What is a microscope?
Theoretically a microscope is an array of two lenses Need of Microscopy
Living organisms, cells and their organelles which are too small to be seen with the human eye, can only be seen with the aid of a microscope.
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(https://web.stanford.edu/group/Urchin/introm.html) Magnification and Resolution:-
Magnification can be defined as capacity of a microscope to maximize the image of an object than its real size.
Magnification is - Magnification by the objective x Magnification by eyepiece Concept of Magnification-
Magnification (M) of the Microscope
M Microscope = M of Objective X M of Eyepiece X M of Intermediate Factor M = Magnification
Example: Objective = 70 x Eyepiece = 10 x Intermediate Factor = 1 x Overall M = 700 x
Maximum Magnification does NOT mean maximum Resolution!
The details seen from the microscope depend on the resolving power of a microscope
(Resolution), Resolution is a capacity of a microscope/ lens to distinguish two separate objects.
Resolution can be defined as the ability to distinguish between two points on an image.
Resolution or resolving power of a microscope is ultimately limited by the wavelength of light (400-600nm for visible light) .
When an object is much smaller than the wavelength of the radiation being used, the waves are not interrupted by an object and so are not detected.
Using a microscope with a more powerful magnification will increase the size of the image, and will not increase the resolution.
To improve the resolution shorter wavelength of light is needed. As blue light has the shortest wavelength (In visible range), microscopes sometimes have blue filters for improvement of resolution.
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy.
• Main branches: optical, electron and scanning probe microscopy. (+ less used X-ray microscopy)
• Optical and electron microscopy involves the diffraction, reflection, or refraction of radiation incident upon the subject of study, and the subsequent collection of this scattered radiation in order to build up an image.
• Scanning probe microscopy involves the interaction of a scanning probe with the surface or object of interest.
• Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample.
• The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally.
The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the basic light
microscope.
Optical microscopy
Light / Optical Microscopy:
Oldest, simplest and most widely-used form of microscopy.
Specimens are illuminated with light, are focused using glass lenses and are viewed using an eye or photographic film.
Specimens can be living or dead, but often need to be stained with a coloured dye to make them visible. Many different stains are available that stain specific parts of the cell such as DNA, lipids, cytoskeleton, etc.
All light microscopes today are compound microscopes, which use several lenses to obtain high magnification.
Light microscopy has a resolution of about 200 nm, which is good enough to see the cells, but are not sufficient to observe the details of cell organelles.
Light microscopy technique is being improved partly due to technical improvements, which has improved the resolution far beyond the theoretical limit.
Example fluorescence microscopy has a resolution of about 10 nm, while interference microscopy has a resolution of about 1 nm.
Types of Microscopy:
Light Microscopy:
Light microscopy is oldest technique which is simple and most widely used technique.
In this the specimen is illuminated using natural sunlight or artificial light source using glass lenses. The image is seen by an eye or can be captured on photographic film.
Most of the times the specimen needs to be stained. Various stains are available that stain the specific parts of the cell such as DNA, lipids, cytoskeleton, etc.
The light microscopes we use now are compound microscopes (several lenses are used to obtain high magnification images).
With the resolution of about 200 nm in Light microscopy cells can be observed, but the details of cell organelles cannot be observed.
For example fluorescence microscopy has a resolution of about 10 nm, while interference microscopy has a resolution of about 1 nm.
• Fluorescence Microscope- When illuminated with the high energy, certain compounds emit light of a lower frequency which is known as fluorescence. This method can be extremely sensitive, allowing the detection of single molecules. Various fluorescent dyes are available which are used to stain different chemical compounds as well as structures.
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Eg. One such powerful method is the combination of antibodies coupled to a fluorophore as in immune staining. Commonly used fluorophores are fluorescein or rhodamine.
The antibodies can be tailor-made for a chemical compound
• It is very useful for examination of biological samples.
• It facilitates identification of particular molecules in complex structure (e.g. cells)
• It can locate the particular molecules in the structure
• It is used to study Biochemical dynamics.
• Fluorescence microscope drawback is it requires chemical labeling.
Advanced example fluorescence microscopy is two-photon or multi-photon imaging.
Two photon imaging- enable greater excitation light penetration and background emission signal is reduced to allow imaging of living tissues up to a very high depth.
Superpenetration Multi Photon Microscopy is a recent developmentand allows imaging at greater depths than two-photon or multi-photon imaging.
In microscopy preparation of slide samples is done using following methods.
Fixation: Chemicals preserve material in a life like condition. Does not distort the specimen.
Dehydration: Ethanol washes are given to remove water from the specimen. This is mainly important for electron microscopy as water molecules deflect the electron beam and interfere with which image formation making it blur.
Embedding: Tissue is embedded in wax or resin to facilitate cutting of samples into thin sections.
