component of the compound light microscope includes a light source that is focused on the specimen by the condenser lens. The light that enters the specimen can either pass through the specimen (transmitted light) or get reflected. These refracted and reflected light are focused by the objective lens into the eyepiece lens. There are two basic types of compound microscope - an upright microscope and an inverted microscope. If the light source is below the condenser lens such a microscope is the upright microscope this is the most commonly used format. The inverted microscope is engineered in such a way that the light source and the condenser lens are above the specimen stage and the objective lens is beneath it.
Advantages and disadvantages of bright field microscope: -
Direct imaging with no need for sample pretreatment. It is the only microscope for real colour imaging.
Fast and adaptable to all kind of sample system (either gas liquid or solid sample system) in any shape or geometry.
Easy to be integrated with a digital camera system for data storage and analysis.
Low-resolution power that limits specimen visualization specifically to only submicron or a few hundred of nanometers.
Use of bright field microscopy the bright field microscope can be used to characterized microorganisms cell and tissue in higher organisms. Bright field microscopy has been used in chemical trials. Typically hair and fibre are identified with the light microscope.
Darkfield microscopy: -
For dark field microscopy, dark field illumination is desired. The specimen usually is illuminated with a hollow cone of light. In the absence of a specimen, illuminating light does not enter the objective lens because of the numerical aperture of substage condenser is larger than the numerical aperture of the objective.
Phase contrast microscope can create dark field microscope by using the 100 X annular ring in combination with 10 x 20 X objective.
Since, the dark field condition required the numerical aperture of the objective be smaller than the numerical aperture of the substage condenser, an objective with the variable aperture or Iris is very useful for dark field microscope. With variable iris, the objective lens lets us adjust the numerical aperture of the objective so that it is just smaller than the numerical aperture of the substage condenser and thus, obtain optimal resolution and contrast. Once an object is inserting into a dark field microscope, the illuminating Light that interacts with the specimen is diffraction.
Darkfield microscope is best suited to levelling outlines the edges and boundaries of an object. It is less useful for ring internal details of the cell unless there is a lot of highly ductile bodies in relatively transparent systole. Darkfield microscopy can be used to visualise weak amplitude and phase information.
Phase contrast microscopy: -
Phase contrast microscope is an important microscopic technique which is being used to observe internal structures of the live organism without staining. Staining can alter or stop specimens life processes like metabolism, division. So, phase contrast microscope provides a facility to study live organism.
The working principle of phase contrast microscope
Phase contrast works
Light can be considered as a wave. When light enters glass which is optical transparent material denser than air, it slows down and the number of waves increases in proportion to both the density (as determined by the refractive index) and thickness of the material. Consider a second beam (from the same source), which moves wholly in airfare to the first without entering the glass. The first Beam will have travelled a greater distance than second. All specimen deflect or scattered light and these deflected beams carry the information about the structure of the object. An image of the object is formed at the primary image plane due to interference between defected beams by the specimen and undiffracted ( zero order) beam.
With a specimen, there is a half wavelength phase difference between undeflected beams and diffracted beam by the specimen. Coloured objects or specimen selectively absorb light of the certain wavelength within the visible spectrum.
If the beam illuminating the field of view is trained by annulus in the first focal plane of the condenser, it will form an annular image in the back focal plane of the objective. The optical path traversed by the undiffracted beam alone can now be selectively advanced (or retarded) and the necessary extra one quarter wavelength phase difference between two beams introduced before combing to form the image at the primary image plane of the microscope. This trench is called the phase ring and it is carried on the phase plate. The phase ring carries an absorbing layer that reduces the amplitude of the undeflected zero order beam, reducing its brightness.
Construction of the phase contrast microscope: -
A special set of objectives fitted with phase plate is needed for phase contrast microscopy. Manufacturers generally provide several different sizes of in condenser to match objects of different magnification and numerical aperture.
