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Dark Specimen, bright viewing field requires staining
Pathologist, histologist, cytochemist
Iden
1. Bright field microscopy/compound light microscopy
isolate the tissue or organ, choose a fixative that cross-links protein (formaldehyde), remove water and the fixative, replacing it with ethanol, replace ethanol with xylene, replace with molten paraffin and let solidify, cut 10-15 micron sections, remove the wax by doing the above steps in reverse, and stain with dye
Typical protocol for bright field microscopy
A chamber that can maintain low temperatures, preserves frozen tissue samples that can then be sliced
1. Cryostat
Mohs surgery
What would a cryostat be used for?
bserves living cells, light changes speed when travelling through extracellular structures, suitable for single cells or thin tissue not thicque. Relies on constructive and destructive interference
1. Phase contrast microscope
cell biologists who want to use living cells and see the outer surface of cells
who would use phase contrast microscopy?
Splits light into two perpendicular components before going through the specimen and creating a interference pattern, used to look at organelles, this and phase contrast can be used in a time lapse
1. Nomarski/Differential Interference Contrast
cell biologists who want to use living cells/neurobiologists who want to position pipettes for intracellular injection of transmembrane voltage recordings
Who would use Nomarski/DIC
Used to study ionic currents of individual isolated living cells, or patches of cell membrane, sucks up a little of the cell
Single cell membrane activity
Patch clamp
Electrophysiology
who would use a patch clamp
Area around cells is dark, used to enhance contrast in unstained living samples, can photodegrade things
Increases contrast but not resolution
Polarizing light micro
i. Neurons are highly organized so no light penetration
Darkfield microscopy
monochromatic laser (diffraction limited), laser can be manipulated to study the specimen spot by spot, stray image is filtered using a confocal pinhole, total summed image can be displayed on a screen
characteristics of confocal microscopy
Images are taken through several holes simultaneously within a millisecond- good for dynamic, moving processes
Spinning Disk Confocal microscopy
faster, requires lower laser intensity, decrease in photobleaching, less heat generated, good for cells undergoing dynamic processes
Advantages of spinning sidk confocal microscope
Collects illuminated light through a pin hole, moves across the focal plane to get the full image, can shoot at different heights to make 3D image, slow and photobleachs
Very bright images, need dead cells
1. (point scanning) Laser-Scanning Confocal microscopy
Can photobleach, and not good for dynamic processes, heat affects the image and resolution- fine for regular work with fixed specimens
Problems with point scanning confocal microscope
Neurobioligists/muscle cell biologists. detects highly ordered parallel structures in a cell
Polarizing light microscope
has a polarizer above the light soure and analyzer above the objective lens. Both rotate to generate contrast. The polarizer filters light into a single plane and the analyzer determines if intracellular structures rotate the plane of polarized light
how does a polarizing light microscope work?
Can be ised for analyzing fibrosis in organs
Applications of polarizing light microscope
decreases stray image, can optically section a gel, can generate stereo images, can use colorimetric and fluorescent dyes, good for double and triple labelling
How is confocal microscopy better than light
absorbs light at one wavelength and re-emits that energy at another- used to visualize a structure/molecule
fluorescent dye
Absorbs light of one wavelength and emits at a longer wavelength, used to observe cells that have been stained with fluorescence
1. Fluorescence Microscopy
Photobleach cells in an area and see how long unbleached cells take to replace them, can view cell dynamics
50% of proteins are mobile
1. FRAP (fluorescence recovery after photobleaching)
study the mobility of the fluorescently labelled molecules in living cells (analysis of molecule diffusion, fluidity of membranes, and protein binding)
Applicaions of FRAP
Pair of fluorescent proteins with similar wavelengths where the energy is transferred, proportional to R^-6
Ability to indicate ligand receptor in living cells
FRET (forster resonance energy transfer)
Separate the two fluoroproteins by something undergoing a conformational change ause after the change they will be close enough to FRET
FRET biosensor
Used to detect and track interactions between proteins, and can measure distances between domains in a protein
What is FRET used for?
Used when fluroescene imaging on a thin focal plane adjacent to a surface where out of focus background must be minimized. Only the part right next to the surface is illuminated
1. TIRF (total internal reflection fluorescene) microscopy
Combines kinetic studies with spatial information in samples (investigating molecule trafficking in cytoskeleton assembly)
What can TIRF be used for?