Such sections are cut with a microtome or an ulramicrotome.
Staining: Most of the biological material is transparent therefore it needs staining.
Staining increases the contrast between different structures. Different types of tissues are used stained using different stains. Methylene blue is often used for animal cells, while iodine in KI solution is used for plant tissues.
Mounting: Mounting is necessary to protect the sample.
Optical Microscopy can only image dark or strongly refracting objects effectively.
Compound optical microscopes have many limition with respect to ability to resolve fine details It is dependant on the properties of light and the refractive materials of lenses.
A lens magnifies by bending light.
Dark Field Microscopy
Dark Field Microscopy is used in case of objects lacking in sufficient contrast and are difficult to observe.
It is used to observe live, unstained samples. In dark field microscopy, underneath the condenser lens an opaque disc is placed to view a specimen due to which only light scattered by an objects on the slide can be reached to eye piece. Due to this instead of coming up through the specimen the light gets reflected by particles on the slide, Everything on the slide gets visible as bright white against a dark background. Pigmented objects are often seen as different color than the color of the object which is called as "false colors". Better resolution can be obtained as compared to bright field viewing. This is the low cost alternative for phase contrast optics. The contrast and resolution obtained with inexpensive dark field equipment may be superior to student grade phase contrast equipment.
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Dark field microscope can be used for initial examination of suspensions of cells (motile cultures, blood cells, bacteria, yeast, cell and tissue fractions, chloroplasts, mitochondria, etc).
This technique is recently been used in computer mouse pointing devices, for allow an optical mouse to work on transparent.
Differential Interference Contrast Microscopy - An excellent mechanism for rendering contrast in transparent specimens, differential interference contrast (DIC) microscopy is a beam- shearing interference system in which the reference beam is sheared by a minuscule amount, generally somewhat less than the diameter of an Airy disk. The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image contrast is dramatically increased.
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Phase Contrast Microscope - was first described in 1934 by Dutch physicist Frits Zernike, This optical technique enhances contrast, which helps in producing high-contrast images of transparent specimens, eg- living cells, subcellular particles, microorganisms etc. very important advantage of this technique is we can observe living cells in their natural state without killing them. Most of the living biological specimens are virtually transparent when observed in the optical microscope under brightfield illumination. One has to reduce the opening size of the substage condenser iris diaphragm, for improvement of visibility and contrast in such specimens.
But this causes serious loss of resolution as well as introduces diffraction artifacts. Phase contrast technique was introduced for testing of telescope mirrors in 1930's. After several years it was adapted by Zeiss laboratories which brought it into a commercial microscope stage.
Large, extended specimens are also easily visualized with phase contrast optics due to diffraction and scattering phenomena that occur at the edges of these objects.
When An incident wavefront of an illuminating beam of light passes through a phase specimen it gets divided into two components viz-1) primary component undiffracted is wavefront, commonly referred to as the surround (S) wave which is undeviated. This does not interact with the specimen but passes through and around the specimen. 2) deviated or diffracted spherical
wavefront (D-wave. This scatteres over in many directions and passes through the full aperture of the objective.
when surround and diffracted light waves enter the objective front lens element after leaving the specimen, they are focused and are combine at image plane to produce a resultant particle wave (P-wave). Detection of the specimen image depends on the relative intensity differences, and on the amplitudes, of the particle and surround (P and S) waves. If the significantly different amplitudes of the particle and surround waves in the intermediate image plane, then the specimen acquires a considerable amount of contrast and is easily visualized in the microscope eyepieces. In other case the specimen remains transparent and appears as same as ordinary brightfield conditions.
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This technique has proved to be an excellent method of improving contrast. Without significant loss in resolution, it facilitates the observation of unstained biological specimens. Due to this property it is widely utilized for examination of dynamic events in living cells. Phase contrast microscopy is useful in observing transparent, unstained, live cells This technique is superior to bright-field optics as fine details which cannot be observed under bright-field optics can be observed here in high contrast. But in case of ideal for thick samples, this technique is not ideal as they may give Halo effect and appear as distorted.
Electron Microscopy
Electron Microscopy- Was developed in the 1930s that uses electron beams instead of o observe the image.
Because of the much lower wavelength of the electron beam than of light, resolution is far higher.
This uses a beam of electrons, rather than electromagnetic radiation, to "illuminate" the specimen. Electrons behave like waves and can easily be produced (using a hot
wire), focused (using electromagnets) and detected (using a phosphor screen or photographic film). A beam of electrons has an effective wavelength of less than 1 nm, so can be used to resolve small sub-cellular ultrastructure. The development of the electron microscope in the 1930s revolutionized biology, allowing observation of organelles such as mitochondria, ER and membranes in detail for the first time.
• The main problem with the electron microscope is that specimens must be dead so that they can be fixed and viewed in a vacuum.
