Applications of microscopy in bacteriology and its advancements
•Download as PPTX, PDF•
7 likes•3,872 views
Exhaustive list of microscopes types, advantages, limitations, applications and newer advances
Recommended
More Related Content
What's hot
What's hot (20)
Similar to Applications of microscopy in bacteriology and its advancements
Similar to Applications of microscopy in bacteriology and its advancements (20)
Recently uploaded
Recently uploaded (20)
Applications of microscopy in bacteriology and its advancements
- 1. Applications of microscopy in bacteriology and its advancements SPEAKER: Dr. AbhishekYadav MODERATOR: Dr.Vikas Manchanda 1
- 2. Scale 2
- 3. History ■ 1590 - Hans Janssen and his son, developed first microscope. ■ Robert Hooke- Micrographia ■ AntonVan Leeuwenhoek (1632-1723) – First person to observe and describe micro-organisms accurately – Discovered bacteria, microscopic parasites, blood cells, sperm cells etc. – ‘Animalcules’ – Father of microbiology – Created simple microscopes with magnification about 200X. – Glass spheres as lenses. 3
- 4. Lenses and the Bending of Light ■ Refraction ■ Refractive index (μ) ■ Focal point (F) ■ Focal length (f) ■ Magnification relation to focal length 4
- 5. Refractive index ■ Refractive index is the light-bending ability of a medium. The light may bend in air so much that it misses the small high- magnification lens. ■ Immersion oil is used to keep light from bending. ■ RI of Air- 1.0 RI ofWater -1.3 RI of Glycerol- 1.47 RI of Glass(avg)- 1.5 , RI of Oil- 1.52 5
- 6. 3 basic functions of Microscope: ■ Magnification? ■ Resolution? (limited by λ not lens aberrations) ■ Contrast? – Absorption- Stains, Fluorescence – Differences in μ 6
- 7. SomeTerms ■ Limit of Resolution (LR) ■ NumericalAperture (NA) ■ NA = μ Sin θ/2 Practically, the highest possible NA of a dry lens is 0.95 and for an oil immersion lens is 1.4. ■ Angle of aperture (θ) ■ Wavelength (λ) LR = 0.61 x λ / NA 7
- 8. 8
- 9. 9
- 10. OtherTerms ■ Visible Light: EM spectrum detected by human eye= 400nm(deep violet)- 800nm(Far red). ■ ColorTemperature- Natural daylight ~5200kelvin ■ Higher color temp= more blue/white = cooler ■ Lower color temp= more red/yellow =warmer ■ Amplitude (i.e. brightness) ■ Energy Level-Visible light- 1electron volt/photon. Soft x-ray portion- 50-100ev/photon 10
- 11. Resolving power ■ The capacity of an objective to render the outline of the object clear and distinct. ■ Depends on elimination of optical aberrations. ■ Optical aberrations – – Spherical aberrations – Chromatic aberrations 11
- 13. ■ Illumination: ■ Critical illumination-when the light source is focused by condensor in the same plane as the object, when the object is in focus. ■ The illuminating light consists of rays or waves that are nearly parallel to one another. ■ This quality of light is called partially coherent light. The ultimate resolving power of the microscope depends on the use of partially coherent illumination. 13
- 14. 14
- 15. MICROSCOPES CAN BE SEPARATED INTO SEVERAL DIFFERENT CLASSES What interacts with the sample to generate the image: 1. Light or photons (optical microscopes) 2. Electrons (electron microscopes) 3. Probe (scanning probe microscopes). Whether they analyze the sample via: 1. Scanning point (confocal optical microscopes, scanning electron microscopes and scanning probe microscopes) 2. Analyze the sample all at once (wide field optical microscope and transmission electron microscopes). 15
- 16. BRIGHT-FIELD MICROSCOPY ■ Dark objects are visible against a bright background. ■ Light reflected off the specimen does not enter the objective lens. Transmitted light enters 16
- 17. ADVANTAGES ■ Fewer adjustments ■ For transparent or colorless specimens, staining needed. ■ Can be used for live and mobile specimens such as bacteria – Hanging drop preparation ■ It is adaptable with new technology and optional pieces of equipment can be implemented with brightfield illumination to give versatility in the tasks it can perform. 17
- 18. Applications and Advances ■ Everywhere ■ Brightfield technique has been mated with cell imaging software to better perform tasks previously delegated to fluorescence microscopy. By using multiple focal levels the cell borders and nuclei can be located in cell populations. ■ The benefit of using brightfield illumination for this task is that it frees fluorescent channels in microscopes and eliminates distortions by the overlapping of the color emissions of the stains and the excitation of the fluorescing materials. 18
