Bright field microscopy/compound light microscopy
fixed and processed tissues/cells.
high magnification (40 - 1000x),
two sets of lenses: the ocular lens (in the eyepiece) and the objective lenses (close to the sample)
low optical resolution due to limitations of light + staining
Bright field microscopy/compound light microscopy PROCEDURE
Process: Isolate tissue/organ → Kill tissue (usually with aldehydes) → Remove water and replace with alcohol (dehydrate) → Replace alcohol replace with xylene → Replace xylene with liquid paraffin, let solidify (tissue is solid in wax) → cut tissue and put in microtome (10-15 µm sections), put on slide → Remove paraffin → By taking slide, put it back in xylene then put it back in ethanol and finally water. Left with tissue section w/o wax but water is back in tissue. Now you can stain them → Stain with dye
Cryostat
A type of Microtome (machines that slice tissue very thinly)
maintains the cryogenic temperature of samples or devices placed inside it. Freezes the tissue and also cuts the frozen tissue for microscopic section.
Cryosections are faster, but integrity of tissue is somewhat compromised (Substitutes for the paraffin embedding process, e.g. makes the tissue rigid)
Used for Mohs surgery - a process used by dermatologists to make sure that the margin of skin cancer like melanoma cells has a “clear margin” not showing cancer cells - can take much of a day to do this
Phase Contrast Microscope
Generates contrast due to differences in refractive indices in cells.
Living cells- not fixed
Constructive and destructive interference is critical to the function of this microscope.
Manipulating light
Differential Interference Contrast Microscope (Nomarski)
Used by cell biologist who want to use living cells and see the outer surface of cells
Produce 3d effect and can do limited optical sectioning
Especially important to neurobiologists for positioning intracellular micropipettes for intracellular injection of transmembrane voltage recordings as well as doing patch clamping
Manipulating light
Dark Field Microscopy
Good for microbio (good for viewing bacteria)
Visualize small objects in cell with high contrast (mitochondria and lysosomes)
No stains necessary but can be used
Works due to a special condenser
The Confocal Microscope
Spinning Disk and Laser-Scanning microscopes (we will go over)
Based on lasers and computers that hadn’t been developed to build the first confocal microscope
Small focal plane creates clearer image
Major components:
Laser (monochromatic (488nm) - diffraction is limited, laser can be manipulated to image specimen spot by spot)
Stray image is filtered using a confocal pinhole, total summed image can be displayed on screen
(photobleaching is a problem with fluorescent dyes)
Vivascope
The Confocal Microscope
Benefits of Confocal vs. conventional
Decrease stray image by 50%
Optically section a cell
Generate stereo images
Use both colorimetric and fluorescent dyes
Excellent for double or triple labeling where all two or three different colored stains can be distinguished from each other
Can be used for art conservation
Laser-Scanning confocal
(first gen)
Slower but generates a bright image
Problem: can photobleach and not good for dynamic interactions due to the time required for imaging
Heat is created that can negatively affect the image and practical limit of resolution
Fine for routine work using fixed specimens
Spinning Disk Confocal
(second gen)- improvements to spinning disc
System that views an image through several holes simultaneously within a millisecond
Good for dynamic, moving phenomena
Faster
Lower laser intensity required
Decrease in photobleaching
Less heat generated
Good for living cells undergoing dynamic processes
Fluorescence Microscopy
Fluorochromes or fluorescent dye - absorb light of one wavelength and emit at a slightly longer wavelength. The longer wavelength is due to energy lost (heat) upon absorption of the excitation light.
Fluorochromes can be used to monitor some changes in physiologics in a living cell
FRAP - Fluorescence Recovery After Photobleaching
Measures membranes fluidity of a particular protein in living cells in many ways
Uses a fluorescently tagged cell membrane protein
A defined laser beam localized on part of plasma membrane and the fluorescence is photobleached
Next one has to determine the amount of time it takes for the fluorescence to re-distribute itself to the bleached area (or not)
Indirect method of monitoring mobility of that protein in the membrane
TIRF - Total Internal Reflection Microscopy
Easily images activities and structures near the cell surface that confocals cannot accomplish
Angles excitation beam
Creates “evanescent wave” 50-100 nm of the surfaces
FRET - Förster Resonance Energy Transfer & FRET Biosensor
Useful to look in living cells when a ligand binds to a receptor
When X (ligand/hormone) binds to Y (receptor) it gives off a different wavelength to make a new color
X gets excited and makes the Y excited, which gives off its wavelength to indicate that they bonded to each other
Biosensor measures muscle proximity
Biosensor is a molecule that can be synthesized that can light up via FRET when it changes shape upon binding with a molecule that you want to measure
Calmodulin is used to indicate if calcium is at high or low levels in cells
Calmodulin is fluorescent red when it’s inactive, and becomes green when it gets excited (calcium makes it excited and activates the fluorescent green color)
Vital Fluorescent dyes
