Microscopy Lecture Notes PDF

The Study of Microbial Structure: Microscopy and Specimen Preparation 1  Microscopy microorganisms range in size from – Smallest=nanometers (nm) – Largest=protists (μm). 2 3  Lenses and the Bending of Light Refraction= bending o...

The Study of Microbial Structure: Microscopy and Specimen Preparation 1  Microscopy microorganisms range in size from – Smallest=nanometers (nm) – Largest=protists (μm). 2 3  Lenses and the Bending of Light Refraction= bending of light when passing from one medium to another Refractive index=measure of how greatly a substance slows the velocity of light Direction and magnitude of bending is determined by the refractive indices of the two media forming the interface (i.e., glass and air) Glass has a higher refractive index than air 4  Lenses focus light rays at a specific place called the focal point distance between center of lens and focal point is the focal length strength of lens related to focal length 5  The Light Microscope Many varieties – bright-field microscope – dark-field microscope – phase-contrast microscope – fluorescence microscope – confocal microscope Modern microscopes are all compound microscopes 6  The Bright-Field Microscope Both stained and unstained produces a dark image against a brighter background has several objective lenses – parfocal microscopes total magnification – product of the magnifications of the ocular lenses and the objective lenses 7  Bright Field Microscope 8  Microscope Resolution Resolution (or resolving power) - ability of lens to distinguish small objects that are close together Wavelength of light used is a major factor in resolution shorter wavelength  greater resolution (blue light 450 – 500 nm can not resolve structures smaller than 0.2 um) 9  Numerical Aperature Numerical aperature – ability of the lens to gather light Refractive index – how much a substance bends a light ray The refractive index of air is 1.00 if we increase this by using immersion oil, we can increase the numerical aperature 10  Numerical Aperature Smaller working distances give better resolution – can better separate close objects because the light spreads out more 11  Using Immersion oil to increase refractive index Replacing Air with Immersion Oil 12 -working distance- distance between the surface of lens and the surface of cover glass or specimen when it is in sharp focus -Resolving power – ability to distinguish close objects as separate 13 The Dark-Field Microscope image is formed by light reflected or refracted by specimen produces a bright image of the object against a dark background used to observe living, unstained preparations 14  Dark Field Microscope Uses a hollow cone of light so that only light that has been reflected or refracted by the specimen enters the lens 15 Examples of Dark-Field Microscopy Treponema pallidum and Volvox 16  The Phase-Contrast Microscope Uses slight differences in refractive index and cell density Uses a hollow cone of light Cone of light passes through a specimen some is retarded (out of phase). Light passes through phase plate brining it back into phase excellent way to observe unstained, living cells 17  Phase-Contrast Microscope 18  Examples of Phase-Contrast Microscopy Pseudomonas sp., Amoeba, Paramecium 19  The Differential Interference Contrast Microscope (DIC) Similar to phase-contrast - creates image by detecting differences in refractive indices and thickness of different parts of specimen Developed out of the phase contrast: developed by George markoski? We don’t use a dark eld stop or a phase plate. Key feature is two prisms and also plane polarized light. Uses two beams of polarized light to create a 3D image of specimen excellent way to observe living cells – live, unstained cells appear brightly colored and three- dimensional 20  Differential Interference Contrast Microscopy Amoeba proteus Generate something pseudo3D. 21  The Fluorescence Microscope developed by O. Shimomuram, M. Chalfie, and R. Tsien exposes specimen to ultraviolet, violet, or blue light Short wavelength light. Will cause molecules in the uorocarbons to jump to the next energy state and as they go back down they emit light. specimens usually stained with fluorochromes (fluorescent dyes) shows a bright image of the object resulting from the fluorescent light emitted by the specimen has applications in medical microbiology and microbial ecology studies 22  Green Fluorescent Protein fused with Mbl cytoskeletal protein of Bacillus subtilis 23  Epifluorescence Microscopy Light that they emit is a longer wavelength light 24 No need to memorize the commonly used uorochrome 25  Immuno-Fluorescence ( refer to Wessner Toolbox 3.1 on p. 82) Not talking about staining with uorochromes. We’re attaching uorochrome to our antibodies. 26  Cells stained with fluorescent dyes Living cells (green) dead cells (red), Streptococcus pyogenes (antibody staining) Causative agent of strep throat Live death stain which could be useful to see if a compound works to kill a species. 