• The specimens can be damaged by the electron beam and they must be stained with an electron-dense chemical (usually heavy metals like osmium, lead or gold). Initially there was a problem of artefacts (i.e. observed structures that were due to the preparation process and were not real), but improvements in technique have eliminated most of these.
• There are two kinds of electron microscope. The Transmission Electron Microscope (TEM) works much like a light microscope, transmitting a beam of electrons through a thin specimen and then focusing the electrons to form an image on a screen or on film. This is the most common form of electron microscope and has the best resolution. The Scanning Electron Microscope (SEM) scans a fine beam of electron onto a specimen and collects the electrons scattered by the surface. This has poorer resolution, but gives excellent 3-D images of surfaces.
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Types of Electron Microscope
• Transmission Electron Microscopy (TEM) is principally quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. As these electrons needs to pass through the sample, taking section of a sample is necessary for the image to produce. After the beam hits the sample section, the electons and X- rays are ejected from the sample. The detector collects these rays and converts it in to the signal and then into an image. The resolution limit (in 2005) is around 0.05 nanometer.
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Transmission electron microscopy- an image is formed when a beam of an electron passes through a sample. The image is captured either on fluorescent screen or layer of photographic film or to be detected by a sensor
Electron beam is created using a cathode (tungsten filament), which involves high voltage.the beam is focused using electrostatic and electromagnetic lenses.
• The partof a sample which is transparent for a beam of electron carries information about the inner structure of the specimen. It gets trasmitted and reaches to the imaging system of the microscope. The spacial variation gets magnified with the help of electromagnetic lenses and recorded by CCD camera, photographic plate or fluorescent screen.
• Scanning Electron Microscopy (SEM) – (https://www.purdue.edu/ehps/rem/search.htm
?q=TEM) It uses electron insted of a light beam. At the top of an instrument electron gun is placed to produce the beam of electron. The verticle path throught wich the electron beam is passed is filled with a vacuum. It travelles through electromagnetic field and lenses, followed by a sample.
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• A beam of electrons is passed over the surface of the sample.
• The sample is coated in heavy metal and the electrons are reflected off the surface of these coated sample.
• Reflected beams of electron are focused on fluorescent screen in order to make the image.
• As it gives the outer structure, larger, thicker structures can also be observed in SEM as the electrons do not have to pass through the sample to form an image.
• SEM has lower resolution than that of the TEM.
• Visualizes details on the surfaces of cells and particles and gives a very nice 3D view and allows researcher to examine a variety of specimen. SEM has allowed researchers to
examine a much bigger variety of specimens. The magnification is in the lower range than that of the transmission electron microscope.
• Advantges of SEM over Traditional Microscope
Has large depth of field allowing more specimen to focus at a time.
Higher rsolution, allowing magnification of cloed spaced specimen at high level Strickingly clear images.
Sample preparation- As vacuum condition is included in the technique, all the water must be removed from the sample by vaporizing it.
X-ray microscopy
X-ray microscopy is used to produce enlarged images of samples illuminated with X-rays. There are two main principles of microscopes-: 1) Full field microscopes and 2) Scanning microscopes.
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In scanning microscopes the sample is illuminated with a bright well focused spot scanning over the sample. The detector then measures the total intensity over time coming from the currently illuminated spot on the sample and the image is calculated from this data when the scanning process is finished.
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Advantages of X-ray microscopes over other types of microscopes
X-rays penetrate matter far easier than visible light, giving the inside image of samples which are opaque for visible light.
X-ray microscopes can achieve higher optical resolution than microscopes using visible light.
As wavelength of X-rays is much shorter than the visible light, limit of the optical resolution (caused by diffraction) of X-ray microscopes is far below than the diffraction limit of microscopes working with visible light.
Scanning electron microscopes in comparison have a high image resolution, but they need vacuum-proof samples with metallic or metallized surfaces and they are unable to image the inner of a sample.
Reference
http://www.biologymad.com/cells/microscopy.htm https://web.stanford.edu/group/Urchin/introm.html
https://www.google.com/search?q=google&ie=utf-8&oe=utf-8 old.lf3.cuni.cz/biofyzika/doc/eq/ImagingMethods.ppt
http://www.biologymad.com/cells/microscopy.htm
https://cw.fel.cvut.cz/wiki/_media/courses/a6m33zsl/microscopic_techniques.pdf https://en.wikipedia.org/wiki/Fluorescence_microscope
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/dfield.html https://en.wikipedia.org/wiki/Dark_field_microscopy
http://www.iitk.ac.in/acms/electronmicroscopylab.html
http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html https://www.purdue.edu/ehps/rem/search.htm ?q=TEM
www.teachers.stanford.edu/links/TypesOfMicroscopy.ppt