Interpreting the phase contrast image: -
The undeflected and diffracted beam are out of phase with one another by half wavelength overall. They will interfere to form a visible image without concerning whether the deflected beam is retarded or advance by one-quarter wavelength with respect to the undeflected Beam. Two forms of phase contrast microscope are therefore possible referred to as positive and negative phase contrast. Positive phase contrast refers to the most widely used system where the phase plate is constructed with a Trench deflected beam( passing outside the phase ring) which travel one-quarter of a wavelength further than zero order beams structure with the refractive index higher than their surroundings. Positive phase contrast is responsible for the commonly recognised appearance of a cell in which nucleus, lysosomal components and the cell membrane appear darker than their surroundings. Phase contrast effect is maximum at regions of sudden changes in optical path differences( edges) and is less pronounced where the changes in optical path difference between adjacent area are not abrupt (wedges). This phenomenon is known as shading- off.
As a consequence, the centre of one structure may appear the same shade of Grey as the another of quite different refractive index. Phase contrast is better suited for structures with end edges rather than structures with edges boundaries.
Types of phase contrast:-
Several types of phase contrast are possible depending on the construction of the phase plate in the objective.
1) Positive (+ve) In positive phase contrast, direct light is an advanced 1/4 wave in phase (-type). This produced destructive interference and create dark detail on a light background. This is the most common form of phase contrast.
2) Negative (–ve) In negative phase contrast, the direct light is retarded 1/4 in phase(+ type). This produces constructive interference resulting in light details on a dark background.
3) Positive (+ve) Negative (–ve) In either positive or negative phase contrast, the phase plate may be one of two types. In which either direct flight can be absorbed( a type) reflected light can be absorbed (b type). The most common type is (a type). Both a and b type plates related by the percentage transmission of ring area 20% is most common.
Limitation of phase contrast:-
Phase contrast is excellent for thin colourless transparent specimens. However, if the specimen is very thick, confusing space image will be produced. These plate does not limit the objectives numerical aperture. Nevertheless, most phase objectives are perfectly suitable for bright field work.
Fluorescence Microscopy :
Fluorescence microscopy is a major tool to monitor cell physiology. Although the concept of fluorescence and its optical separation use similar to the filter element, designs vary with the aim of interesting image contrast and special resolution.
In fluorescence microscopy, the specimen itself act as the light source. The specimen is studied by using either fluorescent materials or stain with fluorescent dyes. A chemical is said to be fluorescent if it absorbs light at one wavelength and emits light fluorescence at the special longer wavelength. A most fluorescent dye (fluorochrome) emit visible light but some emit infrared light. Fluorochromes exhibit distinct excitation and emission spectra that are dependent on atomic structure and electron resonance properties.
The principle of fluorescence: -
The underlying process of fluorescence involves the absorption of light energy( a Photon) by indicator followed by the emission of the sum of light energy (as another Photon) a few seconds later. Because some energy is lost in this process, the emitted Photon has less energy than absorbed Photon.
Light with a shorter wavelength (towards the blue) has higher energy than light with a longer wavelength (towards the red). Therefore, light emitted from indicator usually has a longer wavelength than the absorbed excitation light. This change is called the Stokes shift. The molecular transition explains this process in terms of Jablonski energy diagram. This microscopy is similar to an ordinary light microscope except for the illuminating light that is passed through the set of filters before reaching the specimen. To filter the light emitted from the specimen, Excitation filter pass only the wavelength that excites the fluorescent dye while the barrier filter blocks the remaining light. The conventional microscope uses visible light to illuminate the magnified images of the sample.
High-intensity ultraviolet light excites fluorescent molecule in the sample of interest, following excitation of the fluorescence molecule emit longer wavelength light producing a magnified image of the sample.
Types of different filters present in the fluorescence microscope: -
1) Excitation Filter: -
The filter placed within the illumination path of the fluorescence microscope has the purpose to filter out the wavelength of light source, except for the excitation range of light for fluorophore under inspection.
