microscopy Flashcards
Cynvenio instrument
-designed to analyze and detect rare cancer cells in blood samples by isolating CTC’s and cell-free DNA from the blood
Vortex instrument
-captures CTC’s in a microfluidic vortex, WBC’s/RBC’s move faster, CTC’s are left behind and collected/counted
CellInsight CX5 instrument
-allows analysis of large number of cells under different conditions, detailed info about cell behavior & interactions
Bright Field Microscopy
-used by pathologists
-sections of fixed tissue observed
-paraffin sections vs cryosections
Paraffin sections
-great morphology but long processing
-embedded in wax
Cryosections
-poor morphology (preserves tissue in nat. state) but fast processing
-no paraffin, freezes tissue, cut sections then stains
Phase Microscopy
-used by cell culture biologists
-looks at living cells (unstained, colorless, thin)
-manipulates light to create contrast
Nomarski/DIC microscopy
-living cells, gives 3D like image of cells
-used for single cell electrophysiology & patch clamping
single cell electrophysiology
technique used to measure the electrical activities of individual excitable cells (neurons)
Patch Clamping
measure ion flow through channels, used to understand how channels can be influenced by hormones, etc.
-inside-out (how intracellular ligands activate channels)
-outside-out (how extracellular solutions affect ion channels)
Darkfield Microscopy
-used by microbiologists
-illuminates small structures within cells (mitochondria, lysosomes)
-bright image on dark field
Polarizing light microscopy
-great for specimen with highly ordered structures
-polarized light shines upward on cell that contains moving liquid
point scanning confocal microscopy
-revolutionary, governed by abbe’s law but increases theoretical limit of resolution
-monochromatic lasers, confocal pinholes, point by point image scanning
point scan. confocal microscopy advantages & disadvantages
advantages: less stray marks on image, optical sections of an image, can use mult. dyes at once
disadvantages: uses fluorochromes which are subject to photobleaching
spinning disk confocal microscopy
good for living cells since it captures faster, lower laser intensity means less heat
vivascope
-confocal imaging system
-used by dermatologists for handheld skin diagnoses
-optically sections through skin, non invasive
fluorochromes
dyes that emit light when excited
vital fluorescence microscopy
-uses fluorochromes to measure changes in cell behavior
-dye and AM group attached and brought into the cell. the cell separates the AM and dye causing the dye to fluoresce
vital fluorescence- calcein AM
-calcein with AM makes calcein membrane soluble, dyes cell green
-live-dead assay: if cell dies/membrane is damaged, calcein will leave and propidium iodide will stain the nucleus
vital fluorescence- FLUO3-AM Ca conc.
-level of fluorescence indicates calcium concentration within living cells
microspectrofluorometry
-allows for quantitative measure of fluorescence strength
-intensity appears as different colors
FRAP
-Fluorescence Recovery After Photobleaching
-uses photobleaching as a tool to examine the lateral fluidity of proteins
-1. laser is targeted on one spot of the cell causing it to fluoresce, spot becomes photobleached
-2. if the protein in that spot is mobile, then the cell will be filled in by other material, photobleaching goes away
Lucifer Yellow
-intracellular injection, used to trace neurons in vivo (living organism)
-can indicate electrical connectivity between non neural cells too
-must use some type of fluorescent dye that is membrane insoluble and easy to diffuse
fluorescence immunocytochemistry
-use of antibodies with fluorochromes to identify proteins in cells
-antigen= POI
-epitope= area of antigen the antibody binds to
-antibody
fluorescent immunocytochemistry terms
antibody specificity: antibody only binds to the intended protein
antibody affinity: the strength of binding to the protein
direct technique: antibody has a fluoro attached & lights up cell when in contact w protein
indirect technique: antibody is attached to another antibody containing fluoro, better because direct often sacrifices specificity
plate reading spectrofluorometers
-machines that can sum the fluorescent strength of a signal from a large group of cells, average out the emitted wavelength from fluorochromes