Rhodamine, JC-1, PMF - Good for measuring mitochondrial activity
Calcium-AM & propI - live dead assay
FLOU-3 - measures intracellular calcium
Vital Fluorescent Dyes
acetoxymethyl ester group
What do most vital fluorescent dyes have
Designed to locate specific molecules in or on a cell using fluorochromes that are associated with antibodies
Fluorescence immunocytochemistry
Antibodies produced by a single clone of B lymphocytes and that are therefore identical in structure and antigen specificity- only bind to one epitope
monoclonal antibodies
Antibodies produced by injecting animals with a specific antigen. A series of antibodies are produced responding to a variety of different sites on the antigen. (bind to multiple epitopes)
polyclonal antibodies
fluorescent microscope/confocal microscope and plate reading spectrofluorometer
methods for studying vital fluorescent dyes in cells
good for single cells or portions of cells, but cannot average signals from many cells (generates an image, so it is qualitative)
fluorescent microscope/confocal microscope
Not a microscope, can average signals together, generates a fluorescence intensity level value (number, not an image)
Plate reading spectrofluorometer
Plate-based assay technique designed for detecting and quantifying antibodies using an immobilized antigen
Quantifies he amount of proteins inside and outside of cell
ELISA (enzyme linked immunosorbent assay)
detects antigen concentration in a sample (direct, indirect, or sandwich)
how does ELISA work
The primary antibody is directly conjugated to the fluorochrome
Direct IF
The primary antibody is not conjugated, and a fluorochrome conjugated secondary antibody is directed against the primary antibody and used for detection
Indirect IF
Qualitative images based on wavelength emissions of excitation
Microspectrofluorometry/Cytofluor
Antiboides made by identical immune cell parent "clones"
Polyclonal/monoclonal antibodies
Fluorescent proteins regulatory vs other
GFP, YFP, CFP as reporter molecules
Programmed cell death - annexin V
Non-programmed cell death - PI
Apoptosis/Necrosis/Annexin V/Propidium iodide
enters cell when PM loses integrity; binds DNA and emits fluorescence (stains the nucleus)
propidium iodide
Use of x-ray film to detect radioactive isotopes conjugated to things
3H thymidine stains DNA synthesis in dividing cells
3H leucine used to track protein synthesis
Autoradiography
technique that allows you to search for the presence of certain genes of mRNA in cells using a light or fluorescence microscope
in situ hybridization
Fluorescent probes that bind to only nucleic acids (chromosomes)
FISH (fluorescent in situ hybridization)
A complementary DNA probe is attached to a fluorescent molecule. The probe binds to the DNA of interest, allowing scientists to visualize where on the chromosome a specific piece of DNA is located.
How does FISH work
subtyping cervical carcinomas
Typical application of FISH
Single cell microinjection using a micropipette
i. Somatic cell nuclear transfer
Electroporation
i. Tiny holes in membrane to diffuse
Liposome and nanoparticles
Intracellular injection techniques (4 different types)
TEM transmits electrons through a specimen (resolution is .1 nm)
SEM scans the surface of a structure by electrons being reflected from the surface (resolution is 10 nm)
TEM vs SEM
Electron beam condensed and sent through specimen to the detector on the other side
Cannot be living bc its in a vaccum
Transmission electron microscopy
Similar to TEM but thicker sections can be used, increases the theoretical limit of resolution, but not the practical because a lot of heat is generated
High voltage electron microscopy
The focused beam of electrons scans across the sample
Beam doent penetrate sample
Scanning electron microscopy
Method of cutting specimens thin enough for TEM (fixation, dehydration steps, embed in plastic, ultramirotome, stain with heavy metals)
Plastic thin sectioning
LEad stains membranes, uranium stains everything else
Heavy metal stains
Governed by Abbe's equation and is diffraction limited. The wavelength of electrons can be changed by changing the accelerating voltage (faster electrons=short wavelength=higher resolution)
TEM resolution
Rapid freezing and cracked along the plane, designed to view the internal organization of membranes
Freeze fracture
Cells frozen, split with razor blade, coated with platinum, then carbon, creating a platinum replica
freeze fracture steps
Uses autoradiography to find receptor in the synapses
Ultrastructural autoradiography
Uses gold nanoparticle to tag antibodies à antibodies tag proteins- can do double labelling with 2 different sized gold particles
Ultrastructural immunocytochemistry
3D structuring of sub-cellular macro-molecular objects, extension of TEM
done through serial sections or cryoelectron microscopy
Electron tomography
Better than normal MRI
MRI microscopy
Allows you to take specific set of cells
Melt film over section and cut desired shape
Laser-capture microdissection microscopy
isolates a single cell in a tissue for analysis- ethylene-vinyl acetate is laid on a specimen and melted with a laser (can analyze individual cells in a tumor, but an amplification technique is needed later)
Laser Capture Microdissection
Scanned proximity probe - no microscope, no lenses
Resolution based off tip size
good for examining the surfaces of cells bc they are non-conducting
Atomic Force Microscopy
First technique to see DNA molecules
not useful for cell structure, but has potential for imaging biomolecules without generating heat
Scanning Tunneling microscopy
Expensive
Uses fluorescene
Deeper penetration with less and phototoxicity
resolution is diffraction limited (follows Abbe's equation)
Two photon microscopy
Not diffraction limited
PALM à photoactivated local microscopy
SIM à sturcuterd illuminated micrscopy
SEMD à stimulated emission depletion microscopy
Super Resolution Microscopy (3 types)
yields high-resolution optical sections that can be reconstructed into one three-dimensional image
Deconvolution Microscopy
Surface enhanced laser desorption ionization
Resolution is 1 amino acid
SELDI-TOF/MALDI-TOF
smaller proteins fly faster than big proteins
time of flight
very small sample amount necessary, resolution is one amino acid
advantages of MALDI/SELDI TOF
Measures optial density à useful for fluoresence
Densitometer
Technique for following the movement of a substance through a biochemical or cellular pathway, by briefly adding the radioactively labeled substance (the pulse) followed by the unlabeled substance (the chase).
