Light Microscopy All in One PDF

Light Microscopy  Light Microscopy Bright field Microscopy Dark field Microscopy Confocal Microscopy Phase Contrast Microscopy Fluorescence Microscopy  Bright field Microscopy In bright field microscope, the specimen appears as dark against the b...

Light Microscopy  Light Microscopy Bright field Microscopy Dark field Microscopy Confocal Microscopy Phase Contrast Microscopy Fluorescence Microscopy  Bright field Microscopy In bright field microscope, the specimen appears as dark against the bright background. Bright-field microscope is a widely used microscope in laboratories and it also known as a compound or Light Microscope. Stained, fixed and live specimens are observed under a bright field microscope. A bright-field microscope is consists of A piece of apparatus, consisting of an eyepiece, an objective lens, a condenser lens, stage, and light source, which collects electromagnetic radiation in the visible range.  Principal of Bright field Microscopy The specimen to be observed is placed on the stage of a bright field microscope. The light will transmit through the specimen from the source and then it will enter the objective lens where a magnified image of specimen will form. Then the light will enter an ocular lens or eyepiece, where the image will further magnify a then enter the into the user’s eyes. The viewers observe a dark image against a bright background. In a bright-field microscope, only the scattered lights are able to enter the objective lens and transmitted lights or unscattered light rays are omitted, that’s why the viewer sees a dark image against the bright field.  Light Path A stained specimen or sample is placed over the specimen stage. A condenser lens containing an aperture diaphragm is located under the stage, will focus the light ray on the sample or specimen. The light rays will pass through the specimen sample and then it will be collected by an objective lens located over the stage. The objective lens will form a magnified image of the specimen and then transmit it to the eyepiece, where viewers will observe a dark image against the bright field. During the transmission through the specimen, some of the light rays are absorbed by the stains, pigmentation, or dense areas of the specimen.  Advantages of Bright field Microscopy It is easy to use. Low price. These are compact in size and easy to carry. Both stained and unstained specimens can be observed.  Disadvantages of Bright field Microscopy Aperture diaphragm adds greater contrast which can create distortion. Living specimens of bacteria can not be observed under a bright-fields Microscope. Most of the specimens required to be stained to visualize under this microscope, because of its low contrast. Distort images can be produced during the oil immersion. Specimens can be damaged by the uses of the coverslip. The bright-field microscope required a strong source of light to illuminate the specimen.  Applications of Bright field Microscopy Used in blood counting. Used to examine the bacterial cells. Used to examine the fungal cells. It also used in forensic laboratories. Used in agricultural laboratories. Used to study plant cells.  Thanks for watching this video Like Share Subscribe Dark field Microscopy  Light Microscopy Bright field Microscopy Dark field Microscopy Confocal Microscopy Phase Contrast Microscopy Fluorescence Microscopy  Dark field Microscopy Principal Uses of Dark field Microscopy Advantages Dark field Microscopy Disadvantages Dark field Microscopy  Dark field Microscopy In this microscopy, the specimen is brightly illuminated while the background is dark. Dark-field microscopy is a technique that can be used for the observation of living, unstained cells and microorganisms. It is one type of light microscope, others being bright-field, phase- contrast, differential interface contrast, and fluorescence.  Principal Dark-field microscopy uses a light microscope with an extra opaque disc underneath the condenser lens, or a special condenser having a central blacked-out area, due to which the light coming from the source cannot directly enter into the objective. The path of the light is directed in such a way that it can pass through the outer edge of the condenser at a wide-angle and strike the sample at an oblique angle. Only the light scattered by the sample reaches the objective lens for visualization. All other light that passes through the specimen will miss the objective, thus the specimen is brightly illuminated on a dark background.  Uses of Dark field Microscopy The dark field microscope is used to identify bacteria like thin and distinctively shaped. We Can observe the living and unstained cells by using a dark field microscope. Considerable internal structure in microorganisms can be revealed by the dark field microscope. A biological dark field microscope used to observe the blood cells. Used to identify algae. A metallurgical dark field microscope is used to observe hairline metal fractures. Stereo dark field microscope or gemological microscope used to study the diamonds and other precious stones. A stereo dark-field microscope used to observe the shrimp or other invertebrates.  