2) Emission Filter: -
Emission filter is placed within the imaging path of the fluorescence microscope. Its purpose is to filter out the entire excitation range of transmitted emission range of the Chosen fluorophore.
3) dichroic mirror or beam splitter: -
Dichroic mirror beam splitter place in between the excitation filter and emission filter at 45-degree angle. Its purpose is to reflect the excitation signal towards the fluorophore under inspection, to transmit the emission signal towards the detector.
Fluorescence results when molecules called fluorophores absorbed light, which raises their energy level in an excited state. They emit fluorescent light as they decay from the excited state( known as Stokes shift).
In general, a fluorophore will be excited by a high-frequency light and EMIT light at the slightly lower frequency. There is three most common fluorophore used are DAPI (emits blue), FITC ( emits green), Texas red( emits red).
Wide-field fluorescence microscopy: -
A basic fluorescence microscopy is a common tool of modern cell biologist. In this instrument, a parallel beam of light simultaneously illuminates the whole specimen to excite the fluorophore it contains. Traditionally, excitation light is provided by a Mercury or Xenon high-pressure bulb and the required wavelength is selected with custom optical filters. The laser scanning microscope eliminating only a small point of the specimen at any instant and the laser beam, therefore, needs to scan across the specimen to create an image. The new approach of excitation light that is rapidly being adopted is the use of bright, single wavelength light emitting diode (LEDs). LEDs have the advantage of long life, fast switching and tight wavelength control. HD may be required for different indicators although white LED could be used. LEDs may obviate the need for filter wheels. Mercury/ xenon bulb can also be replaced by longer lasting fibre coupled metal halide lamp system.
The resulting fluorescence in the specimen is viewed by the eye or captured electronically. The simplicity of wide-field microscopy is that all parts of the specimen are viewed simultaneously an image can be easily captured with a camera. However, this simplicity in conjunction with diffraction limited optics and the unavoidable projection of out of focus light on to single image plane of the camera can result in the image of low contrast and spatial resolution.
A confocal microscope is a powerful tool that creates sharp images of a specimen that would otherwise appear blurred when viewed under a conventional microscope. This is achieved by excluding most of the light from the specimen that is not from the microscopes focal plane. The image thus obtained has less haze and better contrast than that of a conventional microscope and represent a thin cross-section of the specimen. Laser scanning confocal microscopy has become a valuable tool for imaging thin optical section in living and fixes specimen, ranging in thickness up to 100 micrometres. In fact, the confocal microscope is often capable of revolving the presence of a single molecule.
Current instruments are highly modified from the earliest version, but the principle of confocal imaging that was developed by Marvin Minsky is employed in all modern confocal microscope. The image in the confocal microscope is achieved by scanning one or more focused beam of light, usually from a laser or Arc discharge source across the specimen. This point of illumination is brought to focus in the specimen by the objective lens and laterally scanned using some form of scanning device under computer control. The sequence of points of light from the specimen is detected by the photomultiplier tube(PMT) through a pinhole and the output from the PMT is built into image and display by the computer. Although, the unstained specimen can be viewed using light reflected back from it. Most of the times, specimens are labelled with one or more fluorescent probes. A laser is used to provide the excitation light.
The laser light reflects by a dichromic mirror hits two lenses mounted on motors. These Mirrors scan the laser across the sample, dyed fluorescent and the emitted light get discerned. The same mirror scans the excitation light from the laser. The emitted light passes through the mirror and is focused on the pinhole. The Light That passes through the pinhole is measured by a detector which is a photomultiplier tube. Thus, there is never a complete image of a sample at any given instant, only one point of the sample is viewed. The detector is attached to the computer which builds up the image at one time.
Components of confocal microscope:-
1) light source (laser system)
3) Acousto optical devices
5) detector (PMT)
1) laser system: -
LASER- light amplification by the stimulated emission of radiation can be defined as materials with altered distribution of atoms as such that there are more excited atoms, ready to emit energy. The process of altering the energy distribution of the atom is required so that most of them are in higher energy state.
