Introduction to microscopy
The technical field of viewing objects through microscopes which are not seen with the naked eye.
Some objects are so small that these are not within the resolution range of human eye. So to study these objects it is necessary to aid the human eye with some instrument. The instrument used to view these small objects is the microscope.
Ancient Greek: Micros=small, Skopἱen= to look or see
Microscope is an optical instrument used to view small sized objects invisible to the unaided eye.
The microscopes can be classified into two types based on the number and types of lens used.
- Simple Microscope:
It consists of a single lens or more than one lens grouped in one unit. This simple microscope is only used to enlarge the object. The lens may range from double convex to two Plano-convex lenses.
Examples are Jewelry eyepiece, Reading glass, pocket magnifiers, loops etc.
Earlier used simple microscopes were of different shapes
- Compound Microscope:
It consists of more than one types of lenses usually one being the objective and the other Eyepiece. A series of lenses of different types is present in the optic tube. It has higher magnification and hence is used to view finer details of the object being viewed.
The objective forms areal image of the object while the eyepiece forms the enlarged virtual image. Moreover the objective lenses are exchangeable to adjust the magnification.
Due to advancement in technology, many types of Compound microscopes have been invented. The major types of microscopes are:
- Optical microscope
The optical microscopes include:
i. Bright field microscope
ii. Dark field microscope
iii. Inverted microscope
iv. Phase contrast microscope
v. Differential interference contrast microscope
vi. Fluorescence microscope(epifluorescence microscope)
vii. Confocal microscope
viii. Interference reflection microscope
- Electron microscope
The electron microscopes include:
- Scanning electron microscope
- Transmission electron microscope
- Scanning probe microscope
The scanning probe microscopes include:
- Scanning Thermal microscope
- Scanning tunneling microscope
- Scanning voltage microscope
- Scanning electrochemical microscope
- Scanning atom probe
- Scanning Hall probe microscope
- Scanning SQUID microscope
- Kelvin probe force microscope
- Recurrence tracking microscope etc.
The history of microscopy started with the optical microscopes also called as light microscopes. Later in the 20th century another technique of microscopy evolved which used electrons instead of light to form images. The microscopes following this technique were named electron microscopes. Later in 1980 have started the development of scanning probe microscopes.
In the optical and electron microscopes, diffracted, reflected or refracted electromagnetic radiations or electron beams interact with the specimen and these scattered radiations or signals are collected to create image. In scanning probe microscope, a scanning probe interacts with the specimen.
- Other microscopes include
- Infrared microscope
- Laser microscope
- Digital Holographic microscope
- 3D printed microscope
- X-ray microscope
- Multifocal plane microscope
- Digital pathology microscope
- Polarized light microscope
- Digital microscope
The type of compound microscope in which light is reflected from or transmitted through the specimen through single or multiple lenses to get a magnified view of the specimen.
The image formed is observed by the eye or photographic plate or captured digitally.
The objective forms areal image of the object while the eyepiece forms the enlarged virtual image. The light rays falling on the specimen are focused by the objective first which forms a real image. These light rays are then transmitted to the eyepiece which forms the enlarged virtual image of the specimen, hence magnifying the object many times its original size.
A light microscope consists of the following basic parts:
i) Body tube
ii) Ocular lens/eyepiece => 10X 0r 15X power magnification
v) Objectives => 4X to 100X power magnification range
viii) Stage clips
ix) Coarse adjustment Knobs
x) Fine adjustment Knobs
xiii) Light Source
A compound microscope may be monocular if it contains one eyepiece and binocular if it contains two eyepieces.
The magnification of a standard compound light microscope is achieved by multiplying the magnification of the objective and the eye piece e.g. 4X power objective multiplied with 10X power eyepiece gives a total magnification of 40X i.e. 40 times enlarged image of the specimen.
Ð The biggest advantage of a compound light microscope is its simplicity and convenience.
Ð Due to its small size it is easy to use, handle and store.
Ð Multiple lenses and adjustable objectives help to reveal many details of the specimen.
Following are the some of the limitations of standard compound light microscope.
a) Dark or strongly refracting objects can only be effectively imaged
b) Resolution is limited to approx. 0.2 micrometers due to diffraction
c) Image clarity is compromised due to the out-of-focus light
To observe the colorless and transparent internal structure of cells, their contrast must be enhanced by using special techniques. With the advancement in technology, a large number of microscopy techniques are developed to increase the contrast of specimen. Some of the techniques are explained in detail here.