Advantages 1. Resolution by dark-field microscopy is somewhat better than bright- field microscopy. 2. It improves image contrast without the use of stain, and thus do not kill cells. 3. Direct detection of non-culturable bacteria present in patient samples. 4. No sample preparation is required. 5. It requires no special setup, even a light microscope can be converted to dark field.  Disadvantages Necessity to examine wet, moist specimens containing living organisms very quickly, because visualization of the moving bacteria is essential to detection. The sample must be very strongly illuminated, which can cause damage to the sample. Besides the sample, dust particles also scatter the light and appear bright. Sample material needs to be spread thinly, dense preparations can grossly affect the contrast and accuracy of the dark field’s image. Thanks for watching this video Like Share Subscribe Phase Contrast Microscopy  Light Microscopy Bright field Microscopy Dark field Microscopy Confocal Microscopy Phase Contrast Microscopy Fluorescence Microscopy  Phase Contrast Microscopy Principal Uses/ Application Advantages Disadvantages  Phase Contrast Microscopy Phase contrast is a light microscopy technique used to enhance the contrast of images of transparent and colourless specimens. It enables visualisation of cells and cell components that would be difficult to see using an ordinary light microscope. As phase contrast microscopy does not require cells to be killed, fixed or stained, the technique enables living cells, usually in culture, to be visualised in their natural state. This means biological processes can be seen and recorded at high contrast and specimen detail can be observed.  Principal of Phase Contrast Microscopy Phase contrast microscopy translates small changes in the phase into changes in brightness, which are then seen as differences in image contrast. Unstained specimens that do not absorb light are known as phase objects. This is because they slightly change the phase of light that is diffracted by them; the light is usually phase-shifted by about ¼ wavelength compared to the background light. Our eyes are unable to detect these slight phase differences as they can only detect variations in the frequency and intensity of light.  Phase contrast enables high contrast images to be produced by further increasing the difference of the light phase. It is this characteristic that enables background light to be separated from specimen diffracted light. The difference of the light phase is increased by slowing down (or advancing) the background light by a ¼ wavelength, with a phase plate just before the image plane. When the light is focused on the image plane, the diffracted and background light cause destructive (or constructive) interference which decreases (or increases) the brightness of the areas that contain the sample, in comparison to the background light.  Application of Phase Contrast Microscopy Phase contrast is used to visualise transparent specimens, to produce high contrast image, Living cells (usually in culture) Microorganisms Thin tissue slices Fibres Subcellular particles, including organelles.  Advantages of Phase Contrast Microscopy Living cells can be observed in their natural state without previous fixation or labeling. It makes a highly transparent object more visible. No special preparation of fixation or staining etc. is needed to study an object under a phase-contrast microscope which saves a lot of time. Examining intracellular components of living cells at relatively high resolution. eg: The dynamic motility of mitochondria, mitotic chromosomes & vacuoles. It made it possible for biologists to study living cells and how they proliferate through cell division. Phase-contrast optical components can be added to virtually any brightfield microscope, provided the specialized phase objectives conform to the tube length parameters, and the condenser will accept an annular phase ring of the correct size.  Limitations The resolution of phase images can be reduced due to the phase annuli limiting the numerical aperture of the system. Phase contrast doesn’t work well with thick specimens as these can appear distorted.  Thanks for watching this video Like Share Subscribe Fluorescence Microscopy  Fluorescence Microscopy Fluorescence microscope is a type of light microscope which uses a higher intensity (lower wavelength) light source that excites a fluorescent molecule called a fluorophore (also known as fluorochrome). Fluorescence is a phenomenon that takes place when the substances (fluorophore) absorbs light at a given wavelength and emits light at a higher wavelength. Thus, fluorescence microscopy combines the magnifying properties of the light microscope with fluorescence technology.  