- 22. PRINCIPLE ■ Unstained/living cells & their components have refractive indices close to medium- (lack of contrast) ■ A specially sized disc, the patch stop blocks some light from the light source, leaving an outer ring of illumination. ■ Only light gathered is the reflected or diffracted by structures within the specimen. ■ This causes the specimen to appear as a bright image on a dark background, the contrast being reversed & increased. ■ Dark field permits the detection of particles smaller than the optical resolution that would be obtained in bright field, due to high contrast of scattered light. 22
- 23. 1 2 3 4 23
- 24. ADVANTAGES ■ The dark field microscopes divert illumination and light rays thus, making the SURFACE details of the specimen appear luminous. ■ Dark field light microscopes provide good results, especially through the examination of live blood samples. 24
- 25. Limitations ■ Prone to degradation, distortion and inaccuracies. ■ The preparation and quality of the slides can grossly affect the contrast and accuracy of a dark field image. ■ Liquid bubbles, dust will cause images degradation, decrease the contrast and details of the specimen. ■ Intense amount of light + scattered light rays, can cause glare and distortion. ■ Poor depth of field (internal structures not seen) 25
- 27. DEFINITION ■ Phase contrast microscopy is an optical illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. ■ The technique was invented by Frits Zernite in the 1930s for which he received the Nobel prize in physics in 1953 27
- 28. 28
- 29. 29
- 30. APPLICATION ■ Unstained/living biological specimens have little contrast with their surrounding medium.To see them clearly involves: – A-Bright field -Closing down iris diaphragm=decreased NA. – B-Dark field illumination, which enhances contrast by reversal, but often fails to reveal internal details. ■ The Phase Contrast microscopy overcomes these problems by controlled illumination using the full aperture & thus improving the Resolution. ■ The higher the refractive index of the structure, the darker it will appear against the light background, i.e. with more contrast. (in positive Phase contrast) 30
- 31. 31
- 33. 33
- 34. PRINCIPLE ■ Polarization can be achieved with the use of substance/crystals which allow vibration only in one plane & called birefringent. ■ Light entering a birefringent crystal such as calcite is split into two light paths, each determined by a different refractive index & each vibrating in one direction only, but at right angle to each other. ■ The higher the RI, the greater the retardation of the ray, so that each ray leaves the crystal at a different velocity. ■ The high RI ray =slow and low RI ray = fast. ■ There is also a phase difference between the rays, so that if they are recombined, interference occurs & various spectral colors are seen. 34
- 35. ■ Numerous crystals, fibrous structures (both natural & artificial), pigment, lipid, protein, bone & amyloid deposits exhibit birefringence. ■ Some substances & crystals can produce plane polarized light by differential absorption & give rise to phenomenon of Dichroism.These absorb the slow rays & are pleochoric (absorbing all colors equally) and are the most useful in microscopy. ■ Such crystals suspended in thin plastic films & oriented in one direction have replaced bulky Nicol Prism. ■ Weak birefringence in biological specimen is increased by addition of dyes or impregnating metals, in a orderly linear alignment e.g. amyloid fibrils. 35
- 36. Applications 36
- 37. DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPE ■ In this complex form of polarised light microscopy two slightly separate, plane polarised beams of light are used to create a 3D-like image with shades of grey. ■ Wollaston prisms situated in the condenser and above the objective produce the effect, and additional elements add color to the image. ■ Interpretation of hills and valleys.The height of a "hill" (e.g. the nucleus) is a product of both the actual thickness of the feature (i.e. ray path length) and its refractive index. ■ Discovered by Nomarski and de Senarmont. 37
- 38. Advantages ■ A primary advantage of differential interference contrast over phase contrast is the ability to utilize the instrument at full numerical aperture without the masking effects of phase plates or condenser annuli, which severely restrict the size of condenser and objective apertures.Axial resolution improved ■ Reduced halo/shade off artifacts ■ Depth of field and optical sectioning ■ Can be applied to both stained and unstained specimens. 38
- 39. 39