In modern life sciences, the detection of single molecules is done by means of a technique in microscopy which uses fluorescence and phosphorescence.
Any light microscope that uses the phenomenon of fluorescence and phosphorescence to study the details of biological molecules is called fluorescence microscope.
The phenomenon of emission of low energy light from a substance which has absorbed a light/electromagnetic radiation of higher energy is called fluorescence.
The type of photoluminescence in which the material re-emits the absorbed radiation very slowly is termed as phosphorescence
TYPES OF FLUORESCENCE MICROSCOPES:
Fluorescence microscope may be any type of microscope that uses fluorescence to create images of the sample .Different types of fluorescence microscopes include:
ð Simple microscope like the wide-field epifluorescence microscope in which the excitation of the fluorophore and the detection of the fluorescence are done simultaneously through the same light path. These microscopes are very commonly used in biology labs.
ð More complicated microscopes like
Ð confocal microscope
Ð Total internal reflection microscope.
Both these microscopes are used where 3D structure is important.
The illumination of specific wavelength is directed towards the specimen through the objective lens. This light is then absorbed by the fluorophore in the specimen and the fluorophore then re-emits a light of longer wavelength. This emitted light then forms the image.
Spectral emission filters are used to separate the illumination light and the weak emitted fluorescence
Major components of fluorescence microscopes are mentioned below:
- Light source (xenon arc lamp, mercury vapor lamp, high power LED’s and lasers etc.)
- Filters (excitation and emission filters)
- DIchoric mirror
Choice of the excitation and the emission filter and the dichoric relates to the spectral properties of the fluorophore used. This makes the observation of a single fluorophore viewed at one time. Multi-colored images are formed by combining many single color images.
Light sources like hydrogen lamps etc. can’t be used in fluorescence microscope as it needs intense, near-monochromatic illumination. For this purpose four main types of light sources are widely used in fluorescence microscopy. These include:
ÐXenon arc lamps / mercury vapor lamps with an excitation filter
Ð Super continuum sources
Ð High power LEDs
In complex microscopes i.e. confocal microscopes and total internal reflection microscopes light source used is the LASERS. While Xenon arc lamps or mercury vapor lamps and LEDs with a dichoric excitation filter are widely used in wide field epifluorescence microscopes
It is a high quality optical-glass filter used for the selection of the excitation wavelength (relatively short wavelength) of light from a light source. It isolates the fluorescence illumination used. The two types of excitation filters are:
- Short pass filters
- Band pass filters
Other forms of excitation filters are
- wedge prisms coupled with a narrow slit
- holographic diffraction gratings
It is a color filter used to separate light of specific colors from the white light. This light is then passed on to the sample
Notch Filters or deep blocking filters are used as emission filters. Allow the reflection of the light from the specimen
"The chemicals used in the fluorescence microscopy are termed as fluorophore”.
These re-emit light upon excitation. Plane or cyclic molecules with several pi-bond and several aromatic compounds combine to constitute fluorophore.
The use of fluorophore in biological sciences started in mid -20th century when DAPI which binds to DNA was used to label specific cell structures. Recent developments include:
- Hoechst (excited by UV wavelength light), DRAQ5 and DRAQ7 (optimally excited by red light). All of these bind to the minor groove of DNA.
- The immunostaining in which antibodies are coupled with fluorophores to recognize specific proteins within a given sample.
- The green fluorescent proteins GFP (developed by gene fusion technique) which are expressed by the living cell itself and are used to study the function of proteins in vivo
Many fluorescent dyes are also used to stain different cell structures.
Ð Highly sensitive and selective (only specific structures can be labeled with appropriate probes)
Ð Versatile (different probes are used for different cellular structures)
Ð Provides excellent contrast as the objects are self-illuminated against a dark background
Ð Absorption spectrum can be separated from emission spectrum with the help of Stoke’s shift
Ð Extremely small number of fluorescent molecules can be detected
Electrons excited during fluorescence cause a chemical damage to fluorescent molecules/fluorophores.-This photo-bleaching limits the observation time of the specimen
With shorter wavelengths, cells are susceptible to photo-toxicity. When using fluorescent reporter proteins. Also under illumination, fluorescent molecules have a tendency to produce reactive chemicals which increases the phototoxic effect
iii. High specificity:
Only structures labeled for study are observed. Fluorescence microscope provides no information about cell morphologies other than the targeted molecule/organelle.