27  Confocal Microscopy confocal scanning laser microscopy (CLSM) creates sharp, composite 3-D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface Take images across multiple planes to get a 3D image. Additional aperture to eliminate stray light and keep it in plane. Specimen is usually fluorescently stained numerous applications including study of biofilms We have microbial communities like bio lms, and they take on complex 3D structures which is why a confocal microscope is used. 28  Confocal Microscope 29  Confocal Microscopy Bio lms are hard to kill and live on medical devices like catheters. If you did only one plane it wouldn’t look at the levels of the bio lm and how well it was killed. 30  Preparation and Staining of Specimens increases visibility of specimen accentuates specific morphological features preserves specimens Can thicken up thin or small portions of an organism. Fixation techniques. 31  Fixation preserves internal and external structures and fixes them in position organisms usually killed and firmly attached to microscope slide Attach microorganisms to the slides – heat fixation – routinely used with bacteria and archaea We use with bacteria and archaea, small volume of liquid and a loop ful of the bacteria and then you move it over the very top part of the ame and evaporate the liquid and that then dries the organisms into place on the slide. Have to make sure you don’t burn your organisms. Preserves morphology inactivate enzymes, and there might be artifacts and break down proteins. – chemical fixation – used with larger, more delicate organisms Ethanol, etc… they help make our specimen insoluble, immobile, and inactive. There might be some artifacts still. 32  Dyes and Simple Staining dyes - Contrast - Have two common features chromophore groups ability to bind cells Help enhance speci c features, little appendages for example. They have to have a chromosphere group, a chemical group with conjugated double bonds. Have to have ability to bind to cells it could be covalent, hydrophobic, or ionic to bind. Majority I’d through ionic 33  Dyes and Simple Staining Dyes ionizable dyes have charged groups They will interact with negative components like DNA, cell – basic dyes have positive charges surface or amino acids with negative charges. Crystal violet, basic fusion, and a bunch of other dyes. – acid dyes have negative charges : used in negative staining we They have negative charges and interact with positively charged parts of the cells. One area use acid dyes is for negative staining. Negative dyes avoids membrane and thus stains background and not the organisms. simple stains – a single stain is used – can determine size, shape, and arrangement of bacteria 34 Crystal violet can stain E. coli. They kinda lump together and have some chains. Are rod shaped cells. Simple Staining Both basic stains 35  Differential Staining divides microorganisms into groups based on their staining properties – e.g., Gram stain – e.g., acid-fast stain differential stain also used to detect presence or absence of structures 36  Gram Staining (refer to Wessner Toolbox 2.1 p55) most widely used differential staining procedure Developed by Christian Gram, 1884 divides bacteria (but not archaea) into two groups 37  Gram Stain Steps Enhances interaction between dye and peptidoglycan If we are gram positive alcohol won’t be able to wash out the stain. 38 Safranin will help gram negative cells to be visualized.  Acid-Fast Staining particularly useful for staining members of the genus Mycobacterium Acid fast bacteria tend to be mycobacterium. If gram stain doesn’t seem to work u could try the acid fast. - High lipid content in cell walls (mycolic acid) - Uses high heat and phenol to drive basic fuchsin into the cells Typical counterstain is methylene blue for acid fast. 39  Differential Staining of Specific Structures endospore staining – exceptionally resistant to staining (e.g. Bacillus sp. and Clostridium sp.) Formed under starvation conditions. So you suspect they’re endospores you want to starve them then stain capsule stain used to visualize capsules surrounding bacteria (India ink or nigrosin) Host has hard time seeing bacteria if have capsules will do negative staining flagella staining – very thin and can only be seen with an electron microscope Stain if you’re interested in looking at agella or use electron microscope. Stain will enhance thickness of agella. 