- 40. 40
- 41. Limitations ■ Expensive setup ■ Not actual 3D 41
- 42. FLUORESCENT MICROSCOPY ■ This method is used for demonstration of naturally occurring fluorescent material and other non- fluorescent substances or micro- organisms after staining with some fluorescent dyes e.g. Mycobacterium tuberculosis, amyloid, lipids, elastic fibres etc. ■ UV light is used for illumination. 42
- 43. ■ Fluorescent Substances absorb UV light (high energy) and emit visible light(low energy). ■ Cells may be stained with fluorescent dyes (fluorochromes). 43
- 44. WHY FLUORESCENCE MICROSCOPY? ■ In all types of microscopes, cell constituents are not distinguishable, staining helps but not totally. ■ In fluorescent microscopy, various fluorescent dyes are used which gives property of fluorescence to only specific part of the cell and hence it can be focused. ■ Fluorescent microscopy depends upon illumination of a substance with a specific wavelength (UV region i.e. invisible region) which then emits light at a higher wavelength (visible region). 44
- 45. PRINCIPLE ■ When certain compounds are illuminated with high energy light, they then emit light of a different, lower frequency.This effect is known as fluorescence. ■ Often specimens show their own characteristic auto-fluorescence image, based on their chemical makeup. ■ The key feature of fluorescence microscopy is that it employs reflected rather than transmitted light, which means transmitted light techniques such as phase contrast and DIC can be combined with fluorescence microscopy. 45
- 46. COMPONENTS ■ Typical components of a fluorescence microscope are: – the light source (xenon arc lamp or mercury- vapor lamp), – the excitation filter, – the dichroic mirror and – the emission filter. 46
- 47. STAINING ■ Many different fluorescent dyes can be used to stain different structures or chemical compounds. ■ One particularly powerful method is the combination of antibodies coupled to a fluorochrome as in immunostaining. (Immunofluorescence) ■ Examples of commonly used fluorochromes are fluorescein or rhodamine. 47
- 48. FUNCTIONING ■ A component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore. ■ The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). ■ Fading? –Photobleaching, Quenching? 48
- 50. Advantages ■ Unlike confocal microscopes, multiphoton microscopes do not contain pinhole apertures ■ The longer wavelength, lower energy (typically infrared) excitation lasers of multiphoton microscopes are well-suited to use in imaging live cells as they cause less damage than short-wavelength lasers typically used for single-photon excitation, so cells may be observed for longer periods with fewer toxic effects. ■ Infrared pennetration is more.(thicker specimen can be focussed) ■ Optical sectioning just like confocal ■ Disadvantage- more expensive 50
- 51. Fluorescence lifetime microscopy (FLIM) ■ The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample. ■ It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy ■ This allows contrast between materials with different fluorescence decay rates (even if those materials fluoresce at exactly the same wavelength) 51
- 52. Light sheet microscopy (LSM) 52
- 53. Total Internal Reflection Microscopy (TIRM) 53
- 54. 54
- 55. Stimulated emission depletion (STED) super resolution microscopy • The resolution enhancement is based on switching off the fluorescence of dye molecules by stimulated emission using intense laser light in the outer regions of the diffraction limited excitation focus. • Fluorescence from the remaining excited dye molecules in the center of the excitation focus is then detected and used to form the high resolution images 55
- 58. PRINCIPLE ■ In conventional microscopy, not only is the plane of focus illuminated, but much of the specimen above and below this point is also illuminated resulting in out-of-focus blur from these areas. ■ Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. ■ It enables the reconstruction of 3D structures from the obtained images. 58
- 59. 59
- 60. ■ Uses fluorochromes and a laser light. ■ The laser illuminates each plane in a specimen to produce a 3-D image. 60
- 61. APPLICATIONS IN CONFOCAL MICROSCOPY ■ The broad range of applications available to laser scanning confocal microscopy includes a wide variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues. ■ In addition, the growing use of new fluorescent proteins is rapidly expanding the number of original research reports coupling these useful tools to modern microscopic investigations. 61