Background signals may compromise the sensitivity of fluorescence microscopes. These signals may be from
ð the endogenous sample constituents
ð non-specific reagents
These unwanted signals cause the blurring of the final image.
Specimen may also show auto-fluorescence due to its chemical makeup. This leads to an unclear image of the target being observed.
To avoid image degradation due to background signals or any other cause, a more advanced technique is used in microscopy which has the capability to control depth of field, increase the optical resolution, eliminate the background signals, increase contrast of a micrograph, form a sharp image of the specimen and reconstruct the 3D-structure from the image already obtained. This technique is named as confocal microscopy. And the microscopes used for this purpose are generally named as confocal microscope.
The microscopes that have a common illumination and detection light-paths which are achieved by same focal plane due to the two pinholes present at equal distances from the sample specimen are called confocal microscopes.
The name CONFOCAL refers to this arrangement of light path in the confocal microscopes. The basic idea of the confocal microscopy is the spatial filtering which eliminates the out-of -focus light in the specimen.
Imaging fixed and living cells and tissues and ease of obtaining high quality images is proving the confocal microscope to be the most advanced tools in optical microscopy and has resulted in its wide applications in cell biology.
The basic principle of confocal imaging was first developed by Marvin Minsky in 1957 which is now used by modern confocal microscopes as well. According to this basic concept focused beams of light from LASERs or arc discharge sources are scanned across the specimen i.e. the light of different wavelengths is sent through the pinhole and is passed to the objective lens and the sample specimen through the beam splitter / dichoric mirror. .Objective lens brings this point of illumination to focus in the specimen. Computer controlled scanning devices scan these wavelengths laterally. The light emitted from the specimen can go to the detection pinhole and the detector. Photomultiplier tube PMT detects the sequence of points of light from the specimen through the pinhole or slit and the computer forms the image of this output from the PMT. Confocal microscopes uses point illumination and thus only the light closer to the focal plane can be detected by this method.
Through this pinhole arrangement, the light coming from the narrow focal plane enhances the z-resolution as compared to other microscopes.
Components of a typical confocal microscope include:
1. The Light source/LASER system
2. Illumination Filters
3. Acousto-optical devices
5. Detector (PMT, APD)
Gas LASERS like Krypton/Argon and Helium/Neon mixed gas lasers are used generally in the confocal microscopy. Mercury and xenon lasers are too weak for confocal microscopes and hence are not used. Other LASER systems used include:
- Semi-conductor LASERS.
- Solid state LASERS
Four types of filters used to transmit or block any desired range of wavelength include:
- Short pass filter: block/cut-off longer wavelengths
- Long pass filters: Transmit/cut-on light of longer wavelengths
- Band pass filters: transmit light in between the cut-on and the cut-off wavelength
- Beam-splitters/di-chrome mirrors: separate the excitation and emission wavelengths
Use of these filters is much reduced now-a-days in the favor of the Acousto-optical devices.
These devices are used for the modulation, deflection, signal processing and frequency shifting of light beams. The Acousto-optical effect controls the working of these devices.
Presence of sound waves in a medium changes its refractive index. Refractive index grating is produced by these sound waves in that medium and this grating is observed by the light waves. Refraction, reflection, interference and diffraction effects help to detect optically the variations in the refractive index due to fluctuation in pressure. This combined effect of sound and light waves forms the base of acousto-optic effect.
Following are some of the Acousto-optical devices used widely in confocal microscopy:
- Acousto-optic tunable filter
ð Help to select the intensity and wavelength on a pixel by pixel basis
- Acousto-optic beam-splitter
ð Replaces the di-choric mirror. More efficient as it deflects the light at specific LASER lens
- Acousto-optic modulator
ð Modulate amplitude, phase, polarization, frequency etc. Must provide maximum light intensity in a single diffracted beam to avoid switching speed and bandwidth limitations.
- Acousto-optic deflector
ð Both the amplitude and frequency of sound waves are adjusted in AODs.
The step-by-step signal collection in confocal imaging requires the scanning of the focused illumination beam through the specimen under observation. For this purpose either the stage or the beam is scanned. Thus the principle scanning variations in confocal imaging include:
Ð STAGE SCANNING: Specimen stage (laterally translated and coupled to a stationary illuminating light beam) is scanned
Ð BEAM SCANNING: Light beam with stationary stage is scanned. In modern confocal imaging, two techniques are developed for beam scanning.
