BIOL 311 Exam 1 Binghamton

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