Fluorescence Microscopy The fluorophore absorbs photons leading to electrons moving to a higher energy state (excited state). When the electrons return to the ground state by losing energy, the fluorophore emits light of a longer wavelength.  Principal Light source such as Xenon or Mercury Arc Lamp which provides light in a wide range of wavelength, from ultraviolet to the infrared is directed through an exciter filter (selects the excitation wavelength). This light is reflected toward the sample by a special mirror called a dichroic mirror, which is designed to reflect light only at the excitation wavelength. The reflected light passes through the objective where it is focused onto the fluorescent specimen. The emissions from the specimen are in turn, passed back up through the objective where magnification of the image occurs and through the dichroic mirror.  This light is filtered by the barrier filter, which selects for the emission wavelength and filters out contaminating light from the arc lamp or other sources that are reflected off from the microscope components. Finally, the filtered fluorescent emission is sent to a detector where the image can be digitized. Fluorescence Microscopy  Working mechanism The specimen to be observed are stained or labeled with a fluorescent dye and then illuminated with high intensity ultra violet light from mercury arc lamp. The light passes through the exciter filter that allows only blue light to pass through. Then the blue light reaches dichroic mirror and reflected downward to the specimen. The specimen labeled with fluorescent dye absorbs blue light (shorter wavelength) and emits green light. The emitted green light goes upward and passes through dichroic mirror, reflects back blue light and allows only green light to pass the objective lens, then it reaches barrier filter which allows only green light. The filtered fluorescent emission is sent to a detector where the image can be digitized.  Applications To identify structures in fixed and live biological samples. Fluorescence microscopy is a common tool for today’s life science research because it allows the use of multicolor staining, labeling of structures within cells. Study of microorganisms who causes the disease and how they caused diseases Study of position of protein within a cell. Study of pathway of protein Chromosomal study in fishes.  Thanks for watching this video Like Share Subscribe. Confocal Microscopy  Confocal Microscopy Modified version of fluorescence microscopy. Use of two sets of pinhole apertures which will focus the light of one plane and not allow the light which is out of plane. This will enhance the resolution and the image formed will be sharp. Give very specific location identity and 3D structure and very sharp image and very detailed specific image.  Principal In confocal microscopy two pinholes are typically used: A pinhole is placed in front of the illumination source to allow transmission only through a small area. Another fluorescence of background area is blocked by second pinhole , fluorescence of specific region of a cell emitted and a sharp and clear image visualise. Laser Laser light is much focused on a specific image plane and can reach depth of cell. We can change the intensity of laser according to Cell’s depth.  Confocal Microscopy  Applications Stem cell research DNA hybridization confocal microscopy has been adapted into numerous fields – such as cell biology, life sciences, semiconductor inspection, materials science, neuroanatomy, and neurophysiology.  Advantages 1. The advantage of the Confocal microscope is that it improves the outcome of the image because it analyses the image from one optical point to another, therefore there is no interference with scattered light from other parts of the specimen. 2. They have better resolution and each point of interest is visualized and captured. 3. It can be used to study live and fixed cells. 4. It generates 3D sets of images.  Limitations of confocal Microscopy 1.Pin hole size: Strength of optical sectioning depends on the size of the pinhole. 2.Fluorophores a) The fluorophore should tag the correct part of the specimen. B)Fluorophore should be sensitive enough for the given excitation wave length. Polarized Microscopy  Telegram Channel: SCIENCE WORKSHOP Twitter: Kusum Chaudhary  Polarized Microscopy : Principal Polarized Microscopy is a contrast enhancing technique to allow to evaluate the composition and 3D Structure of Speciemen. The light Beam vibrates in all the direction. In this microscope a polarizer is used to allow only one direction vibration. Polarizer : Makes light vibrate in one direction. Analyser : This device makes light detectable. Both polarizer and Analyser are aliened parreler to each other.  