- SINGLE BEAM SCANNING: specimen is scanned at the rate of 1 frame/sec with the help of a pair of computer controlled galvanometer mirror. As the optical system is spatially invariant, this technique is more advantageous.
- MULTIPLE BEAM-SCANNING: For this purpose, confocal microscopes have the following equipment.
ð NipKow disk with an array of pinholes
ð Arc discharge lamp light source instead of lasers(to reduce damage of specimen and enhance low fluorescence level detection)
ð Detectors like CCD camera system and PMT etc. ((capture image readily)
This technique is faster and does not involve moving of sample. But the use of galvano-scanner mirror limits its applications as the light must be scanned to avoid its movement relative to the pinhole.
In confocal microscopy, light through the focal plane is very much reduced which necessitates the use of highly sensitive photon detectors to detect the exceedingly low levels of light with a highly quick response to the continuous flux of varying light intensity. Several classes of photon detectors used in confocal imaging include:
v Photomultiplier detector: No spectral information is required to measure the density. The electrical impulse is used to tell the position of logical circuits. Principal of amplification of signal includes the conversion of photons into electrons, multiply electron and then the signal read-out.
v Assigning pseudo-color detector: Grey-scale images are assigned different indexed colors. Gain and offset adjust the detector signals such that resulting image/output includes maximum grey level.
Gain: input signal is multiplied which results in higher grey level value, bright features are saturated and general brightness of the image is increased.
Offset: Grey level of a selected background is set to zero. Darkest features in the image are adjusted to black.
Optical sectioning in confocal microscopy depends on the pinhole and its capability of rejecting the out-of-focus light rays from the specimen. And the strength of optical sectioning depends on the size of the pinhole but up to a certain limit. To get a Fine Approximation, the pinhole must be of the size of Airy Disk. For further accuracy, the number of photons arriving at the detector is increased along-with the use of highly sensitive photon detectors
Confocal imaging is a two-step process.
First -> excitation light focused by the objective on the specimen is initially passed through the pinhole or slit. An optical fiber can be used alternatively to introduce a narrow beam of light in to the system.
This controls/minimizes the fluorescence not in the focus.
Second -> another aperture/slit in front of the detector blocks the fluorescence emission originating above/below the focal plane.
The size of this aperture determines the strength of optical sectioning. The smaller this aperture is, the higher the rejection of out-of-focus light and in turn the thinner the optical section which improves the contrast and axial resolution but affects the brightness of the specimen.
In confocal microscopes, high sensitivity and target specificity in the chemically fixed as well as living tissues and cells is achieved with the help of fluorophores. The fluorophores used in confocal microscopy include:
ð Fluorescence proteins and quantum dots which have a higher target specificity and photo-stability.
ð Synthetic aromatic organic chemicals which bind with biological macromolecules within specific structural regions like mitochondria, Golgi apparatus, cytoskeleton, endoplasmic reticulum and nucleus etc.
Many other probes are used for
- Monitoring dynamic processes
- Locating environmental variables such as pH, reactive oxygen species, concentration of inorganic metallic ions, and membrane potentials.
- Monitoring cellular integrity, signal transduction, exocytosis, endocytosis, protein activity, activity of enzyme and fluidity of membrane etc.
- Molecular genetics (Genetic mapping and chromosome analysis)
TYPES OF CONFOCAL MICROSCOPES:
Four types of confocal microscopes are available commercially.
a) Confocal laser scanning microscope: use multiple mirrors which scan the lasers across the sample and de-scan the image across a fixed pinhole and detector.
b) Spinning disk (NipKow disk) confocal microscope: scans spots of light with the help of series of moving pinholes on a disc. The arrangement of pinholes decreases the excitation energy which reduces photo-toxicity and photo-bleaching.
c) Micro-lens/Dual spinning disc confocal microscope: another disc containing micro-lenses is placed before the NipKow disk containing the pinholes. These micro-lenses capture a broad and of light and focus it in to each pinhole. This makes it more sensitive than the standard spinning disk microscopes.
d) Programmable array microscope: use spatial light modulator which produces a set of moving pinholes. The image is captured by a CCD camera (charged couple device )
Confocal microscopes have many advantages over the wide-field microscopes. Few basic advantages are mentioned below:
Ð Serially producing 0.5 to 1.5 micrometer thin optical sections from a 50 micrometer thick specimen is the primary advantage of the confocal microscopes.