- 62. ■ Confocal reflection microscopy can be utilized to gather additional information from a specimen with relatively little extra effort, since the technique requires minimum specimen preparation and instrument re-configuration. ■ In addition, information from unstained tissues is readily available with confocal reflection microscopy, as is data from tissues labeled with probes that reflect light. ■ Can be easily combined with fluorescence techniques. 62
- 63. Spinning disk confocal Microscopy (SDCLM) 63
- 64. ELECTRON MICROSCOPY ■ Electron microscope is a type of microscope that uses a particle beam of electrons to illuminate a specimen & create a highly-magnified image. ■ Co- invented by Germans, Max Knoll and Ernst Ruska in 1931 64
- 65. Parts ■ ELECTRON GUN ■ 2 types of guns are used in electron microscopy- Thermionic EmissionGun & Field Emission Gun. – Thermionic – Field Emission ■ ELECTROMAGNETIC LENS – An electromagnet designed to produce a suitably shaped magnetic field for focusing & deflection of electrons in electron optical instruments. – This field acts as a convex lens in case of electron microscope 65
- 66. CONDENSER LENS ■ First lens: (controlled by "spot size knob") largely determines the "spot size"; the general size range of the final spot that strikes the sample. ■ Second lens: (controlled by the "intensity/ brightness knob" changes the size of the spot on the sample; changing it from a wide dispersed spot to a pinpoint beam. 66
- 67. OTHER PARTS ■ The other parts include: - Condenser aperture, – Objective lens, – Objective aperture, – Selected area aperture(to examine diffraction patterns), – Intermediate lens – Projector lens. 67
- 68. TYPES There are 2 types of electron microscopes: ■ TRANSMISSION ELECTRON MICROSCOPE (TEM) ■ SCANNING ELECTRON MICROSCOPE (SEM) 68
- 69. Transmission Electron Microscopy (TEM) ■ Ultrathin sections of specimens. ■ Light passes through specimen, then an electromagnetic lens, to a screen or film. ■ Specimens may be stained with heavy metal salts. 69
- 70. PRINCIPLE ■ This transmitted portion is focused by the objective lens into an image. ■ The Objective & SelectedArea metal apertures restrict the beam. ■ The image is passed down the column through the intermediate and projector lenses, being enlarged all the way. ■ The image strikes the fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera to generate image. 70
- 71. ■ The darker areas represent areas that fewer electrons were transmitted (thicker or denser).The lighter areas represent areas that more electrons were transmitted (thinner or less dense) ■ Transmission electron microscopes produce two- dimensional, black and white images. ■ Resolution limited by spherical and chromatic aberration, but a new aberration correctors have limited these aberrations. 71
- 72. 72
- 73. Applications 73
- 74. Scanning Electron Microscopy (SEM) ■ An electron gun produces a beam of electrons that scans the surface of a whole specimen. ■ Secondary electrons emitted from the specimen produce the image. 74
- 75. ■ The1st SEM debuted in 1938 byVon Ardenne with the first commercial instruments out around 1965. ■ Unlike theTEM, where the electrons in the primary beam are transmitted through the sample, the SEM produces images by detecting secondary electrons which are emitted from the surface due to excitation by the primary electron beam. ■ In the SEM, the electron beam is scanned across the surface of the sample in a raster pattern, with detectors building up an image by mapping the detected signals with beam position. 75
- 76. 76
- 77. TEM vs SEM ■ Transmitted electrons ■ 2D imaging ■ Specimens should be very thin sections (10nm) ■ Whole sample at a time ■ Cannot tell surface contours ■ Scattered electrons ■ 3D Imaging ■ Thicker sections can be viewed ■ Part of sample ■ Surface contours 77
- 78. SPECIMEN FIXATION ■ Fixation- 4% glutaraldehyde at 4°C for 4 hours. ■ Dehydration - Organic solvents such as ethanol or acetone for SEM specimens or infiltration with resin and subsequent embedding forTEM specimens. ■ Embedding - Resin (for electron microscopy) such as araldite or LRWhite, which can then be polymerised into a hardened block for subsequent sectioning. 78
- 79. SPECIMEN ■ Sectioning -Typically around 90nm cut on an ultramicrotome with a glass or diamond knife. ■ Staining - uses heavy metals such as lead and uranium to scatter imaging electrons and thus give contrast between different structures 79