Pulse-chase
EGTA - chelating agent used to disassociate cells
Binds to calcium and stops cell adhesion molecules
EGTA/Protease
Sorts cells based on fluorescence protein expression
Cells flow past a laser which can detect the light emitted
The water droplets they're in are given a charge and sorted
FACS/Flow cytometry (fluorescence activated cell sorter)
using annexin V and PI to detect apoptosis from necrosis
Applications of FACS
Flow cytometry machine
Easy for quantifying cells- counts fluorecently tagged cells in multiwell plates but it does not separate them like FACS
Guava
A method of separating subcellular components in a dilute salt solution. The tube is centrifuged, and the components sediment throughout the tube based upon size (largest at the bottom)
Velocity sedimentation
Magnetic beads used for cell sorting
Dynabeads/Speedbeads etc
Machine uses selective surface
Veridex CellSearch System
Use of syringe to mechanically lyse cells
Trituration
Procedure for separating cellular components according to their size and density by spinning a cell homogenate in a series of centrifuge runs. After each run, the supernatant is removed from the deposited material (pellet) and spun again at progressively higher speeds.
Differential centrifugation
After cells are homogenized the RER breaks up into small closed vesicles with ribosomes on the outside termed rough microsomes. Contain most of the biochemical properties of the ER.
rough microsomes
have a low density and stop sedimenting and float at low sucrose concentration (originates from thesmooth ER, plasma membranes, and other organelle membranes)
smooth microsomes
Separates the particles based on density
Equilibrium Density (Rate Zonal) centrifugation
Separates molecules based on charge
DEAE à positive
CM à neg
DEAE and CM Ion exchange chromatography
Chromatographic technique used to separate and purify proteins in a mixture on the basis of size differences; also known as size exclusion chromatography.
Gel filtration
uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest- best for isolating the insulin receptor
Affinity Chromatography
Uses antibodies to purify PI
Protein A - staph aures band
Protein A immunoprecipitation
Ability to change solvent based on concentration gradient
Dialysis
Protein molecules are attracted to a charged end of a gel (usually the positively charged end) and are sorted into distinct bands based on how well they can move through the gel (based on either size or charge) Stronger/more charged or smaller proteins move fastest through the gel
Native gel electrophoresis
Proteins are pretreated
High resolution- denatures them so there is no native activity (better resolution)
SDS gel electrophoresis
A specialized method of separating proteins by their isoelectric point using electrophoresis; the gel is modified to possess a pH gradient- very powerful technique
Isoelectric focusing
the greatest resolution of the electrophoresis techniques
1. isoelectric focusing done in one dimension for better resolution
2/ isoelectric bands form the samples for the next step- SDS gel phase
2D gel electrophoresis
-used to detect a particular protein in a mixture of proteins
-antibodies are commonly used
-A primary antibody specific to the protein of interest is placed on a membrane.
-Primary antibody binds to protein of interest
-A secondary antibody-enxyme is added to bind to the primary antibody and marks it with an enzyme that can be visualized because the reaction cataylzed by enzyme produces a colored product.
Western blots
combination of a gel electrophoresis and blotting technique- used to identify a protein based on its ability to bind to an antibody
can be qualitative (molecular weight) and quantitative (indicate amount)
what are western blots