Ð It can control the depth of the field, eliminate out-of-focus fluorescence and reduces background information
Ð Collection of a series of images to reconstruct a 3D-image
Ð Compensates auto-fluorescence
Ð Multi-dimensional views of living tissues and cells
Ð Adjusting the magnification electronically instead of changing the objective.
i. Co-localization: overlapping of fluorescence emission signals in digital images due to their close proximity with the specimen. It occurs when fluorescent molecules bind with the target.
ii. Number of excitation wavelengths in common lasers is very limited
iii. Excitation wavelengths occur over very narrow laser bands and are very costly to produce in UV region
iv. Laser irradiation is highly intense to living tissues and cells
v. Limited use of multi-user confocal microscope systems in small laboratories due to their high cost of purchasing and operating.
With the conventional upright microscope, only small and preserved cell and tissue cultures could be observed. To observe living and large specimen, special modifications in the light microscope was required. This need was fulfilled by Inverted microscope.
This is the optical compound microscope which allows the specimen to be observed with the light source from above the specimen.
It was invented by J. Lawrence in 1850. This was an important invention in the field of microscopy as it allowed observing large and living specimens rather than the preserved specimen used earlier.
In this microscope the light source and the condenser lens are pointing downwards and are above the stage while the rotating turret and the objective lens are pointing upwards and are present below the stage. The condenser lens’ function is to concentrate the light while objective lens is used to focus the light to produce a real image.
The inverted microscope is composed of the following parts:
1) Phase slider
2) Filter holder
3) Iris diaphragm
4) Long working distance condenser
5) Eyepiece with diopter
6) Tri-nocular port for camera
7) Coarse and fine focus
8) Objective lens
The stage is usually fixed and the movement of objective lens along the vertical axis can bring the specimen closer or farther from the lens. For the coarse and fine adjustment the inverted microscope consists of dual concentric knobs. This is called the typical focusing mechanism. The rotating turret is also called nosepiece. On the rotating turret four to six objective lens of different magnification power can be fixed depending upon the microscope size. For more resolution and high magnification the accessories e.g. video camera, fluorescence illuminations, confocal scanning etc. can also be fixed.
Inverted microscope is used for wide applications but the most common are:
i. Biological adaptation:
It is an excellent microscope to observe the living specimen, valuable life processes, in a more natural environment than a glass slide for a longer time period. The specimen could be placed in a large petri dish or a container than on a glass slide. The large container provides more natural environment, more gas exchange, thus maintaining the integrity of life and can be observed under an inverted microscope
Inverted microscope can also be used for micromanipulation and observing metallurgical samples. In micromanipulation analysis, the specimen is placed above the stage and micro-tools are used to hold the specimen and then the specimen is viewed by using the refractive objectives from underneath.
Ð It is an excellent microscope for biological applications and micromanipulation.
Ð It is advantageous because of its capacity to observe living specimens in large container
Ð The specimen can be observed for longer time period
Ð It enables the observer to view the large number of samples in short time period
i. It is very costly and expensive microscope.
ii. Because of its high price there are very few engineers which are manufacturing this microscope.
iii. There is a disadvantage that it is of limited magnification power and sometimes is to be used with the conventional microscope.
Phase Contrast Microscopes
Changes in the amplitude and phase of the light wave occur when it travels from medium other than vacuum. These changes depend on the properties of the medium. Human eyes and photographic equipment cannot detect the phase change without special arrangements.
The optical compound microscope in which the phase shift of light passing through the transparent specimen is translated into brightness of the image and high-contrast images are formed is called phase contrast microscope.
Phase contrast microscopes have a phase-contrast condenser and phase contrast objective that can help translate these changes into brightness of the image.
Phase contrast microscopes can be of upright or inverted.
It was invented by Dutch Physicist Frits Zernike in 1934 and was built during the World War II. In 1953 he won the Noble prize.
It is used in the field of cellular biology as it helps to study the living cells and even their proliferation through cell division without any damage to the cell caused previously by fixing and staining etc.
The basic principle involves the segregation of specimen scattered light from the illuminating background light to make the phase changes visible.
The phase contrast microscopy is achieved by the following components:
i. Light Source
iii. Objective lens
iv. Condenser annulus
v. Phase plate
vi. Phase shift ring
vii. Gray filter ring
The condenser focuses the illuminating light on the specimen and some of this light is scattered by the specimen. The background is formed by the remaining un-scattered light. For an unstained specimen the scattered light is weak and has a phase shift of 90 degree relative to the background. This leads to a same intensity of foreground and background, resulting in a low contrast image.