- 80. Limitations ■ Very expensive to buy and maintain. ■ Require extremely stable high voltage supplies, extremely stable currents to each electromagnetic coil/lens, continuously-pumped high/ultra-high vacuum systems and a cooling water supply circulation through the lenses and pumps. ■ As they are very sensitive to vibration and external magnetic fields ■ Proper training needed. ■ Specimen preparation (dead) ■ Grayscale images 80
- 81. Scanning Probe Microscopy (SPM) Developed in 1981, Gerd Binnig and Heinrich Rohrer, Nobel Prize in 1986 Scanning tunneling microscope – steady current (tunneling current) maintained between microscope probe and specimen – up and down movement of probe as it maintains current is detected and used to create image of surface of specimen Disadvantages: - Difficult alignment. Extremely Sensitive to surface conditions -Thermal drifts, local changes in barrier height affect force measurements But it opens the idea to develop a wide variety of SPM techniques. Atomic force microscope 81
- 82. Atomic Force Microscopy (AFM) 82
- 83. Cantilever modes ■ Contact mode (left): the deflection of cantilever is kept constant . ■ Non - contact mode (right): the tip is oscillated at the resonance frequency and the amplitude of the oscillation is kept constant . ■ Tapping mode : somewhere between the contact and non - contact mode. 83
- 84. Scanner types ■ Constant Height (of Scanner): ■ spatial variation of the cantilever deflection is used directly to generate the topographic data set because the height of the scanner is fixed as it scans. ■ Constant Force ■ In this mode, the image is generated from the scanner’s z – motion, with force constant. 84
- 86. Applications 1.Used in metallurgy 2.Examination of cultures in flat bottom dishes 3.Micro dissection 4.Aquatic specimens 5.Examination of parasites 6.Observation of agglutination in serology 86
- 87. IMAGE ANALYSERS AND MORPHOMETRY ■ In these techniques, microscopes are attached to video monitors and computers with dedicated software systems. ■ Microscopic images are converted into digital images and various cellular parameters (e.g. nuclear area, cell size etc) can be measured.This quantitative measurement introduces objectivity to microscopic analysis. 87
- 88. TELEPATHOLOGY (VIRTUAL MICROSCOPY) ■ It is the examination of slides under microscope set up at a distance.This can be done by using a remote control device to move the stage of the microscope or change the microscope field or magnification called as robobic telepathology. ■ Alternatively and more commonly, it can be used by scanning the images and using the highspeed internet server to transmit the images to another station termed as static telepathology. ■ Telepathology is employed for consultation for another expert opinion or for primary examination 88
- 89. Still Newer technologies (The sub diffraction techniques) ■ Time lapse ■ Deconvolution microscopy ■ FRET (fluorescence resonant energy transfer)- TIRM utilises this. ■ BRET (bioluminescence resonant energy transfer) ■ FRAP (fluorescence recovery after photobleaching) ■ BiFC (biomolecular fluorescence complementation). 89
- 90. Summary ■ Microscopic study of bacteria ■ Morphology of bacteria ■ Study of Nuclear components ■ Physiology of bacterial cells ■ Protein interactions, diffusion properties ■ Cell membrane receptor study 90
- 91. THANKYOU 91
Editor's Notes
- Good evening everyone. The topic of my seminar today is---
- This figure shows Scale of micro organisms and what all can be covered with what microscope. Light microscopy- 200nm LR with useful magnificn- below 2000x Electron - LR- 0.2 nm with 10*5 - 10*6 useful magnification
- Robert Hooke- used compound microscope to 1st view microbes. But of low quality. He published his findings in micrographia. Leeuwenhoek was the 1st one to see single celled life forms from a high quality simple microscope. Animalcules- a microscopic animal
- Light is refracted (bent) when passing from one medium to another having different indices. refractive index-a measure of how greatly a substance slows the velocity of light direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface focus light rays at a specific place called the focal point distance between center of lens and focal point is the focal length short focal length more magnification
- I will touch upon why oil is used in oil immersion.
- Magnification- Degree of enlargement ---no. of times the length, breadth or diameter, of an object is multiplied. Resolution-Ability to reveal closely adjacent structural details as separate and distinct Contrast- object stands out from the background. Most specimens are transparent at visible wavelengths. Empty Magnification.