Depending on the material used to the medium laser, these are of three types
1) Gas Laser
2) Solid State Laser
3) Semiconductor Laser
Laser safety: -
Two major concerns to safe laser operation are exposed to the beam, the electrical hazards associated with a high voltage within the laser and its power supply. While there are no known cases of laser beam contributing to a person's death, there have been several instances of death due to contact with high voltage laser-related components. The beam of sufficiently high power can burn the skin, or in some cases create a hazard by burning or damaging other materials. But the primary concern with regard to the laser beam is potential damage to the eyes which are most sensitive to light.
Filtering devices: -
In the fluorescence microscope, filtering devices are used to separate a light beam on the basis of their wavelength. Four types of filters are used to selectively transmit or block a desired range of wavelength.
A) Short pass filters:- the cutoff wavelength longer than a certain wavelength eg. : heat filters are used to exclude infrared light to reduce specimen heating by illumination.
B) Long pass filters:- example fluorescent filter that transmits light longer than the certain wavelength.
C) Bandpass filter:- that transmit light only between a cut-off and cut on wavelength. It is especially useful when one is trying to image signal from more than one fluorochrome simultaneously.
D) Dichrome Mirrors:- that separate the emitted light from excited light.
Acousto optical device:-
This device works on the principle of the acousto optic effect. In general, acousto optic effect is based on the changes in the refractive index of a medium due to the presence of sound waves in the medium. Sound waves produce a refractive index grating in the material and this grating is seen by the light wave. These variations in the refractive index due to the pressure fluctuations may be detected optically by refraction, diffraction, interference and reflection effect. The acousto - optic effect is used in measurement and study of ultrasonic waves.
Various acousto optic device available : -
1) Acousto-optic Tunable Filters (AOTFs)
2) Acousto-optic Beam Splitter (AOBS)
3) Acousto-optic Modulator (AOM)
4) Acousto-optic deflector (AOD)
Confocal imaging relies upon the sequential collection of light from spatially filtered individual specimen points, followed by electronic signal processing and eventually the visual display as corresponding image points. Point by point signal collection process requires a mechanism for scanning the focus illuminating beam through the specimen. Specimen volume under observation which is achieved by scanning the stage or the beam scanning variation commonly employed to produce confocal microscope images includes: -
1) Scanning is literally translating specimen stage coupled to a stationary illuminating light beam (stage scanning).
2) Scanning light beam with a stationary stage (beam scanning). In the modern confocal microscope, two fundamentally different techniques for beam scanning have been developed.
Single beam scanning: It uses of pair of computer and controlled galvanometer to scan the specimen at the rate of approximately One Frame per second.
Multiple beam scanning: In confocal microscope that is equipped with the spinning nipkow disk containing an array of pinholes, here the light source is arc discharge lamp instead of a laser to reduce specimen damage and enhance detection of low fluorescence level during real-time image collection. Apart from this, multiple Beam microscope can readily capture the image with an array of detectors such as CCD camera system, PMT etc.
Detectors: - As light emitted by fluorophores in the sample is mainly unpolarised, polarisation sensitive beam splitter can only separate half of it from polarized excitation light. Instead, the emitted light is either sequentially split by the combination of a short long pass or long pass dichrome mirror, before being passed through a bandpass. In confocal microscopy, fluorescence emission is detected through a pinhole aperture positioned near the image plane to exclude light from a fluorescent structure located away from the objective focal plane. Thus, reducing the amount of light available for image formation.
Pinhole: - The optical sectioning capability of confocal microscope depends on pinhole and its capability to reject out of focus light rays. i.e. The strength of optical sectioning depends strongly on the size of the pinhole. Thus one can assume that making the pinhole as small as possible is the best way to enhance optical sectioning. However, as the pinhole size is reduced, a large number of photons that arrive at the detector from the specimen is blocked. This may lead to a reduced signal to noise ratio.
Types of confocal microscope: - There are two types of confocal microscope
1) Confocal laser scanning microscope.