polyclonal antibodies
-come from several B cells
-1. inject an antigen into living animal, animals b-cells will make polyclonal antibodies
-2. get serum from animal containing antibodies
monoclonal antibodies
-fuse b-cells and myeloma cells together to make them immortal, combination is a heterokaryron cells with two nuclei that fuse together
-resulting cell can infinitely produce mAbs that are identical
-clinical applications: cancer, cardiac disease, immune diseases, infections
ELISA
-Enzyme Linked Immunoosorbent Assay
-used to quantify amount of POI
-have a control and experimental group, isolate POI
necrosis
pathological cell death
-outside source like poison (ricin), cell expands then blows up
apoptosis
programmed cell death
-how most cancer therapies work, it shrinks
-Annexin V tags apoptic cells and results in death, detects phosphatidylserine
GFP (green fluorescent protein)
-originates from jellyfish
-reporter molecule
-protein expression:
–isolate the gene of interest, take GFP sequence and turn it into a chimera gene by combining w GOI
–transfect the chimera DNA into a living cell, if GFP is expressed then GOI is also expressed
reporter molecule: regulated marker
expression is controlled by specific regulatory element (off/on)
reporter molecule: constituent marker
always on
FRET
-Forster Resonance Energy Transfer
-can determine the proximity of 2 POI
-pair of fluorescent proteins with similar wavelengths where the energy is transferred
-ability to indicate ligand-receptor in living cells
-uses proteins in GFP family
-can be used to identify phosphorylation
FRET biosensors
calmodulin- can detect changes in intracellular calcium with time
ionomycin- calcium transporter across cell membrane
autoradiography
ability to trace a molecule or process in a cell with a radioactive probe
-cell division with 3H-thymidine
–probe enters cell & film is layered over cells, spots appear on film when hit w radiation indicating s-phase occuring
FISH
-Fluorescence in Situ Hybridization
-in situ=inside cell
-label DNA or mRNA by using a complimentary fluorescent-tagged oligonucleotide
-application:diagnostics; unknown cell type and want to know does it have the gene for HPV
-positive control, negative control, unknown
intracellular injection- single cell microinjection
somatic cell nuclear transfer (swapping nuclei), can only do one cell at a time
intracellular injection- electroporation
injects many cells at once, electric plates make a charge difference & molecules diffuse into cells, then it is turned off
intracellular injection- liposomes & nanoparticles
several cells at once, never 100% effective
-liposomes: small body made of lipids containing desired molecule, binds to cell membrane
-nanoparticles: designed to bind to molecules of interest you want to introduce into the cell
intracellular injections- viral transfection
very good for research, not great for clinical applications. uses viruses to transport genes
transmission electron microscopy (TEM)
-very thin sections (50-90 nm)
-adheres to abbe’s equation
-faster electron stream yields better resolution, electrons travel through specimen
scanning electron microscopy (SEM)
-scans image from the outside
-fix cells then spray metal on top of them, viewing emissions of secondary electrons
-lower resolution than TEM
plastic thin sectioning
-select tissue of interest
-fixation
-dehydration
-infiltration of epoxy plastic
-ultra-thin sectioning w an ultra microtone
-staining with heavy metals that reflect electrons (lead, uranium)
freeze fracture
-cells are frozen then fractured with razor blade
-spray w platinum & carbon to stabilize
ultrastructural immunocytochemistry
-uses gold particles indirectly attached to mAbs
-allows for double labeling with different gold sizing
laser capture microdissection microscope
takes sections of a single cell
atomic force microscopy
no lense, a laser puts pressure on a node that outlines a sample
two-photon microscope
-thicker and deeper tissue imaging
-uses light more efficiently leading to less photo toxicity
scanning tunnel microscope
-first to image native DNA & see structure
-not useful for cell structure, but great potential for imaging biological molecules w/o problems of heat generation
super resolution microscopy
- not diffration limited
-many different forms