- Limit of res- The min distance between two visible bodies at which they can be seen as separate and not in contact with each other. NA- measure of how much light can be collected by an optical system VIBGYOR- wavelength increases
- working distance-- distance between the front surface of lens and surface of cover glass or specimen lower the working distance= more is the light collected
- Energy is directly proportional to freq (1/wavelength) 1 wavelength = 1 crest + 1 trough Amplitude/Intensity of light wave= height of the crest or trough
- Human eye doesn’t see much beyond 700nm(deep red). The Amplitude (i.e. brightness): Decreases as light gets further from source because of absorption in media it passes
- Spherical A- Light rays hitting periphery are more refracted. Chromatic A- White light of passing through simple lens, each wavelength will be refracted to different extent. Blue brought to a shorter focus than red, results in a un-sharp image with color fringes Corrected with Achromat or Apochromat lens (convex and planoconcave doublet)
- Condensor= condenses light, so that all light passes to the specimen Coherence= explain with next image.
- Optical mic includes- light, fluorescence, UV and all
- Resolution is determined by certain physical parameters like wavelength of light and light generating power of the objective & condenser lens. Higher N.A =Better light generation=Better Resolution, Shorter the Wavelength =Better Resolution.
- Fewer adjustments to specimens.
- Applications- used everywhere. In routine practices
- Has single lens and simple assembly In pathology- for viewing gross structures. In parasitology- for veiwing structures of parasites. Not much use in bacteriology
- It is 2 lenses system. Condenser- filters out high wavelength rays, allows only short wavelength. Binocular, many headed microscopes, stereo microscope (binocular with both having different views giving 3d effect) vs trinocular(3rd for video feed)
- The aperture of the hollow cone of light is more than the aperture of objective lens- so the lens doesn’t catch that light. The lens only catches the diffracted or scattered light. Thus if no specimen= no light reaches the eyepiece
- High contrast- as we have black background.
- APPLICATIONS: Dark field microscopy is a very simple yet effective technique and well suited for uses involving live and unstained biological samples: Fig 1 Spirochaetes Fig 2 Schizont of vivax on dark field microscopy Fig 3 Fungi attacked by wbc Fig 4 Spirillum bacteria (type of motility)
- Images of internal cellular structure is inaccurate because of missing interference with undiffracted light.
- 2 additional components: Condensor annulus Phase plate- 2 types: positive and negative.
- S- surrounding wave, D= diffracted wave, P= resultant particle wave Positive – advances the surrounding by ½ wavelength or 90 degree if we talk in terms of vector, thus destructive interference, = dark objects, light surroundings Negative contrast plate does the opposite.
- diff refractive indices - create diff degrees of partial phase changes giving different shades of gray
- Closing down iris diaphragm = decreases the NA. producing diffraction effects & destroy the resolving Power of the Objective
- Fig 1: mouth epithelial cell with internal structures and bacteria. Fig 2: Mechanics of phagocytosis
- Light is a series of pulse of energy radiating away from a source & shown diagrammatically as sine curve, with wavelength & amplitude. Natural light vibrates in many directions but polarized light in only one direction. The polariser and analyzer are perpendicular to each other, thus cancel the light totally if no specimen is there. Result = dark
- Birefringent objects rotate the light rays & therefore appear bright in a dark background
- Two phenomenon detected in polarized light- Birefringence & Dichroism. Dichroic mirrors have now replaced prisms
- Apple green birefrengence on congo red staining of amyloid Gout crystals Geological sciences Limitations- bulky and heavy setups coz of prisms.
- Basically its same as polarising microscope , just 2 special condensor and objective prisms added. Basically its phase contrast plus polarising mic. As the prisms detect the phase changes and thus perform destructive and constructive interferences. Very good for Pseudo 3D effect..= tells shape/surface, (due to oblique illumination)
- Upper ones are DIC , lower ones are PC Epithelial cell- a,b Kidney tissue section- c,d
- Light source- mercury lamp- Disadv- 1. ultraviolet (UV) light that is highly deleterious to living biological samples. There is finite leak thru filters .2. the lifespan of a mercury discharge lamp is usually only 200–300 hours, and the intensity of these lamps decays progressively during this time. 3. gas discharge lamps require at least several minutes to reach an operating equilibrium after being turned on and lamp intensity can fluctuate during use. Therefore, once mercury discharge lamps are turned on, they are usually left on for hours to enable fluorescence measurements as needed without delay, thereby shortening lifetime. 4. these lamps generate a significant amount of heat and therefore introduce complications when used in a confined space. 5. mercury discharge lamps can explode, thereby damaging lenses and/or mirrors within the lamp housing. excitation filter- allows short wavelength light to pass thru, blocking long wavelength (thus high energy rays pass) Emission filter- blocks excitation rays(low wavelengths) but only allow selected emission rays of high wavelength. Dichroic mirror- can efficiently reflect excitation wavelengths while allowing emission wavelengths to pass through.
