Light Microscopy - Summary - PDF

 The Light Microscope many types – bright-field microscope – dark-field microscope – phase-contrast microscope – fluorescence microscopes are compound microscopes – image formed by action of 2 lenses  Lenses and the Bending of Light Light is refracted (bent) when pass...

 The Light Microscope many types – bright-field microscope – dark-field microscope – phase-contrast microscope – fluorescence microscopes are compound microscopes – image formed by action of 2 lenses  Lenses and the Bending of Light Light is refracted (bent) when passing from one medium to another 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  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 – short focal length more magnification The Bright-Field Microscope Produces a dark image against a brighter background Parfocal microscopes remain in focus when objectives are changed Parcentral ?: Total magnification – product of the magnifications of the ocular lens and the objective lens Field of View  Bright-Field Common multipurpose microscope for live and preserved stained specimens, specimen is dark and field is white/bright Magnification: 2000X Resolution: 0.2um (200nm)  Microscope Resolution Ability of a lens to separate or distinguish small objects that are close together Wavelength of light used is major factor in resolution shorter wavelength  greater resolution 200nm  Working Distance Distance between the front surface of lens and surface of cover glass or specimen What is happening to working distance and field of view as you increase the magnification?  The Dark-Field Microscope Produces a bright image of the object against a dark background Used to observe living, unstained preparations Magnification: 2000X Resolution: 0.2um The Phase-Contrast Microscope Enhances the contrast between intracellular structures having slight differences in refractive index Excellent way to observe living cells Magnification: 2000X Resolution: 0.2um The Fluorescence Microscope Exposes specimen to ultraviolet, violet, or blue light Specimens usually stained with fluorochromes Shows a bright image of the object resulting from the fluorescent light emitted by the specimen DNA stained blue with DAPI. Microtubules and centromeres stained with fluorescently tagged antibodies Immunofluorescence  The Differential Interference Contrast Microscope Creates image by detecting differences in refractive indices and thickness of different parts of specimen Excellent way to observe living cells  Preparation and Staining of Specimens Increases visibility of specimen Accentuates specific morphological features Preserves specimens  Dyes and Simple Staining Dyes – make internal and external structures of cell more visible by increasing contrast with background – have two common features chromophore groups –Chemical groups give dye its color ability to bind cells by ionic, covalent, or hydrophobic bonding  Two Types of Ionizable Dyes Basic Dyes: Methylene blue, basic fuchin, crystal violoet, safranin, malachite green-positively charged (Positive Staining) Acid Dyes: Eosin, rose Bengal,Nigrosin and acid fuchin- possess, India Ink, negatively charged group (Negative Staining) *Bacterial Cell Surface has net negative charge  Smear A thin, uniform film of bacterial growth on a glass slide in order to proceed with further staining for microscopic examination.  Fixation Process by which internal and external structures are preserved and fixed in position Process by which organism is killed and firmly attached to microscope slide – heat fixing preserves overall morphology but not internal structures – chemical fixing protects fine cellular substructure and morphology of larger, more delicate organisms  Dyes and Simple Staining Simple staining –a single staining agent is used –basic dyes are frequently used dyes with positive charges e.g., crystal violet  Differential Staining Divides microorganisms into groups based on their staining properties –e.g., Gram stain –e.g., Acid-fast stain  Gram Staining Most widely used differential staining procedure Divides Bacteria into two groups based on differences in cell wall structure  Gram Staining 1. The smear is heat fixed and then first stained with crystal violet, which stains all cells purple 2. Iodine is used as a mordant to increase interaction between the cells and the dye 3. Ethanol or acetone is used to decolorize 4. Safranin is then added as a counterstain to turn the gram-negative bacteria pink while leaving the gram-positive bacteria purple  Acid-fast staining Particularly useful for staining members of the genus Mycobacterium e.g., Mycobacterium tuberculosis – causes tuberculosis e.g., Mycobacterium leprae – causes leprosy – high lipid content in cell walls is responsible for their staining characteristics ACID FAST STAINING  Staining Specific Structures Negative staining: Widely used to visualize diffuse capsules surrounding the bacteria; those capsules are unstained by the procedure and appear colorless against a stained background *Acid dyes are used, such as Nigrosin or India Ink  NEGATIVE STAINING Morphology Bacterial Capsule  Spore Staining: Double staining technique by which bacterial endospores are left one color and the vegetative cell a different color Flagella Staining: Mordants are applied to increase the thickness of flagella to make them easier to see after staining  Electron Microscopy Beams of electrons are used to produce images Wavelength of electron beam is much shorter than light, resulting in much higher resolution  Specimen Preparation Analogous to procedures used for light microscopy For transmission electron microscopy, specimens must be cut very thin Specimens are chemically fixed and stained with electron dense material  Other Preparation Methods Shadowing – coating specimen with a thin film of a heavy metal Freeze-etching – freeze specimen then fracture along lines of greatest weakness (e.g., membranes)  The Transmission Electron Microscope Electrons scatter when they pass through thin sections of a specimen Transmitted electrons (those that do not scatter) are used to produce image Magnification: 1000000X Resolution: 0.5nm The Scanning Electron Microscope Uses electrons reflected from the surface of a specimen to create image produces a 3-dimensional image of specimen’s surface features Magnification: 100000X Resolution: 10nm  Newer Techniques in Microscopy confocal microscopy and scanning probe microscopy have extremely high resolution can be used to observe individual atoms Figure 2.20