In a phase contrast microscope, the contrast of image is improved in two steps:
1) The illuminating background light is passed through a phase shift ring to phase shift it by 90 degree. This is done to remove the phase difference between the scattered light and the background light and hence the intensity of the foreground and the background is improved.
2) The background is further dimmed by a gray filter ring to increase the contrast.
Both the scattered light and the background light are dimmed and phase shifted by the rings but the background light is more affected which creates the phase-contrast effect.
This is technique is termed as the negative phase contrast.
In positive phase contrast the background phase shift is +90 degree which makes it 180 degree out of phase to the scattered light. This results in the darker foreground as compared to the background.
Ð The capacity to observe living cells in their natural state.
Ð High contrast images are formed. Phase contrast microscopy has high resolution
Ð Thin specimens can be easily studied
Ð Can also combine with other microscopes like fluorescence microscope etc.
Ð CCD and CMOS computer devices attached with phase contrast microscopes can also capture images /photos.
Ð Helps observe internal structure of a molecule and can detect a mere small number of molecules e.g. proteins in a small number
i. Aperture is limited to some extent by the ring/annuli, as a result of which resolution is decreased.
ii. Thick specimens or particles cannot be observed.
iii. Poor photomicrographs are produced by using white or green light
iv. Shade-off effect caused by larger particles decreases the contrast steadily from the center to the edges
v. Halo effect due to brighter surroundings obscures details of the specimen along the perimeter
Differential Interference Contrast Microscope
Differential interference contrast microscope is the application of optical microscopy in which the transparent sample is enhanced by the illumination technique through phase contrast.
It is also called as Nomarski Interference contrast Microscope. It was invented by Nomarski and Smith in 1960's.
The basic principle of DIC is Interferometry which helps to analyze the optical path length of the sample to view the invisible features. A black or white image with grey background appears with the use of complex light lightening system.
It works by dispersion of polarized light into two orthogonally polarized mutually monochromatic parts which are displaced spatially at the sample plane and combined before observation. The recombination produces an interference which depends upon optical path difference. By adjusting the offset phase the path difference would be proportional to the phase difference thus producing the 3D image.
DIC consists of the following major parts.
- Prism Shear Axis
- Objective Prism
- Condenser Prism
The unpolarized light entering the microscope is polarized at 45 degree.
The polarized light enters the prism and is separated at 90 degree into two rays i.e. the sampling and reference rays. This is a type of prism made up of two layers of crystalline substances e.g. quartz, which causes the splitting of light into two rays depending upon variation in refracting index.
3).Condenser Nomarski Prism:
The condenser prism causes the focusing of the two rays through the sample to the two adjacent points which are 0.2 micrometer apart.
4).Prism Shear Axis:
The rays travel through the specimen adjacent areas, separated by shear. The separation is proportional to the resolution of microscope.
5).2nd Nomarski Prism:
The rays after passing through the objective lens is further modified through 2nd Nomarski prism. It recombines the two rays to produce an interference pattern which functions as “optical Interference” which allows the image to be visualized with the relative brightness and darkness.
A 3D image is produced with the light and dark shadows of the sample because of the difference in the refractive index and the thickness. This 3D image is produced with the constructive interference with the small path difference. When the path difference is greater than 90 degree the result is destructive interference and an anomalous dark region.
Following are the major advantages of DIC.
Ð It is of wide use in observing life and unstained biological samples e.g. in tissue culturing, in smear formation techniques etc.
Ð The main advantageous fact among standard optical microscopy technique is its resolution and clarity.
Ð The image formed by DIC microscopy is bright with the dark background.
Ð This technique can also be used with the digital imaging systems to add further definitions to the image.
i. The main limitation of the DIC is the need of a fairly transparent sample and similar refractive index to the surrounding and environment.
ii. For thick samples and highly pigmented cells DIC is not suitable.
iii. For non-biological samples DIC is unsuitable as it depends upon polarization which could affect physical samples.
The type of compound microscope in which the source of illumination is accelerated beam of electrons is called electron microscope.
The 100000 times shorter wavelength of electrons than the photons results in higher resolving power which leads to revealing the structure of very small objects. In 1938, the first electron microscope was built by Eli Franklin Burton and students.
The electron microscope uses accelerated beams of electrons produced by electron gun to generate the image and the electromagnetic field brings about the magnification
General structure of an electron microscope comprises of the following main components.
- Electron gun
- Condenser lens
- Objective lens
- Projector lens
- Viewing screen
Magnification of the specimen by electron microscope is up to 10000000X due to the 100000X shorter wavelength of electrons as compared to photons.