- Non protein : Xanthene derivatives: fluorescein, rhodamine, Oregon green, eosin, and Texas red Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine Squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes Naphthalene derivatives (dansyl and prodan derivatives) Coumarin derivatives oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole Anthracene derivatives: anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange Pyrene derivatives: cascade blue, etc. Oxazine derivatives: Nile red, Nile blue, cresyl violet, oxazine 170, etc. Acridine derivatives: proflavin, acridine orange, acridine yellow, etc. Arylmethine derivatives: auramine, crystal violet, malachite green Tetrapyrrole derivatives: porphin, phthalocyanine, bilirubin Nucleic acid dyes: acridine orange, ethidium bromide, thiazole orange, Mithramycin Proteins- GFP, YFP, RFP
- The general term for a reduction of fluorescence emission intensity is Fading, Photobleaching- Fluorescence emitted by almost all fluorescent dyes fades during observation. This phenomenon is called photobleaching or dye photolysis and involves a photochemical modification of the dye resulting in the irreversible loss of its ability to fluoresce. It is an irreversible phenomena. This is due to interaction with oxygen. Basically the fluorophores are consumed after each image forming process. Quenching refers to any process which decreases the fluorescence intensity of a given substance
- Instead of exciting the fluorophore with 1 photon, 2 photons with half the energy (twice the wavelength) can achieve the same excitation of the fluorophore. It uses a pulsed infrared laser that is focused to a spot in the specimen. The illumination intensity falls off rapidly above and below the focal point, and since the chance for absorption of 2 photons increases with the square of the intensity, the probability of absorption falls steeply away from the focal point. Thus, only fluorophores in a thin volume are excited. Whereas, in a confocal microscope, fluorophores out- side the focal plane are excited but prevented from being imaged by a pinhole, in a 2-photon microscope, absorption is confined to a small volume in the focal plane. Thus, photobleaching and phototoxicity is restricted to the plane of focus, and the sample as a whole is exposed to much less potentially damaging radiation, making the technique well suited to live-cell imaging.
- To study EGF receptor (epidermal GF) signaling FLIM has been used in clinical multiphoton tomography to detect intradermal cancer cells as well as pharmaceutical and cosmetic compounds
- The optical axes for light delivery and collection are oriented at an angle to one another, usually 90 degrees The use of sheet illumination leads to a large reduction in background fluorescence, which improves the attainable imaging depth by partially mitigating the loss of contrast with depth Sheet illumination also reduces photobleaching occurring at locations not being imaged, which in turn improves sample viability, such as for time-lapse imaging If the plane of illumination is rapidly scanned in tandem with the focal plane of the imaging lens, then light sheet fluorescence microscopy can enable fast functional volumetric imaging
- Principle is FRET (foster/fluorescence resonant energy transfer) Specimen Evanescent wave range Cover slip Immersion oil Objective Emission beam (signal) Excitation beam Only a few nm (100nm) of the sample just adjacent to the slide is excited.( in fluorescence mic, surrounding fluorophores also excited) In this only bound molecules excited.
- Is useful for examining events happening at cell membrane- receptors etc
- (STED) microscopy is one of the techniques that make up super-resolution microscopy. Developed by Stefan W. Hell 1994,[2] Nobel Prize in in 2014
- Highly improved axial resolution. The improvement in resolution is achieved by using two opposing objective lenses, which both are focused to the same geometrical location Basically we are doubling the amount of light captured (NA)
- Most popular among confocal is CLSM-confocal laser scanning mic.
- So basically the illumination is also pin point and the focus is also pin point.
- Fluorescent proteins like GFP
- Optical Sectioning.
- With CLSM(confocal laser scaning microscopy), there is a trade-off between image resolution and speed. Because one point is scanned at a time, there is time skew between first and last points of the scan. To compensate for the brief illumination of each pixel, an intense laser beam is required, and if the specimen is dynamic the time skew can lead to errors in observation. (SDCLM) overcomes this problem by exploiting the multiplex principle- light is detected at multiple points simultaneously – faster and no time skew, good for dynamic scans
- Electrons have very low wavelngth.
