Microscopy PDF

10/20/2024 MICROSCOPY A.P. Dr Intan Shameha Abdul Razak Dept. of Vet. Preclinical Sciences, Fac. of Vet. Medicine [email protected]...

10/20/2024 MICROSCOPY A.P. Dr Intan Shameha Abdul Razak Dept. of Vet. Preclinical Sciences, Fac. of Vet. Medicine [email protected] TYPES OF MICROSCOPE MICROSCOPY Light / compound PRINCIPLE Bright field* Dark field* Basic units Dissecting/ stereo* Quality Phase contrast UV Magnification Fluorescence Resolution CLSM Electron SEM TEM 2 1  10/20/2024 MICROSCOPY Micro = small, scopeos = to look (view) Microscopy is the technical field of using microscopes to view samples & objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye). 3 A microscope is an optical instrument consisting of one or more lenses in order to magnify images of minute objects. 2  10/20/2024 PRINCIPLES OF LIGHT MICROSCOPY The light is the primary source on which magnification is based in light microscopes. The magnification is obtained by a system of optical lenses using light waves. Magnification refers the number of times a specimen is appeared to be larger than its original size. BASIC UNITS FOR MICROSCOPE 1 meter 1000 millimeter 1 millimeter 1000 micrometer (µm) = 10-6 meter 1 micrometer 1000 nanometer (nm) = 10-9 meter 1 Angstrom (1 A) 10-10 meter 1 nanometer 10 Angstrom QUALITY FOCUS BRIGHTNESS CONTRAST RESOLUTION Image: well defined or How light or dark image is How best specimen is The ability to blurry (out of focus) Depends on illumination differentiated from distinguish 2 objects background / adjacent close to each other Focus: adjust the coarse system area of microscopic & fine adjustment knobs Adjust by: Depends on resolving field to adjust focal length power (minimum Lamp voltage Depends on brightness distance between 2 Other factors: thickness changing of illumination & objects which can be of specimen, slide & Condenser diaphragm & specimen distinguishable coverslip diaphragm colour 6 3  10/20/2024 MAGNIFICATION: Lens x Obj. power 10 X 4 = 40 10 X 10 = 100 10 X 40 = 400 10 X 100 = 1000 (oil immersion) https://microbenotes.com/parts-of-a-microscope/ 7 MAGNIFICATION RESOLUTION The total magnification the ability of microscopes to of compound microscope distinguish two objects close to each other is magnifications of objective lens and it depends on resolving power, which refers the eyepiece. minimum distance. Magnification of about Eg. Human’s resolving power 1000 - 1500x is the upper = 0.2 mm (can distinguish 2 limit of compound objects with a distance of 0.2 microscopes. mm close to each other). If he want to see beyond the limit This limit is set because of his resolving power, further of the resolution. magnification is necessary. 8 4  10/20/2024 TYPES OF MICROSCOPES 9 TYPES OF MICROSCOPES LIGHT / COMPOUND MICROSCOPE ELECTRON MICROSCOPE 1. BRIGHT FIELD 1. SCANNING EM 2. DARK FIELD 2. TRANSMISSION EM 3. STEREO / DISSECTING 4. PHASE CONTRAST 5. UV 6.FLUORESCENCE 7.CONFOCAL LASER SCANNING MICROSCOPE 10 5  10/20/2024 BRIGHT FIELD MICROSCOPE to view fixed and live specimens, stained with basic stains which gives a contrast between the image and the image background. with magnifying glasses (lenses) that modify the specimen to produce an image seen through the eyepiece. the specimen must pass through a uniform beam of the illuminating light. Through differential absorption and differential refraction, the microscope will produce a contrasting image. The colored specimens will have a refractive index that will differentiate it from the surrounding, presenting a combination of absorption and refractive contrast. The specimen which is placed on a microscopic slide is viewed under oil immersion (x100 mag.) or/and covered with a coverslip. MICROSCOPE PARTS Eyepiece (Ocular lens) – 2 eyepiece lenses at the top which focuses the image from the objective lenses. The objective lenses which are made up of 4 or more glass lenses, which make a clear image clear from the specimen or the object that is being focused. Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob, found on the microscopes’ arm, which can move the stage or the nosepiece to focus on the image. Their function is to ensure the production of a sharp image with clarity. 6  10/20/2024 MICROSCOPE PARTS The stage is found just below the objectives and this is where the specimen is placed, allowing movement of the specimen around for better viewing with the flexible knobs and it is where the light is focused on. The condenser: It is mounted below the stage which focuses a beam of light onto the specimen. It can be fixed or movable, to adjust the quality of light, but this entirely depends on the microscope. MICROSCOPE PARTS The arm: a sturdy metallic backbone of the microscope, used to carry and move the microscope from one place to another. They also hold the microscope base which is the stand of the microscope. The arm and the base hold all the microscopic parts. Don’t drag! It has a light illuminator or a mirror found at the base or on the microscope’s nosepiece. 