Types of electron microscopes:
Electron microscopes can be of the following types:
i). Transmission electron microscope TEM
ii). Scanning electron microscope SEM
iii). Reflection electron microscope REM
iv). Scanning transmission electron microscope STEM
Electron microscope has many advantages over the traditional light microscopes. Some of these are mentioned below:
Ð Powerful magnification up to 10000000X
Ð It has wide applications in scientific fields such as biology, nano-technology, gemology, Forensic science, medicine, and metallurgy etc.
Ð It is also widely used in industry for quality control, inspection of semiconductors and computer chip manufacturing etc.
The major disadvantages associated with electron microscopy are:
a) High cost, large size, difficulty to maintain and problems in research training along with the image artifacts caused during preparation of sample.
b) Sensitivity to vibrations and external magnetic fields.
c) Requirement of vacuum for specimen viewing.
d) Specimen should be conductive or must be coated with a conductive material to view in high-vacuum environment
Transmission Electron Microscope
TEM is one of the types of electron microscope in which a magnified image of the specimen is obtained by transmitting a beam of electron that interacts with the specimen.
In 1931, Max Knoll and Ernst Ruska made the first transmission electron microscope and the first commercial TEM was made in 1939.
Transmission electron microscope is an electron microscope in which a magnified image of the specimen is obtained by transmitting beam of electron that interacts with the specimen.
Specimen in illuminated and a magnified image of the specimen is obtained by using beam of electron. Beams of electrons are produced by electron gun, which are then focused on the specimen by using electron lenses.
Principle of TEM
TEM consists of the following components:
- Vacuum system
- Stage for specimen
- Electron gun
- A biasing circuit
- Wenhelt cap
- Extraction anode
- Electric lens
It is used to provide high or low vacuum depending upon the need.
It is a platform where the specimen is placed and held by the help of specimen holder. The specimen is placed on the grid that must be of a standard size to be held by the specimen holder.
Beams of electron are produced by heating the filament of the electron gun. These beams are then accelerated towards anode plate. α is the divergence angle at which electron exit from the assembly. Wehnelt Cylinder is negatively charged which forces the electrons into converging pattern.
These control the intensity of electron beams and filter the electrons.
Lenses are used to focus the beam of electrons on specimen. Lenses can be operated by using electric field or by magnetic field. These are made from the alloys such as nickel-cobalt, depending on their magnetic properties.
Ð Used in chemical analysis
Ð Used in study of semiconductors
Ð It has a resolution higher than the resolution of light microscope
Ð Can be used to view the minor details of the specimen
Due to the techniques used in TEM, it has many limitations.
i. It consumes a lot of time in preparing ultra-thin specimen.
ii. While preparing the sample, its structure may change
iii. Electron beam might cause damage to the sample especially biological materials.
Scanning Electron Microscope
Scanning electron microscope is one of the electron microscopes which produces image through scanning by using electron beam.
Accelerated beam of electrons produced from the electron gun possess kinetic energy. Thus accelerated beam is incident on the sample which excites the electrons of the atoms of sample. During de-excitation X-rays are produced having definite wavelength. Each type of element of the sample produces characteristics X-rays.
SEM consists of the following main components.
- Electron gun
- Stage/Sample chamber
- Vacuum Chamber
Electron gun is the source of producing beam of electrons that is essential for operation of SEM. These guns are of two types:
i).Thermionic gun: It is a common type of electron gun that provides thermal energy to the filament to produce electrons which are focused on specimen.
ii).Field emission gun: In this type electric field is used to produce electrons.
Electron guns are located at the bottom of SEM or at the top of microscope.
In SEM, detailed and clear image of the specimen is obtained by using lenses. As compared to the optical microscope, the working of lenses in SEM is different. They are formed from magnets and are used in controlling and focusing the beam of electrons exactly on the specimen.
It is a platform where the specimen to be examined is placed.
Usually SEM contains one detector but some have more than one detector. The capacity of an instrument is based on the type of detectors use d in microscope. The beams of electrons that interact with the sample are detected by using detectors and form the detail image of the specimen
For the proper operation of SEM a vacuum chamber is required.
Ð It has a high resolution power.
Ð It produces a detailed image of the shape of the specimen.
Ð SEM is used in chemical analysis.
Ð Depending upon the atomic number it can be used to differentiate the phases.
