Microscopy
Microscopy
The foundation of cell biology is the microscope
In 1655, Robert Hooke invented the microscope and was the first man to see cells
He called them cells because they reminded him of the unit monks lived in, cells
In 1674, Antoni Van Leeuwenhoek recorded protozoa and bacteria
He is known as the Father of Microbiology
In 1838, Schleiden and Schwann developed the cell theory
Choosing a microscope to use depends on the methodology and what is being looked for
If the cells are living or dead, if the cells are fixed, how the cells are processed, etc
Reticular Theory vs the Neuronal Theory
The Reticular Theory thought the brain worked as a vascular network, not cells
The Neuronal Theory believed that the brain worked with cells, just unlike any of the other cells
Resolving Power
Light Microscope capabilities:
100 microns, the size of a plant cell
10 microns, the size of a typical animal cell
1 micron, the size of mitochondria and bacteria
0.2 micron limit of resolution
Electron Microscope capabilities:
100 nanometers, the size of viruses and ribosomes
10 nanometers, the size of a protein
1 nanometer, the size of molecules
2.4 angstroms, Ao
There are two major choices for microscopy:
Which microscope to select
How to process cell/tissue
Living cells or fixed/stained cells
Resolution is the ability to see two dots as two dots
Ernst Abbe, working with Carl Zeiss, came up with Abbe’s equation
A mathematical description of resolution
d=0.61nsin()-=400 nm
It comes in two different forms
Theoretical Limit of Resolution
The best possible resolution it could ever achieve according to Abbe’s equation
Practical Limit of Resolution
The resolution that is actually achieved
This is usually less than Abbe’s equation
This is due to the fact that cells are poor candidates for microscopy
They are mostly water, giving them very low inherent contrast
They have organic compounds, which absorb light and heat up causing movement
Super Resolution Microscopy is not limited by Abbe’s equation
Eric Betzig, Stefan W. Hell, and William E. Moerner were awarded the Nobel Prize in chemistry for breaking past Abbe’s limit using the fluorescence of molecules
There are various methods used to improve contrast
Sometimes dyes are used
Colorimetric, they absorb different wavelengths and transmit others
Hematoxylin, used to find the nucleus, and eosin, used to find the cytoplasm
Fluorochromes (fluorescent dyes)
However, dyes are somewhat toxic and can damage cells
Manipulate the light, such as by using a phase microscope or DIC
Not as good as dye, but very good for not perturbing the cells
Computer enhancement
Types of Microscopy
Bright Field Microscopy
Oldest and most often used microscope
What Zeist and Abbe worked with
Used mainly by pathologists to see tissue samples
Used mto view dead cells
Process:
Fixation
Use chemicals like formaldehyde to cross-link the proteins
This kills (fixates) the cells
Dehydration
Remove water from the sample and replace it with ethanol
Replacement
Replace ethanol with xylene
Infiltration
Remove xylene and place the sample in a paraffin (wax) cube
Microtone
Slice the cube into 10-15 micrometer sheets
Stain
Stain the sample with hematoxylin and counterstain with eosin
Counterstaining just stains everything else
Apply sections to slide
If samples are needed quickly and there is not enough time/people to perform the entire process, cryosectioning can be done
Quickly freeze the sample instead of using paraffin
Not normally used by pathologists
Used mainly for Mohs surgery, the surgery to remove skin cancer
Samples are needed quickly to determine exactly where the cancer stops so the surgeon knows where to stop
Phase Microscopy
Looks at living cells
“Non-fixated” cells
Uses light interference for better contrast
Used mainly by cell culture biologists
Differential Interference Contrast (DIC) Microscopy
Nomarski optics
Looks at living cells
Provides a 3D image
Used for single cell electrophysiology
Such as looking at a neuronal response to a drug
A very slender pipette enters the cell without killing it and helps measure membrane potential
Patch clamping comes from this
Monitors inside-out and outside-out ion flow through a single membrane channel
Dark Field Microscopy
Used mainly by microbiologists
Uses a dark background and a bright image for better contrast
Resolution is not changed for better contrast
Polarizing Light Microscopy
Light passes through light polarizers so only light from one plane gets through
Used to detect highly ordered parallel structures
Neurobiologists use it for detecting microtubeoles
Muscle cell biologists use it for detecting actin/myosin
Deconvolution Microscopy
Uses an algorithm to create a 3D view of fluorescently stained cells
Confocal Microscopy
Can be used on living cells
Governed by Abbe’s equation, but enormously increased the theoretical limit of resolution
The original patent was filed in 1957
The idea started maturing in the 1970s, but the technology, computers, necessary for it had yet to be developed
1988 saw the first commercial launch of the microscope
It has four main components:
Lasers
1 frequency per laser
The laser can be chosen
Confocal pinholes
Narrows the laser to make it more precise and focused
Point by point scanning
The lasers reflect of the structure point by point, gradually gathering an image
A computer displays the summed image of all the points
Advantages:
Less stray images because of the point by point approach