7  10/20/2024 MICROSCOPE PARTS The nosepiece has about 2-4 objective lenses with different magnifying power. It can move round to any position depending on the objective lens to focus on the image. An aperture diaphragm (contrast): It controls the diameter of the beam of light that passes through the condenser. When the condenser is almost closed, the light comes through to the center of the condenser creating high contrast and when the condenser is widely open, the image is very bright with very low contrast. DARK FIELD MICROSCOPE specimen is brightly illuminated against a dark background. Have a special type of condenser, which prevents the parallel and the oblique rays entering in to the objective and thus making the microscopic field dark. In the absence of specimen the entire field will appear as dark. In the presence of specimen, which differs in refractive index, the oblique rays are scattered by reflection and refraction and the scattered rays enter the objective making the specimen brightly illuminated. Maximum mag. of 1500x and resolution of 0.1 – 0.2 μm. Dark field makes it easy to obtain the correct focal plane at low magnification for small, low contrast specimens. https://microbenotes.com/darkfield-microscopy/ 8  10/20/2024 DARK FIELD MICROSCOPE The uses: 1. Morphology and motility of microorganisms e.g.amoeba 2. Initial examination of cell suspensions e.g. yeast, bacteria, cell and tissue fractions including cheek epithelial cells, chloroplasts, mitochondria 3. Initial survey and observation at low powers of pond water samples 4. Examination of lightly stained prepared slides. Initial location of any specimen of very small size for later viewing at higher power. 5. Determination of motility in cultures BRIGHT FIELD MICROSCOPE 9  10/20/2024 DISSECTING / INVERTED MICROSCOPE an optical microscope designed for low magnification observation of a specimen uses the reflected light rays from the specimen surface instead of transmitted light rays. The magnification power: ranges from 5x-80x. It produces a three-dimensional image of the specimen rather than a flat image. contains two separate objective lens and eyepiece, which creates two separate optical paths for each eye. As a result, it creates a 3D image of the specimen. contains two light sources, one from the upper portion of specimen which is reflected in the eyepieces, and the second one from the below portions of the sample for illumination through thinner samples. Use: dissection, parasitology PHASE CONTRAST MICROSCOPE Principle: Phase contrast microscopy takes advantage of fact that structures with different refractive indexes bend the light differently. With special phase optics, this difference in the ability to bend light translates into a difference in contrast. In phase contrast, a phase plate slows down the highly refracted light rays and puts them "out of phase". This is seen as a difference in contrast between the cell and its background. The background is generally seen as dark, with the organism in sharp contrast. 21 10  10/20/2024 PHASE CONTRAST MICROSCOPE This is extremely useful for microbiologists (can view microorganisms alive, without physically or chemically altering the cell). Simple wet mounts can be made so such properties as motility and growth can be observed. Advantages: Can view live samples and observe motility and responses to stimuli Disadvantage: Phase optics are more expensive than bright field, and must be properly aligned. THE DIFFERENCES 22 UV MICROSCOPE Resolution of a microscope depends upon the wavelength of light used. If, longer the wavelength of light used, lower will be the resolving power while shorter the wavelength, more will be the resolution. The principle: UV rays of shorter wavelength are used as light source. Since UV rays can’t penetrate the glass, quartz lenses are used. Since the UV rays are invisible, photographic plates should be used to record the image or special type of filters should be used to eliminate the UV rays from reaching the eyepiece. This is used in conjunction with fluorescent microscopy. Upon illumination with UV light certain fluorescent dyes emit light in visible range, which can be directly viewed. 11  10/20/2024 ADDITIONAL INFO MICROSCOPES MAINLY USED IN RESEARCH FLUORESCENCE MICROSCOPE Certain chemical compounds absorb light and reemit part of the radiant energy as light of longer wavelength are called fluorescent and the phenomenon is termed as fluorescence. In FM, a high intensity mercury lamp = as the light source, which emits white light. The exciter filter transmits only blue light to the specimen and blocks out all the colours. The blue light is reflected downward to the specimen by the dichroic mirror. The specimen is stained with fluorescent dye (acrydine orange). Only certain portions of the specimen retain the dye, others do not. The stained portion of the specimen absorb blue light and emit green light, which passes upwards, penetrate the dichroic mirror and reaches the barrier filter. This filter allows only green light to pass through and the eye receives only green light emitted from the specimen against the black background whereas unstained portions are invisible. Also, ultraviolet light is used to excite molecules so that they release light of a different wavelength. 