Microscopy and Cell Visualization Flashcards

Question

What is required for immunohistochemistry staining to occur?

Answer 1

the cells must be chemically FIXED and SLICED. common fixatives or preservatives are formaldehyde, glutaraldehyde, ethanol . Fixation will preserve shape, while the membrane is treated with DETERGENTS (create holes) to allow protein detecting antibodies to go inside cell. after fixation, tissues are embedded in harder material (ice wax, resin) and will be cut with MICROTOMES.

Answer 2

The shorter the wavelength, the higher the energy of em waves, higher frequency; longer the wavelength, the lower the energy of EM waves; gamma/violet has shortest wavelength and radio/red has longest wavelength

Answer 3

Light microscopy- uses beam of light to illuminate, work on dead or live cells, needs to be fixed, longer wavelength than EM. Electron microscopy- uses beam of electrons, only works on dead cells, expensive tool, shorter wavelength, higher resolution and magnification

Answer 4

TEM (transmission electron microscopy)- a beam of electrons is transmitted (passed through) (1 direction) a specimen to form an image. Uses electron gun and allows visualization of internal cell structures on tissue cross-sections. Must be thin tissues. SEM (Scanning electron microscopy)- scan the surface with a beam of electrons to detect the secondary e- reflected off the region (different directions). used to study Surface and shape of multicellular structures.

Answer 5

TEM- only have 2D images, internal structures, highest resolution (0.2 nm) SEM- 10 times lower resolution (0.2nm ) than TEM, 3D images, see surface of solid objects must be preserved, dried without shrinkage (critical point drying) and and be coated with gold or platinum.

Answer 6

Tissue sections must be very THIN (50 nm or 500 Angstrom). materials must be embedded in very hard (epoxy) RESINS, and cut with ultramicrotomes with diamond knives. Use microscopic COPPER grids to hold the samples and stain them with URANIUM and LEAD SALTS for enough contrast.

Answer 7

Electrons are both negatively charged particles and waves, hence they have a shorter wavelength and higher resolution.

Answer 8

method of visualizing SPECIFIC molecules using TEM and immunostaining with GOLD BEAD-CONJUGATED ANTIBODIES. process- thin sample cut, stained with antibody that will bind molecule of interest, then secondary antibody attached with gold beads attach to primary antibody, and e- dense gold particle seen under microscope as black dot, indirectly labeling molecule of interest.

Answer 9

Method of rapidly freezing a frozen bio sample in and making it solid, stuck in one place, in order to break apart (fracture) the sample and view the plane or surface, details of structure as it creates replica. It is very useful to image fluid structures like CELLULAR MEMBRANES,

Answer 10

method of using fluorescence to visualize internal structures of cells. highly specific, sensitive and reliable. process of absorbing light at excitation spectrum (e- to high energy state) and emitting light at lower wavelength, to lower energy state.

Answer 11

The part of the molecule that is responsible for its color and defines their fluorescent properties. They are all derived from proteins found in MARINE ORGANISMs(polyps, corals). The first chromophore was green fluorescent. specific color and technical characteristics.

Answer 12

GFP (Green Fluorescent Protein)- intrinsic fluorescent protein from Jelly fish that has greatly revolutionized biomed research. GFP absorbs blue light and emits green fluorescent light. GFP used for whole body imaging, highlight tumors, cancers, lipid. GFP has amino acids serine, tyrosine and glycine form chromophore. GFP co-discovered with fluorescent protein aequorin (emits light in presence of Ca+)

Answer 13

Immunofluorescence is method of antibodies conjugated to fluorophore to make a single protein fluoresce. normally requires FIXATION and MEMBRANE PERMEABILIZATION. label cells with precision.

Answer 14

allows proportional imaging of objects as small as 20 nm. Uses STED (Stimulated-emission Depletion) phenomenon- when excited fluorophore reaches photon that is closer in energy to normal photon emitted, it will prevent fluorophore from emitting original photon (emit lower energy photon that is eliminated in visual field). STED uses two layers to simultaneously excite an object (one beam inside another). enhances resolution (sharper image than confocal)

Answer 15

1. fluorescence not limited by light wavelength, since the light of molecule is not reflected. Object is source of light. 2. Higher contrast and sensitivity because the only fluorescence light is detected (not light from background surface)

Answer 16

FRET (Fluorescence or Foster Resonance Emission Technology)- used to detect protein-protein interactions in vivo with molecular precision when protein interacts with fluorescence fused protein, instead of emitting and being fluorescent itself, it will excite another fluorescent protein in close proximity (so it will fluoresce without direct excitation of additional light)

Answer 17

Fusion proteins used to visualize proteins in live cells. Fluorescence proteins may be light emitting indicators (sensors) to rapidly measure changes in intracellular ion concentration, pH and membrane potential.

Answer 18

FRET (Fluorescence or Foster Resonance Emission Technology)- used to detect protein-protein interactions in vivo with molecular precision when protein interacts with fluorescence fused protein, instead of emitting and being fluorescent itself, it will excite another fluorescent protein in close proximity (so it will fluoresce without direct excitation of additional light) Excite violet light and emit blue light- no interaction. If excite violet light and emit yellow light- there is an interaction .

Answer 19

method to achieve images of living things that are high in thickness. Simultaneously excites 2 photons with longer wavelength than emission and are lower in energy to penetrate deep tissues also creating less tissue damage and imaging farther in samples reduces out of focus emission, since only objects in focus have enough red light intensity to be emitted. 2 photons of low energy are coherent (same frequency) have same excitation of fluorophore that is achieve with one photon

Answer 20

``` Red, longer wavelengths are able to penetrate tissues, because wavelength that are equal or larger can bypass the object. However, Em waves of shorter wavelength (high energy) reflect better off objects. Near Infrared (far red) light can penetrate deeper in tissues in living organisms non-destructive form of red light can detect near infrared FP's that measure size of tumor and its location without harming organism. ```

Answer 21

Human naked eye- 0.2 mm or 200 um, Light microscopy- no smaller than 0.2 um or 200 nm. Super resolution microscopy- 20 nm. EM microscopy- 0.2 nm.

Answer 22

Human naked eye- 0.2 mm or 200 um (cells), Light microscopy- no smaller than 0.2 um or 200 nm; cells and organelles. Super resolution microscopy- 20 nm; cells-molecules. EM microscopy- 0.2 nm, cells- atoms.

Answer 23

Difference (shift) between excitation and emission and wavelengths maximum of absorption wave and max of fluorescence wave. Defines fluorescent emission occurs at longer wavelength than absorbed light.

Answer 24

Swelling and redness caused that occurs when histamine dilates (widen) blood vessels. Histamine is found in Basophilic granules and is a mediator of allergic and inflammatory responses.

Answer 25

most common WBC. neutral staining (neither hemtoxylin, nor eosin). They are part of innate immune system, nuclei are hematoxylin positve, and release cytokines at site of inflammation.

Microscopy and Cell Visualization Flashcards

(49 cards)