2) Spinning disc(nipkow disc) confocal microscope.
Applications of confocal microscope:
A) Fluorescence resonance after photobleaching
B) Fluorescence resonance energy transfer
C) Fluorescence lifetime imaging
D) Fluorescence loss in photobleaching
E) Fluorescence localisation after photobleaching.
F) Fluorescence in situ hybridization
G) Living cell imaging.
Advantages of confocal microscopy:-
1. The primary advantage of laser scanning confocal microscopy is the ability to serially produce thin (0.5 to 1.5 micrometre) optical section through fluorescent specimens that have a thickness ranging up to 50 micrometres or more.
2. The ability to control the depth of field:-
The most important feature of a confocal microscope is the capabilities of isolating and collecting the plane of focus from within a sample. Thus, eliminating the out of focus “haze” normally seen with the fluorescence sample. Fine detail is often obscured by the haze and cannot be detected in the non-confocal fluorescent microscope.
Disadvantages:- Colocalization of the fluorophore in the confocal microscope - two or more fluorescence emission signal can overlap in digital images recorded by confocal microscopy due to their close proximity within the specimen. This effect is known as colocalization and usually occurs when the fluorescently labelled molecule binds to target that lie in very close or identical spatial position.
The fundamental principle of the electron microscope is similar to light microscopy except for one major difference of using the electromagnetic lens, rather than an optical lens to focus of high-velocity electron beam light instead of visible light. The relationship between the limit of resolution and the wavelength of illuminating radiation holds true for both a beam of light or a beam of electrons. Due to the short wavelength of the electron, the resolving power of the electron microscope is very high.
There are two types of electron microscope
a) Transmission electron microscope (TEM)
b) Scanning electron microscope (SEM).
The most commonly used type of electron microscope is transmission electron microscope because it forms an image from electrons that are transmitted through the specimen being examined. The scanning electron microscope is fundamentally different from transmission electron microscope because it produces images from electrons read from specimens outer surface (not transmitted through the specimen).
Scanning electron microscope
In the scanning electron microscope, the image is formed by detection of the secondary electron which is generated by the specimen. It shows topological features of the specimen.
Components of scanning electron microscope: -
a) Electron source:- electron source are meant to emit electrons by which image of the specimen is obtained.
Commonly used electron sources: -
1) tungsten filament
2) field emission gun
3) solid state crystal.
Electrons are generated from these sources by thermionic heating and bombarded on the specimen as a narrow beam having voltage 1 - 40 kilovolt.
1) Tungsten filament:- Approx 100 micrometre is used as an electron source. This filament is inverted V-shaped. On thermionic heating it produces electrons.
2) Field emission gun: - This source also consists of tungsten wire but electrons are released from a very sharp tip of about 100 nm that uses field electron emission to produce an electron beam. The radius of the electron beam and focusing ability is dependent on tip of tungsten wire.
3) Solid state crystal: - Lithium hexaboride or Cerium hexaboride crystals are used as thermionic emission gun. These are high brightness source which can provide 10 times more brightness than tungsten. It also posses longer lifetimes.
Lenses:- In the scanning electron microscope, a series of condenser lens are used to focus the electron beam. When the electron beam falls on the specimen surface, it produces a 'spot'. Size of this spot is determined by with of electron beam.
Scanning coil:- Function of scanning coil is to deflect the electron beam in two dimensions so as to scan the whole surface quickly.
Sample Chambers:- it is the chamber in which the specimen or sample is placed for analysis. Alignment and adjustment of the sample within the Chamber can be carried out with the help of tilting and rotating devices.
Detectors:- The electrons which fall on the specimen can produce secondary electrons, backscattered electrons or characteristic X-rays. On the basis of voltage and density of the sample, the signals from different penetration depth are recognized by detectors.
