Microscopy Techniques PDF

❖ A microscope is Greek word :  Mikron = small + Scopeos = to look. ❖ MICROSCOPE: Is an instrument for viewing objects that are too small to be seen by the naked eye. ❖ MICROSCOPY: The science of investigating small objects using microscope, it is the technical fi...

❖ A microscope is Greek word :  Mikron = small + Scopeos = to look. ❖ MICROSCOPE: Is an instrument for viewing objects that are too small to be seen by the naked eye. ❖ MICROSCOPY: The science of investigating small objects using microscope, it is the technical field of using microscopes to view samples & objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye) ❖ MAGNIFICATION: ✓ Degree of enlargement; number of times the length & diameter, of an object is multiplied. ✓ It depends upon: 1. Optical tube length 2. Focal length of objective 3. Magnifying power of eye piece ✓ TOTAL MAGNIFICATION: magnification of the eyepiece x magnification of the objective. ❖ RESOLUTION : Ability to reveal closely adjacent structures as separate and distinct. ❖ LIMIT OF RESOLUTION (LR): The minimum distance between two visible bodies at which they can be seen as separate and not in contact with each other. NUMERICAL APERTURE(NA): Ratio of diameter of lens to its focal length DEFINITION: Capacity of an objective to render outline of the image of an object clear and distinct. WORKING DISTANCE: Distance between the front surface of lens and surface of cover glass or specimen. CONTRAST: Differences in intensity between two objects, or between an object and background.  Helps in measurement of the size of microscopic objects.  Achieved by the use of  Stage micrometer  Micrometer eye piece 1. Optical microscopy 2. Electron microscopy. 3. Scanning probe microscopy. Eye piece the lens at the top that you look through, usually 10x or lens 15x power. Connects the eyepiece to the Tube objective lenses. Supports the tube and connects Arm it to the base. Base The bottom of the microscope, for support. Illuminator A steady light source (110 volts) The flat platform where you place your slides. Stage clips hold the slides in place. If your Stage with microscope has a mechanical stage, you will be able to move the slide around by turning two Stage Clips knobs. One moves it left and right, the other moves it up and down. Revolving This is the part of the microscope that holds two or more objective lenses and can be rotated to Nosepiece easily change power.  Usually are 3 or 4 objective lenses on a microscope. Objective They almost always consist of 4x, 10x, 40x and 100x powers. Lenses When coupled with a 10x (most common) eyepiece lens, total magnification is 40x (4x times 10x), 100x , 400x and 1000x. Condenser The purpose of the condenser lens is to focus the light onto the specimen. Condenser lenses Lens are most useful at the highest powers (400x and above). Rotating disk under the stage Diaphragm has different sized holes Is used to vary the intensity and size of the or Iris cone of light that is projected upward into the slide  To have good resolution at 1000x, you will need a microscope with an “Abbe condenser”.  An Abbe condenser is composed of two lenses that control the light that passes through the specimen before entering the objective lens on the microscope.  The shortest lens is the lowest power, the longest one is the lens with the greatest power. Lenses are color coded  The illumination in light microscopes can be: Direct or transmitted illumination, as in bright field microscope Indirect illumination  Is one of the simplest optical microscopy.  Principle:  In bright-field microscopy, illumination light is transmitted through the sample and the contrast is generated by the absorption of light in dense areas of the specimen producing a dark image against a brighter background.  2 types: 1. Simple 2. Compound SIMPLE : Contain a single magnifying lens. compound: contain Series of lenses for magnification. Light passes through specimen into objective lens Oil immersion lens increases resolution Have one or two ocular lenses. Resolution=200nm  The compound microscope has two systems of lenses for greater magnification: Ocular eyepiece lens to look through. Objective lens, closest to the object.  Bright field microscope is the standard method by which cells, tissues, and organs are examined in both normal and pathological histology.  Has several objective lenses.  A working knowledge is assumed as to the manipulation of the source of light, condenser, iris diaphragm, objectives, and eyepieces.  Used to view live or stained cells. Advantages: Simple setup with very little preparation required Biological specimen are often of low contrast and need to be stained Disadvantages: Resolution is limited to 200nm  For observation of: Capillary circulation in solid organs Cytological changes  This type of is best obtained with the “Ultrapak” or similar apparatus which projects light from a ring-shaped mirror surrounding the objective lens , nearly vertically down on the object.  It is optical system to enhance the contrast of unstained bodies.  Object appears bright (gleaming) against dark background.  In bright-field microscopy most of the light from the condenser lens enters the objective lens ”after interacting with the object (absorption)” to participate in image formation. This system generally results in an image with a bright field or background. ✓ The dark field microscope creates a contrast between the object and the surrounding field, such that, the background is dark and the object is bright. ✓ The objective and the ocular lenses used in the dark field microscope are the same as in the ordinary light microscope, however, a special condenser is used ✓ The condensing system for dark-field microscope: Utilizes a central circular disk (annular ring) The annular ring prevents direct condenser rays from entering the objective lens. Only those rays that have been scattered by the object enter the objective lens to generate the final image. Object detail responsible for the scattering appears bright on a dark background or field. 