12  10/20/2024 FLUORESCENCE MICROSCOPE This technique is especially important in immunology in which the reactions of antigens and antibodies are studied in great detail. Fluorescent antibody staining is now widely used in diagnostic procedures to determine whether an antigen is present CONFOCAL LASER SCANNING MICROSCOPE Light source and illumination = laser The microscope works in epi-illumination mode. The laser beam is spread by a diverging lens so as to fill the back aperture of the objective lens which functions as condenser as well. The expanded laser light is reflected by the dichroic mirror on the objective that focuses the light as an intense diffraction-limited spot on the sample. The fluorescence from the illuminated spot is collected by the objective and sent to the eyepiece/camera/detector through a pinhole aperture. https://youtu.be/QFtZFbug1SA?si=394w2if0HnN52HmC 13  10/20/2024 ELECTRON MICROSCOPE Electron microscopy 14  10/20/2024 LIGHT MICROSCOPES VS ELECTRON MICROSCOPES 31 Character Light microscope Electron microscope study of the external surface, the ultrastructure of Application study of detailed gross internal structure. cells and very small organisms. Principle The image is formed by the absorption of light The image is formed by scattering or transmission of waves. electrons. Uses light (± wavelength 400-700 nm) to illuminate Uses a beam of electrons (± wavelength 1 nm) to lluminating source the objects under view. make objects larger for a detailed view. Structure Light microscopes are smaller and lighter. Heavier and larger in size. Lenses used Lenses are made of glass. Lenses are made of electromagnets. Vacuum Not used under a vacuum Operates under a high vacuum Magnification Low magnification of up to 1,500x. High magnification of up to 1,000,000x. power Viewing of the Images can be viewed directly by the eyes through Images are viewed on a photographic plate or zinc image formed the eyepiece. sulfate fluorescent screen. grayscale (sometimes called “black and white”) Image Color Colored images. images (except “false-color” electron micrographs). TEM: 2D Image dimension Image plane “flat” (2D). SEM: depth information which seems like 3D. 15  10/20/2024 Character Light microscope Electron microscope involves harsher processes, e.g. using corrosive Specimen Less tedious and simple. chemicals. More skill required – both to prepare preparation specimens & to interpret EM images (due to artifacts). Fixed or unfixed, stained or unstained, Fixed, stained, and non-living. Specimen type living or non-living. Only dead specimens are possible to be observed. Both live and dead specimens can be observed. Preparation time a few minutes to hours. a few days. Thickness of 5 micrometer or thicker Ultra-thin, 0.1 micrometers or below specimen Dehydration of Specimens need not be dehydrated Only dehydrated specimens are used. Specimen before viewing. Coating of Stained by colored dyes for proper Coated with heavy metals to reflect electrons. specimen visualization. Mounting of Mounted on the glass slide. Mounted on the metallic grid (mostly copper). specimen ELECTRON MICROSCOPE In EM, short beam of electrons and magnetic condenser lenses are employed to produce the image. The electrons have short wavelength, which helps in better resolution. It is possible to resolve objects as small as 10°A, which is 100 times more than that of light microscope. It can magnify object up to 200,000X. In EM, a hot tungsten filament forms the source of electrons. The object is placed in the path of moving electrons. Since electrons move only in the vacuum, the entire path of electrons should be kept under vacuum. There are two types of electron microscope. Scanning electron microscope (SEM) Transmission electron microscope (TEM) 16  10/20/2024 SCANNING ELECTRON MICROSCOPE The specimen is coated with a thin layer of heavy metal and subjected to a narrow beam of electrons, which rapidly moves and scans the surface of the specimen. The irradiated specimen depending upon its physical and chemical composition will release secondary electrons. These secondary electrons are then collected by anode detector, which generates an electronic signal. Then the electronic signal is scanned in TV system to produce an image on a cathode ray tube. Magnification on SEM is about 75,000 to 100,000 times. Limitations Specimen is kept under high vacuum on the path of electron beam. So, living cells can’t be examined. Electrons have low penetration capacity, hence ultra thin section and staining should be done which is time consuming and also sometimes alter or distort the structures of microorganisms. High cost and specialized techniques prevent its use in all microbiology labs in spite of greater magnification and resolution. TRANSMISSION ELECTRON MICROSCOPE A high voltage established between the filament and the anode accelerates the electrons from the hot tungsten filament. Ultra thin sections of the specimen must be prepared since electrons can penetrate matter only a short distance. This is done by embedding or freezing the specimen and sectioning it with a diamond or a glass Knife. In TEM, the differential scattering of electrons by the specimen makes the contrast. TEM has a projector lens that project the image onto a fluorescent viewing screen or film plate, because the beam cannot be viewed directly. With TEM greater resolution and higher magnifications than light and Scanning Electron Microscope can be obtained. 17  10/20/2024 A SEM LM C B D Fluorescence TEM SUMMARY TYPES OF MICROSCOPE PRINCIPLE OF EACH MICROSCOPE TISSUES PREPARATION 18