Optical sectioning
The point by point method being used in stacked sections allows for a 3D image
Stereo imaging
Multiple wavelengths allow for multiple labeling
If using fluorescent microscopy, the dyes would all blend together
However, fluorochromes can photobleach
Can also be used with spinning disk
Easier to use for living cells
Has faster imaging, less laser intensity, less heat, and is more dynamic
There are many microscopes that use fluorescent dyes
For fluorescent dyes the excitation wavelength is less than the emission wavelength
Wavelength is lost due to heat
Vital Fluorescent Microscopy
Keeps cells alive (vital)
Uses fluorochromes to measure and analyze changes in cell behavior
The dye itself is used to fluoresce the cell but does not permeate through the cell
An AM group is added to the dye so it can permeate through the cell and fluoresce from within the cell
Once the dye is in the cell, the AM group will detach and will cause the dye to fluoresce
Many uses
Mitochondrial activity uses JC-1 dye
red/orange mitochondria are healthy, green ones are sick
Live dead assay uses calcein-AM and propidium iodide
Calcein-AM turns healthy cells green
When the cell is about to die, the membrane is more permeable so calcein-AM leaves and the propidium iodide enters the cell and dyes the nucleus red
Intracellular calcium levels uses fluoro 3-AM
This dye fluoresces way brighter in response to high calcium levels and can better indicate the concentration of Ca in the cells
Plate Reading Spectrofluorometers
Sums and averages the fluorescent signals from all the cells in a sample
A quantitative assessment of fluorescent probes
Importance comes from cell variability, some will produce more proteins than others
Fluorescence Recovered After Photobleaching (FRAP)
After fluorescing an area of the cell membrane, intense lasers photobleach the fluorochromes, which move around the membrane
FRAP is used on parts of the cell with high membrane fluidity
To track how fast the proteins move around the membrane
Lateral fluidity
Over time, the fluorochromes will be recovered but will not be on the same spot
They will have moved around the membrane
Total Internal Reflection Fluorescence (TIRF) Microscopy
Intracellular injection
Uses lucifer yellow
One singular cell is injected with the dye, and the dye will travel throughout the cell via charged ions
Mainly used by neurologists to see the connectivity of a neuron and exactly where it branches off and which other neurons it is connected to
Can also be used between two non neuronal cells to see if they are connected
Gap junctions (electrical junctions) serve as an opening for ions between two cells
If the dye starts at one cell and travels to the other then that means the two are electrically connected
This can only be done without killing the cell, the dye must fluoresce and must diffuse easily without being membrane soluble
Or else it would leave the cell via the membrane and not junctions
Fluorescence Immunocytochemistry
Uses antibodies to identify proteins in a cell
Limitations:
Has to fluoresce
Has to only bind to one specific target protein
Can only bind onto epitope
Antibodies must be bivalent
One branch for fluorescent dye and the other for binding to the antigen
Has to have high specificity
Only binds to one protein
Has to have high affinity
Binds well to the one specific protein
Can be direct or indirect
For the direct method, antibodies with fluorescent dyes bind to the protein
For the indirect method, the antibodies will detect the antigen and bind to other antibodies with the dye which will bind to the protein
This method is preferred because it increases affinity and can bind to more proteins
When attaching the dye to the antibody, it slightly changes the shape and can decrease affinity, which is why indirect is preferred
Antibodies can be made in two ways
Polyclonal antibodies
Made by many different B (spleen) cells in the body
Typically an animal is injected with the antigen and has several different B cells produce antibodies, blood is collected, and antibodies are collected
However, there is not much specificity because several different antibodies with slightly different structures bind to different epitopes on the antigen, which would mark the wrong protein, have a high chance of cross reactivity, and the supply ends when the animal dies
Monoclonal antibodies (mAbs)
Mouse is immunized with antigen
The mouse spleen cells will produce antibodies
The spleen cells are isolated and fused with myeloma and whichever produces the most antibodies is replicated
Fused cells are called heterokaryon for having two nuclei
Myelomas are cells that are genetically engineered to not be able to make any proteins, preventing antibodies from being produced by other cells
Cells are placed into HAT medium
This kills any spleen or myeloma cells that are not fused together
The fused cells will be tested to see which ones produce the most antibodies and work best
Hybridomas (hybrid cells) are able to be reused as they can be cryopreserved
Can be used to determine cell polarity
Cell polarity is intrinsic asymmetry based off shape, structure, or cellular components
Enzyme Linked Immunosorbent Assay (ELISA)
Used to quantify the protein of interest
For example, cells in a culture naturally produce protein X. When we add drug Y, the cells produce more protein X than when the drug Y was not there. But how much of drug Y is used to make protein X?