Ð Minor features of the object can be measured up to 50nm using SEM.
i. Specimen used for analysis must be solid.
ii. Specimen has to be fixed in the sample chamber.
iii. At low pressures, samples under examination may outgas, that becomes unsuitable for study using SEM.
iv. Very light elements cannot be easily detected.
v. X-ray detectors in most of the SEM that are in solid state are fast but have poor resolution and are sensitive.
A stereomicroscope having low magnification used in examination and dissection of biological samples is called the dissection microscope.
It is a special type of light microscope which is actually to compound microscopes together focusing at the same point from different angles to view the specimen in three dimensions.
It gives a 3D image of the object under examination by the use of light that reflects from the surface of the material instead of using the light that passes through it.
Dissecting microscopes consist of the following components.
- Objective lens
- Light switch
Upper portion in most DM is moveable having two eyepieces for adjustment.
Two lenses called eyepieces are used for viewing at the specimen. Each of the eyepiece has a magnification of 10X.
Ring of rotating diopter is located on the ocular lenses which is used for the adjustment of ocular lens: Focus the eyes on image
Objective lens is located towards the stage. Both types of lenses i.e. the eyepiece and the objective determine the magnification of microscope.
Focus knobs are used in up and down movement of the head of microscope, which gives the sharp view of the image.
At the base of microscope just below the objective lens there is a stage for the placement of the specimen. Clips on the sides of the stage are used to hold the slides. By inserting white or black stage, the background color can be changed.
source of illumination or lighting are present on the top as well as on the bottom side. Lighting rom the top helps in lightening of the specimen and lighting from the bottom pass through the specimen.
The light switches are used for controlling the illumination level. It is located on the top or at the back of microscope.
Ð They are used in the labs in dissecting operations
Ð Used in different microscopic activity
Ð They can be used at the elementary level or high school labs.
Ð It can be named differently based on the type of use.
Ð Use in forensic labs
Ð Also used in biological applications and in surgery. Depending upon the kind of surgery it can be modified.
- They have low magnification.
- They are not easily affordable.
DIGITAL HOLOGRAPHIC MICROSCOPE
Digital holographic microscopy is the application of digital holography applied to microscopy.
The microscope which involves the application of holography (3D photographic recording of light field) to microscopy and is then computed digitally is the digital holographic microscope.
Holography was first applied by Dennis Gabor to improve the electron microscopy.
In DHM, the light wave-front originates information from the specimen and is recorded digitally as a hologram, which is then observed through computer using reconstruction algorithm.
To create the interference pattern, the light source should be coherent. The light wave should be monochromatic e.g. LASER. The monochromatic light source split into two waves i.e. the object beam and a reference beam. The object beam creates an object wave front thus illuminating the sample. The object wave-front is gathered by the microscope lens. The object and the reference wave-front are joined by the beam splitter creating a hologram. Sing a digital hologram, the computer functions as digital lens and calculates the image by using numerical reconstruction algorithm.
Typical optical set up of a DHM
DHM has the following main parts.
- Condenser lens
- Microscope lens
- Tube lens
- Pin hole
- Beam splitter
- CCD Camera
- Image sensor
It functions to split the light which is monochromatic into two beams i.e. object beam and reference beam.
It functions to collect the object beam in the form of wave-front for illuminating the specimen.
3). Tube lens:
It expands the object beam to create the object wave-front.
It collects the object wave-front from the sample and the reference wave-front which is necessarily used to create an interference pattern and a hologram.
It is a computer which acts as a digital lens and calculates an image of the object wavefront with the help of numerical reconstruction algorithm.
DHM is being successfully used in wide range of application areas.
i. DHM makes it possible to perform cell counting in adherent cell cultures.
ii. DHM is also used in the study of apoptotic process in different cells.
iii. The phase shift has been found to be correlated to cell dry mass as the refractive index changes in cell dry mass area thus providing valuable information.
iv. DHM makes it possible to study and observe undisturbed processes in nerve cells.
v. Phase shift during analysis made it possible to study RBCs dynamics.
Following are some of the major advantages by DHM.
Ð DHM involves phase shift image besides from bright field image.
Ð This phase shift image gives valuable information about cell dynamics.
Ð Through this optical thickness of the specimen is also observable.
Ð This microscopy gives 3D image and information
Ð DHM has no image forming lens so optical aberration correction doesn’t apply to it.
Low spatial resolution of electronic devices involved in hologram formation
Comparing light microsope,transmission electron electron microscope and scanning electron microscope
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