Polarized light: Plane Different Directions line or one Direction  If polarizer and Analyser are set prependiculer to one another are crossed no light penetrates and it will result total darkness. Polarized Microscopy  Polarized Microscopy : Working Mechanism There is a light source , light source produce the white light. The polarizer is placed between the light source and the sample stage. It polarized the light before it passes through the specimen. After this penetrating the specimen it must pass through the analyser before it reaches to eyepiece. Where we can see the virtual image and all the fine details of our Speciemen.  Birefringent Speciemen Birefringent or doubly-refracting sample has a unique property that it can produce two individual wave components while one wave passes through it, those two components are termed as ordinary and extraordinary waves. an illustration of a typical construction of Nicol polarizing prism, as we can see, the non-polarized white light are splitted into two ray as it passes through the prism. The one travels out of the prism is called ordinary ray, and the other one is called extraordinary ray. So if we have a birefringent specimen located between the polarizer and analyser, the initial light will be separated into two waves when it passes though the specimen. After exiting the specimen, the light components become out of phase, but are recombined with constructive and destructive interference when they pass through the analyser. Now the combined wave will have polarized light wave. Anisotropic Substances Anisotropic Substances are birefringent. Anisotropic substances have difficult physical properties which has different values, when measured in different directions. Birefringent Substances  Applications of Polarized Microscopy Polarized microscopy is best suited in study of crystals , pigments, lipids , glasses etc. That exhibit birefringence. Interference Microscopy  Telegram Channel: SCIENCE WORKSHOP Twitter: Kusum Chaudhary  Interference Microscopy The Interference Microscopy or Quantitative Interference Microscopy is one of these techniques that derive from Phase Contrast Microscopy but is more sensitive than this technique and make possible the easy and clarify viewing of living organisms This technique is used by taking light from a condenser and using a prism to separate the light into two beams. Thus, one beam (object beam) goes through the specimen and the objective and the other (reference beam) goes through another objective without touching the specimen. Interference occurs when a light beam is retarded or advanced relative to the other. These beams allow a specimen to be seen through the difference in the fields caused by the two beams and the differences of the two images allow details to be seen. Wave interference The phenomenon in which two or more waves superpose to form a resultant wave of greater, lower or the same amplitude.  Differential Interference Contrast Microscopy There is a variation of interference microscopy called Differential Interference Contrast microscopy (DIC), also known as Nomarski Interference Contrast microscopy (NIC) or simply Nomarski microscopy. This optical microscopy illumination technique used to enhance the contrast in unstained or transparent samples and also uses two beams produced by a single polarized light. Initially the polarized light is divided into two rays polarized to each other (sampling and reference rays) when enters in the first Nomarski-modified Wollaston prism. Then this two rays are focused by the condenser for passage through the sample and travel to adjacent areas of the sample, divided by the shear. After that, they will face different optical way lengths where areas differ in refractive index or thickness which will cause a change in phase of one ray relative to the other according to the delay experienced by the wave in the more optically dense material.  Lastly the rays go through the objective lens and are focused for the second Nomarski-modified Wollaston prism which joins the two rays into one polarized which make an image with a three- dimensional appearance. This final junction of rays leads to interference, brightening or darkening the image at that point according to the optical way difference. Differential interference contrast microscopy  Applications These interference techniques have advantages in uses involving living or unstained biological samples, specially their applications in biology, crystallography, mineralogy and chemistry; in standard optical microscopy techniques its resolution and clarity is also visible.  Limitations On the other hand these techniques also have limitations as its requirement for a transparent sample of similar refractive index. Differential Contrast Microscopy is inadequate for thick samples (tissue slices, pigmented cells) and for most non biological samples because of its polarization dependence.  Telegram Channel: SCIENCE WORKSHOP Twitter: Kusum Chaudhary