Vital = alive
Ex) JC-1 - mitochondria - mitotracker red, Rhodamine 123, stains mitochondria red and shows activity shown by emission profile, see healthy or unhealthy cells
JC-1 indicates how active mitochondria is in the cell
Mitochondria has proton motor force (PMF)
Higher PMF (red)= more ATP it can make
Lower PMF (green)= less ATP
Vital Fluorescent dyes
Ex) Calcein-AM, propidium iodine - monitors plasma membrane integrity and indirectly whether cells dead or not
Determined whether the membrane is there or not (living/dead assay) (Live/dead assay)
Cells that contain the Calcein are seen as green = alive
Cells that don’t have Calcein are dyed red because the dye penetrates into their compromised membrane = dead
Ex) Fluo 3-AM - measures intracellular calcium
Qualitative assessment of fluorescent probes - one color dye is used that shows the difference in concentration of Calcium in cells (different concentrations show different colors)- Calcium indicator dye (used in neuron assessment)
Ex) BCECF-AM - measures pH in organelles
Lysosomes are very acidic
Fluorescence immunocytochemistry
Locate specific molecules (proteins) in or on cell using fluorochromes associated with antibodies (uses antibodies to identify location of protein of interest in the cell)
Antigen - capable of eliciting an immune response
Antigenic determinant - epitope - specific portion of an antigen molecule to which the antibody is capable of responding/binding
Antibody - molecule that is bivalent (has two binding sites), recognizes antigens and binds to them in the variable domain region
Fab - antigen binding end
Fc - end region that fluorochrome attaches to
Specificity - ability of a single antibody to bind to only one antigen
Fluorescence immunocytochemistry
Affinity - degree to which an antibody to bind to a given antigen
High affinity means that it binds tightly (preferred) versus a low affinity antibody
Indirect vs. direct
Indirect
is more common, uses primary antibody and then a secondary antibody
Preserves the integrity of the antibody
Secondary antibody binds to the fc region of the primary antibody
Direct
uses one antibody and can cause the antibody to change its degree of affinity and specificity - not good
Presence integrity of antibody
Monoclonal vs. Polyclonal Antibodies
Mono
Expensive
Single antibody species
Will only bind single specific site
is
Used to treat diseases such as cancer
Cells grown in culture are now immortal (stored forever)
Can be used to determine which antibody is most specific and has highest affinity for specific antigen
Bispecific antibodies are generated using “quadroma” cell lines (have two different Fab ends) - used for treating cancer. Biologic drug b/c it’s made out of cell lines
T-cells bind and target tumor cells to kill them
Monoclonal vs. Polyclonal Antibodies
Poly
Cheap
Mixed population of antibodies
May bind to different areas of the target molecule
Could be a problem bc they might cross-react with other non-target proteins
Inject antigen
Antigen is processed and presented to B cells
Antigen associates with the antibodies on the cell surface, causing B cells to divide
Clones of B cells are produced -more b cells means less specificity
Antibodies then shed and can be collected from the blood serum due to ammonium sulfate precipitation
Hybridomas - fused nuclei of two cells
Cancer cells (immortal) are fused with moral cells (from animal)
6. Put in culture dish to grow different cell types
Unfused cells die (selected against), fused cells grow (selected for)
ELISA - Enzyme Linked Immunoassay
Not a microscope, but similar
Detects antigen concentration in a solution sample
Direct - antigen is detected with enzyme-labeled primary antibody
Indirect - antigen is detected with enzyme-labeled secondary antibody
Sandwich - antigen is captured through a pre-coated “capture antibody”
Often used to determine the relative amount of a protein present in a cell under two conditions
Add primary enzyme, secondary enzyme with antigen, then add substrate
Micro Spectrofluorometry/Plate Reading Spectrofluorometers
Micro Spectrofluorometry
Plate reading spectrofluorometers
Single cells
Cant average signals from large numbers of cells
Generates images: qualitative
Plate reading spectrofluorometers
Not a microscope
Averages signals of thousands of cells together
Generates fluorescent intensity level values (# not image): quantitative
Gives a graph that reveals exchange rates or membrane fluidity
First one - CytoFluor (our lab and Millipore Corporation in 1980’s)
GFP, YFP, CFP as reporter molecules
GFP - green fluorescent proteins
Extracted from jellyfish
GFP completely absorbs this blue light and emits it as a green light.
reporter for monitoring gene expression in vivo, in situ and in real time
YFP - Yellowish-green Aequorea fluorescent proteins
CFP - Cyan Aequorea fluorescent proteins
Transmission Electron Microscopy
Transmits electrons through a specimen = resolution is 0.1 nm
Resolution is governed by Abbe’s equation (diffraction is limited)
The faster the electrons, the shorter the wavelength, the higher the resolution
TEM techniques and applications:
Plastic thin sectioning techniques:
Fixation - glutaraldehyde which cross-links (fixes) proteins followed by osmium tetroxide (fixes phospholipids)
Need to embed cells in something hard so they can be easily cut into sections
Series of dehydration steps - removes water
Embed in plastic, not wax - need harder resin for ultrathin sectioning
Use ultramicrotome to cut sections - float on water