1. Applied to fresh tissues, is used for the detection of micro-organisms in body fluids-e.g., trypanosomes in blood, Treponema pallidum , Leptospira & Endospore ✓ It is in use in every venereal disease clinic, and is hence a method of particular importance. 2. Much has also been learnt about the colloidal nature of protoplasm by the application of this method to large cells such as leucocytes: ✓ The use of the dark-field technique, particularly for the study of protoplasm, need experience in the interpretation of the images 3. In the study of materials of low contrast such as small particles or internal inclusions.  Simple setup Advantages Provides contrast to unstained tissue, so living cells can be viewed. Specimen needs to be strongly illuminated which can damage delicate samples. Disadvantages Much experience is required to interpret the appearance of cells by this technique  The theory of phase contrast is mathematical & the basis of the method is:  the exaggeration of slight differences in refractive index (R.I) and converting them into degrees of light and shade.  It manipulates the light paths through the use of phase rings to illuminate transparent biological samples  If (2) unstained structures of almost the same refractive index (R.I.) are examined by ordinary light microscope, it will be noted that they are indistinguishable from each other e.g., small and colorless granules lying in the cytoplasm of living cells  Both dark field and phase contrast microscopes can be used in the observations of cell culture & tissue culture Hence, they are called cell culture microscope, tissue culture microscope, and culture microscope  This enables unstained objects to be examined in details : cell constituents as mitochondria, granules, nucleoli and so on may be seen clearly (great advance in microscopy).  Living cells and organisms may be studied and photographed during division.  It is a research instrument , but now is being widely used in routine laboratory work (in cytology, bacteriology, and virology).  It should be noted that the technique is not usefully applicable to fixed and stained material; this is because these processes alter both the R.I. and the colour of cell components.  Compare between phase- contrast microscope & dark-field microscope  is a contrast-enhancing technique that improves the quality of the image obtained with birefringent materials when compared to other techniques such as bright-field microscope, phase contrast.  Polarized light microscopes have a high degree of sensitivity and can be utilized for both quantitative and qualitative studies targeted at a wide range of anisotropic specimens (biréfringent).  Ordinary light consists of electromagnetic waves vibrating in all directions “more than one direction” , is called ‘unpolarised light’  A light wave that vibrates in a single direction is called ‘polarised light’.  How unpolerized light is converted into polerized light ? ✓ The process of transforming un polarized light into polarized light is known as polarization. ✓ There are a variety of methods of polarizing light. ✓ The most common method of polarization involves the use of a Polaroid filter.  Polaroid filters: Are made of a special material that is capable of blocking parts of vibration planes of an electromagnetic wave , it serves as a device that filters out one-half of the vibrations upon transmission of the light through the filter. A Polaroid filter is able to polarize light because of the chemical composition of the filter material  In a polarized light microscope, a polarizer intervenes between the light source and the sample. The polarized light is converted into plane- polarized light before it hits the sample. This plane-polarized light falls on a doubly refracting object (biréfringent) which generates two wave components.  These two waves are called ordinary and extraordinary light rays. The waves pass through the object in different phases. They are then combined using an analyzer. This leads to the final generation of a high-contrast image.  A polarizing microscope is valuable for the detection of biréfringent or anisotropic objects such as certain foreign bodies, crystals, certain lipids, cross-striated muscle and myelinated nerve fibres.  They rotate the plane of polarized light and appear bright against a dark background.  Objects classified into two groups in regard to their polarity: 1) Isotropic or singly refractile:  These do not divide the beam and hence when examined with polarized microscope are not illuminated. 2) Anisotropic (birefringent or doubly refractile):  These emit two beams when examined with polarized microscope, that combined using the analyzer, causes the object to glow against a dark background.  A further characteristic appearance is shown by small globules of fatty substances: the black cross of polarization.  Examples of anisotropic substace (of biological importance): 1. Normal myelin (degenerated myelin is isotropic). 2. Also striated muscle 3. Cholesterol 4. Some fatty substances 5. red blood-corpuscles (particularly after Zenker fixation). 6. Certain pigments such as:  The formalin pigment  Artifact pigment  Malarial parasite pigment  A) Bright field microscope  B) Polarized microscope  The interference microscope incorporates certain principles of both the phase-contrast and polarizing microscopes, and enables one to see unstained objects in great detail.  In phase contrast microscopy the specimen retards some of the light rays with respect to those passing through the surrounding medium.  The resulting interference of these rays provides image contrast but with an artifact called the phase halo.  In the interference microscope the retarded rays are entirely separated from the direct or reference rays, allowing improved image contrast and color graduation.  