By detecting and analysing these emitted electrons from the specimen, imaging of the sample takes place. These are three different types of electrons detectors:-
1. Backscatter electron detector:- Backscattered electron detector detect elastically scattered electrons. These electrons are emitted from the atom below the sample surface. Backscattered electrons are emitted in an irregular manner depending on the composition and superficial distribution of the specimen. The contrast of the backscatter electron image depends on the multiple factors:-
* Atomic number of sample material
* Acceleration voltage of primary beam.
High atomic number material produces more negative electron than lower atomic number.
2. Secondary electron detector
Secondary electron detector in the microscope gives an image with a high resolution of the sample. It gives an image with the help of "Inelastically scattered electron" of sample surface which contains surface topological information.
Principle:- the electron beam from electrons source scan the specimen surface with the help of deflection/ scanning coils in Roster pattern.
For the formation of the image, the secondary electron emits from each point of specimen analysed to form two dimensional image.
Secondary electron detectors attract low energy secondary electron by positive potential. High energy backscattered electron is detected by the backscattered detector.
The backscattered electron is not able to give information about sample topology because the electron comes from the depth of the sample. It is Helpful to obtain information about sample composition. High atomic number material gives a bright image.
Sample preparation:- Biological samples are moist. Thus cannot be analysed as such in an electron microscope. For analysis, its moisture content must be removed by dehydration process but dehydration may cause structural changes in the specimen, to avoid this are specimen fixed to preserve their structural features. Fixation of the specimen can be done by using Chemicals like glutaraldehyde or by the physical method like cryofixation in liquid nitrogen. After the specimen is dehydrated by using an increasing gradient of ethanol and dried by the critical point method. For effective emission of a secondary electron, specimen coated with conducting material usually gold to make conducting surface of the specimen.
Transmission electron microscope:-
Credit for developing the first electron microscope goes to Knoll and Ruska in 1930. By this electron microscope, a thin specimen can be analysed and this, based on the electrons which have been passed through the specimen to make the image of specimen. This analysis was a microscope is called a transmission electron microscope.
Electron source:- The electron source is of higher voltage of about 100 to 300 kilovolt.
Vacuum system:- In the vacuum system, electrons behave like light. Different vacuum pump used to maintain low pressure at (10–8) Pa.
Electromagnetic lenses:- The electromagnetic lenses are made up of coils. It works when electric current flow in these coils. Due to the flow of current, an electromagnetic field is generated and control the magnification by the strength of the field. Typically there are three types of lenses are present in transmission electron microscope:-
1) Condenser lenses
2) Objective lenses
3) Projector lenses
Work of condenser lenses is to form the primary beam which is focused on the specimen by the objective lens. After passing through the specimen, transmitted electrons fall on the projector lens which expends the electron beam and allows it to fall on Phosphorus screen or film (imaging device)
Imaging device like Phosphorus screen or film serves as the base upon which image is obtained.
Specimen stage: -
Specimen stage is a place where the sample is the place for examination. It is a small mesh-like structure of about 2.5 mm diameter made up of gold or Platinum, controlled by the sample holder.
With the help of specimen holder, rotation of specimen allowed in the desired angle which is helpful for multiple views of the specimen.
Imaging device: - A fluorescent screen is used in the transmission electron microscope. It is affected by the transmitted electron to Emit The Light. A high-resolution camera used for real-time image and permanent record.
Sample preparation and work for transmission electron microscope: -
A very thin (less than hundred nm thickness) specimen is required for analysing under the transmission electron microscope and that also need to be fixed and dried like as for scanning electron microscope.
In the transmission electron microscope, the specimen is fixed using a combination of glutaraldehyde and paraformaldehyde. After fixation, secondary staining done by Osmium tetroxide (OsO4). It acts as an electron stain and fixes the unsaturated lipids. The process of dehydration is the same as the scanning electron microscope. The dehydrated sample is sectioned to obtain an ultra-thin section of less than a hundred nanometers. This process is done by fixing the sample firmly in a mould with the help of acrylic acid and Epoxy. After the process of sectioning, it will stain using heavy metal like uranyl Acetate and phosphotungstic acid. Then deposit on Carbon coated grade for analysis.
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