- Thermionic: Electrons emitted from heated filament ( tungsten, Lanthanum Hexaboride). Most common, cheap & ultra high vacuum not required. Field Emission: Strong electron field used to extract electrons from filament. High vacuum needed
- Intermediate lens- magnifies initial image formed by objective lens Projector- to project electron beam onto the eyepiece
- SEM- Detailed structure of Ecoli Structures of parasites studied with it Ultrastructure of cells
- Fixation has several goals: (1) to stop metabolism; (2) to fix structures of organelles and molecules in their current position; and (3) to make material accessible and stable during further processing. Fixation - In chemical fixation for electron microscopy, glutaraldehyde is often used to crosslink protein molecules and osmium tetroxide to preserve lipids.
- Glass knives can easily be made in the laboratory and are much cheaper than diamond, but they blunt very quickly and therefore need replacing frequently. By staining the samples with heavy metals, electron density is added to it which results in there being more interactions between the electrons in the primary beam and those of the sample, which in turn provides us with contrast in the resultant image.
- Need special housings Vaccuum needed as the molecules that make up air would scatter the electrons. This means that the samples need to be specially prepared by sometimes lengthy and difficult techniques to withstand the environment inside an electron microscope.
- Scanning probe microscopy (SPM) is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen. SPM was founded in 1981, with the invention of the scanning tunneling microscope, an instrument for imaging surfaces at the atomic level. It has countless types based on different types of contact force-dynamics used. Electrostatic, electrochemical, photon, photothermal, thermal, fluid force, chemical force, ballistic electron emission, conductive atomic force etc Popular is AFM
- Uses a diamond and metal probe that scans surface of specimen. Higher resolving power than electron microscopes No special specimen preparation required. Used to observe structure and surface of biomolecules.
- Contact has advantages over- dust/water droplets are ignored while non contact cannot ignore them.
- Constant force preferred for most applications. Applications- for atomic level structuring.. (biofilms) Also it gives great data about the z axis.
- Light source above and objective below. This microscope model is a very good choice if your sample to be viewed is in suspension or is very large and heavy.
- Telepathology is the practice of pathology at a distance. It uses telecommunications technology to facilitate the transfer of image-rich pathology data between distant locations for the purposes of diagnosis, education, and research. 3 types :static image-based systems(images only), real-time systems (video feed), and virtual slide systems(whole virtual slide is constructed and shared) Uses: intraop pathologist consultation without on site pathologist.
- Optical microscopy is diffraction limited- means limited by the diffraction of light. Sub diffraction techniques- overcome these. – STED(stim emission depletion mic., FRET, etc) Timelapse- shots are taken in long intervals and combined in a video form. Deconvolution microscopy consists of acquiring a stack of images separated by a small interval (0.1 to 0.4 mm) in the z-axis. An algorithm based on the PSF (point spread function) of the optical system-the intensity profile created by a point- then uses a Fourier transform deconvolution function to deblur the images acquired.(stitching)- It does not use laser as light source still quality is comparable to confocal without the photobleaching. FRET occurs when a donor molecule is excited by a photon of a specific wavelength and transmits its energy through a non-radiative mechanism to the other molecule, the acceptor, which in turn looses its energy by emission of a fluorescent photon at a lower wavelength. The efficiency of this depentednt on 1/r6 (r= distance r between the two molecules and is proportional to the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor and the relative orientation of their dipole moments. = spectroscopic ruler In BRET (bioluminescence resonant energy transfer), the donor photon is provided by a chemical reaction, and thus light excitation is not necessary. Fluorescence recovery after photobleaching (FRAP) is used to assess the mobility or to obtain the diffusion coefficient of a fluorescence-emitting protein in cells BiFC- To detect interaction between two proteins ,GFP is cut in two halves, and each is fused to one of two proteins. If these proteins interact, the two GFP pieces will be close enough to reconstitute the protein & produce fluorescence.
- Specific to bacteriology: we can do Microscopic study: Bright field, dark field etc Morphology: electron M, AFM, fluorsecent, confocal Nuclear components: Electron M Physiology: TEM, x ray spectroscopy Protein interactions, diffusion properties – FRET, BRET, FLIM Cell membrane receptor study- Total internl reflection mic.