By measuring the difference in color between cell components, or the change in the wave front caused by particular objects, their weight may be calculated  Being virtually a microscopical balance, the weight of objects, such as single mitochondria, may be estimated with great accuracy  Types of interference microscope: 1. Classical interference microscopy 2. Differential contrast interference microscopy 3. Fluorescence contrast interference microscopy  There is no phase halo, comparing to phase- contrast microscope With interference microscopy quantitative Advantages measuring of phase change (optical path difference), refractive index, dry mass of cells (optical weighing), and section thickness are also improved. Its adjustments are critical and complicated, and it is unlikely in its Disadvantages present form to become an instrument for routine use.  This involves the conversion of short wave-lengths of light into longer ones by certain substances and tissues (fluorophores).  Fluorophores are microscopic molecules, which may be proteins, small organic compounds, or synthetic polymers that absorb light of specific wavelengths (invisible spectrum i.e ultra-violet/ short wave lengths) and emit light of longer wavelengths (visible spectrum) e.g fluorescein & rhodamine (fluorescent dyes)  Fluorescent material examined by this technique will appear bright on a dark background.  Certain elements are naturally fluorescent {innate fluorescence), but the use of fluorescent dyes allows the specific demonstration of many tissue elements and bacteria. ❖ Application of fluorescence microscopy: 1. The identification of the bacilli of tuberculosis and leprosy (clinical diagnostic microbiology) 2. Immunofluorescence “Cell Labeling” 3. Protein Characterization ❖ Limitation of fluorescence Microscope: Photobleaching  Resolution of the normal compound microscope is limited by two factors: 1. The numerical aperture of the objective used 2. The wavelength of the light source employed. ✓The numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light ✓Numerical aperture is used in microscopy to describe the acceptance cone of an objective (and hence its light-gathering ability and resolution )  An increase in resolution is therefore possible by using ultra-violet light which has a shorter wavelength than white light.  As ultra-violet light is not in the visible spectrum: ✓ The image must be recorded photographically. ✓ The lenses employed must be made from quartz.  Confocal microscopy: ✓ is an optical imaging technique for increasing optical resolution and contrast of a micrograph. ✓ Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three- dimensional structure within an object.  PRINCIPLE OF CONFOCAL MICROSCOPE : ✓ Confocal microscope uses fluorescence optics, instead of illuminating the whole sample at once, laser light is focused onto a defined spot at a specific depth within the sample. This leads to the emission of fluorescent light at exactly this point. ✓ By scanning many thin sections through a sample, one can build up a very clean three-dimensional image.  Advantages: Optical sectioning ability. 3D reconstruction. Excellent resolution (0.1-0.2 μm). Very high sensitivity  Disadvantages: Expensive. Complex to operate. Chemical labeling High intensity laser light  USES OF CONFOCAL MICROSCOPE: ✓ Study of biofilms ✓ Observing cellular morphology in multilayered specimen for example: It used in diagnosing cervical cancer Evaluation and diagnosis of basal cell carcinoma of skin  An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce magnified images of objects.  Samples are commonly analyzed in a crystal form.  The use of electrons in place of light rays, and electrical lenses in place of glass lenses has greatly increased the degree of magnification and resolution.  An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination.  Very high magnifications : progressive and continuous magnifications from 1,000 to 100,000 times are obtainable  Many ultra-microscopic particles, such as the smaller viruses, were seen for the first time by this technique 1. Transmission electron microscope (TEM). 2. Scanning electron microscope (SEM).  In the electron microscope there is:  A source of electrons, not of light rays: a tungsten electron gun  A magnetic condenser  A magnetic objective  A magnetic projector Light rays do not enter at all into the formation of the image, but only electrons.  Tungsten electron gun which projects a stream of negatively charged electrons through A magnetic condenser focuses a convergent beam through the object into A magnetic objective forming a magnified intermediate image which in its turn is projected into Magnetic projector like the eyepiece, still further magnifies the image  Direct visualization is impossible, but the electron image can be seen on a fluorescent screen or recorded on a photographic plate.  The above components are all mounted in a metal tube, and the interior of this is maintained at a very high and quite constant vacuum. 1. The apparatus is expensive and delicate to manipulate 2. Material has to be dried and examined in vacuum, introducing certain artifacts also making study of living cells impossible (TEM). 3. Heat: as electrons pass through the specimen they lose energy and this produces heat (TEM)  Application of (TEM): cancer research Virology Nanotechnology.  Applications of (SEM): Use in ultra high vacuum, air, water and various liquid environment. Use for the live specimen examination. Use for visualization of intracellular change  The family of SPM uses no lenses, but rather a probe that interacts with the sample surface.  Modes of Scanning Probe Microscopy:  The most common modes in SPM are: Atomic Force Microscopy (AFM) Scanning Tunneling Microscopy (STM).  How Scanning Probe Microscopy Works? ✓ SPM scans an sharp probe over a surface, usually at a distance of a few nanometers or angstroms. ✓ The contact between the sharp probe and surface produces a 3D topographic image of the surface at the atomic scale.  Advantages: Simple design. Low cost. Easy to handle. Automatically resolve image.