40 Examples of Differential Stains Negative staining Basic fuschion dye, and if pink then mycolic acid is there and has retained the dye. The bluer is because they didn’t have mycolic and picked up 41 counterstain of methylene blue. With light microscope and you want to look at stu that is smaller than 0.2 micrometers, you wont be able to resolve those things Resolution depends on Wavelength and Electron Microscopy aperture. For light microscope The best light microscope has a resolving limit of 0.2um (max. mag of 1500X) Electrons as source of illumination (resolution of 0.5 nm, max mag of 100,000X) allows for study of microbial morphology in great detail 42  Limits of Resolution Angstrom level of resolution, to see amino 43 acids and DNA  Light vs Electron Microscopy Rhodospirillum rubrum Internal detail, nucleotide area, internal membrane vesicle. If you want morphology you probably want to use light microscope. Light microscope brings you in very close and gives you very detailed structures. 44  The Transmission Electron Microscope (TEM) Electrons generated and focused on specimen by electromagnents TEM was rst electron microscope developed. Beaming electrons at specimen, using di erential electron scatter. Electrons scatter (gives darker appearance) when going through denser parts. As electrons pass through specimen they form an image Denser areas of specimen will scatter some electrons 45  Transmission Electron Miscroscope Beams the electrons down at sample 46  Comparison of Light Microscope and TEM 47 48 F Putting image on metal grid  Specimen Preparation for TEM analogous to procedures used for light microscopy specimens must be cut very thin If a specimen is very thick you wouldn’t get di erential electron scatter. specimens are chemically fixed and stained Staining using heavy metals, which will enhance the electron scatter and give better contrast. 49 Other TEM Preparation Methods negative stain-specimen spread out in a thin film with heavy metals Generate the same time of image as we saw in light microscope. – heavy metals do not penetrate the specimen but render dark background Coat metal Grid that the specimen is sitting on with heavy metals so background appears dark – used for study of viruses, bacterial gas vacuoles shadowing – coating specimen with a thin film of a heavy metal only on one side – useful for viral morphology, flagella, DNA 50  TEM Staining Methods Negative stain Shadowing, only half of specimen is coated in heavy metals, and the other half is left alone. 51 Other TEM Preparation Methods freeze-etching More for bacteria imaging – freeze specimen then fracture along lines of greatest weakness In liquid nitrogen, it helps reduce artifacts. Organism fractures along lines of greatest weakness tend to be membranes and then you can crack o the top to look at intracellular features – 3-D observation of shapes of intracellular structures Freeze etching is Combined with shadowing often – reduces artifacts 52  Freeze-Etching If ymm.name 53  Disadvantages of TEM Electrons can only penetrate very thin specimens If its thick it will probably just look dark. Usually gives only 2D image Specimens must be viewed under high vacuum Specimens are dead/artifacts 54  The Scanning Electron Microscope Electrons reflected from the surface of a specimen Only external structure visualized. Beaming electrons knocks o secondary electrons o the surface of the specimen. 3D image of specimen's surface features can determine actual in situ location of microorganisms in ecological niches We can take samples from environment to see what’s there, what shapes etc are in the location. – dried samples coated with a thin film of metal Enhance secondary electrons that get knocked 55 o.  SEM 56  SEM Mycobacterium tuberculosis 57  Electron Cryotomography Rapid freezing technique developed in the 1990s Came a little bit later then TEM and SEM. Freezing in Vitreous ice Tilt series created Creates 3D image composite. Provides extremely high resolution of ultrastructure 58  TEM vs Electron Cryotomography Caulobacter crescentus This is just one of the sections High resolution image from cryotomography. Slime layer Peptidoglycan layer Vacuole Darker dots are ribosomes. cant see ribosomes in TEM but can in Cryotomography) 59  Scanning Probe Microscopy scanning tunneling microscope (1980) – magnification 100 million times Foundations in quantum mechanics. We have very ne atomic tip, tunneling current between tip and specimen. Taking the ow of charge between tip and space in and converting it to image – steady current (tunneling current) maintained between microscope probe and specimen – up and down movement of probe maintaining constant current creates image of surface 60  Scanning Tunneling Microscopy of DNA 61  Scanning Probe Microscopy atomic force microscope -sharp probe moves over surface of specimen at constant distance We dont change distance so when there are thicker or denser parts of the space in there will be more force on the tip, and vice versa if the space in part is less dense. -up and down movement of probe as it maintains constant distance -Used to study surfaces that do not conduct electricity well 62  Atomic Force Microscope Can do live imagine. 63  Atomic Force Microscopy Aquaporin membrane protein 64