Process:
The protein being tested is isolated
Primary antibodies attach to secondary antibodies
Secondary antibodies have enzymes that react in the presence of a substrate
Sometimes the enzymes will turn a different color
This is the colorimetric approach
Sometimes the enzyme will fluoresce
This is the fluorescent approach
The sample is placed in the spectrometer and OD adjusted to a certain value
The brighter or more vibrant the coloring, the higher the concentration of the protein
Cell Death
There are two causes of cell death
Necrosis, pathological cell death
An external cause such as poison
Causes the cell the explode
Apoptosis, genetic cell death
An internal cause
Chemotherapy, radiation, T-cells
The cell implodes
Annexin V tags apoptotic cells
Bonds to phosphatidylserine which is normally on the inner membrane of the cell
When cells undergo apoptosis, it goes to the outer membrane allowing annexin V to bind to it
When annexin V binds the membrane can fluoresce
Fluorescent Proteins
Fluorescent proteins act like dyes but because they are proteins they are easily synthesized in vitro
Roger Tsien introduced the green fluorescent protein, GFP
Found in jellyfish
Serves as a reporter molecule
Fluoresces when something happens in the cell
There are two types of report molecules
Continuous
Will always fluoresce
For example, putting human cells with GFP into a pig blastocyte to grow human organs in the pig
All the human organs will fluoresce green because the GFP serves as a continuous reporter here
Regulated
Will turn on and off depending on what is happening in the cell
For example, if a gene of interest has the GFP coding gene added to it, if the cell fluoresces that shows that the gene of interest was coded well
Can be used to tell if a certain protein is being expressed
Can be used alongside other fluorescent proteins with other colors
Forster Resonance Energy Transfer (FRET)
Tells how far apart two proteins are from each other using fluorescent proteins
If protein A has a fluorescent protein that fluoresces blue and it is close to protein B with a yellow fluorescent protein, the fluorescence emitted would be yellow
If they were not close together, it would be blue
Can also be used to measure enzyme activity
Some enzymes work by tinkering around proteins
Can connect or distance proteins
FRET can determine how far apart two proteins are from each other and this enzymatic activity
If an enzyme is supposed to bring proteins closer together but FRET is not working then the enzyme is not working like it should
Kinase is a protein that phosphorylates proteins
Phosphatase dephosphorylates proteins
Two proteins might only come together if phosphorylated
These proteins are marked with two different fluorescent proteins
FRET can be used to see how far apart they are
If there is a high concentration of the two proteins together that means kinase is very active and vice verse for phosphatase
A biosensor is a protein that reveals a change in behavior
Calmodulin coils up in the presence of calcium
Calcium is essential for neurotransmission
Fluorescent proteins are placed at either end of the calmodulin
FRET measures the distance between them
The higher the distance, the less calcium
Other Methods for Non-Electron Microscopy
Autoradiography
Uses a radioactive probe to track cell processes
During the synthesis phase in mitosis, DNA is replicated
To see which cells are undergoing synthesis, radioactive thiamine is placed into the cell culture and binds to cells in the synthesis phase
Film placed on the culture and radioactive thiamine will release beta particles that will show on the film as a black dot
This can also be used to detect certain types of cancers
Radioactive leucine and methionine (amino acids) can be used to track protein synthesis and protein travel around the cell
Fluorescence in Situ Hybridization (FISH)
Uses fluorochromes to identify mRNA
Probes with a complementary RNA strand with a fluorochrome attached to it
RNA binds to the mRNA, and the mRNA can now be tracked
Can also use fluorochromes to identify DNA
DNA is degraded using the same steps as mRNA
Used to track the telomeres of chromosomes
In Situ Hybridization for Detecting HPV
HeLa cells have HPV
Serves as a positive control, a group that will show up positive for whatever is being tested
HPV negative cells serve as negative control
Uses fluoresced genetic sequencing probe to see if HPV genetic material is present
If the sample fluoresces and so does positive control, it is HPV positive
Single Cell Intracellular Injection
Single Cell Micropipette Intracellular Injection
A single cell micropipette is used to inject something into a cell
Same idea as lucifer yellow
Used in somatic cellular nuclear transfers
A cloning technique where the nucleus of a somatic egg cell is placed in an egg cell that does not have a nucleus
Can only do one cell at a time
Electroporation
A sample is charged with an electromagnetic field
Electromagnetic pulses cause pores to open up in the cell membrane and anything from the outside can enter for a brief time
Can only be used for tough cells
Weaker more fluid cells could fully rupture
Liposomes
Uses lipid vesicles to transfect genetic material or other molecules into the cell
The vesicle is positively charged, and the phosphate heads of the membrane are negatively charged
Liposomes are used in mRNA vaccines
However, lysosomes in the cell can destroy the liposome
Viral Transfection
Genes are added to a viral capsid
The virus infects cells and transfects genes, inserting genes into the cell genome
Retrovirus is used for this method
This is not FDA approved
Deliberately injecting oneself with a virus
The immune system also might just kill the virus before it reaches the cell
Electron Transmission Microscope (TEM)
One of the two main types of electron microscopes
The other is the Scanning Electron Microscope, SEM
Works by “scanning” the sample and giving surface imaging
Resolution follows a new equation
The wavelength of electrons = 12.3voltage
The shorter the wavelength the better the resolution
Works by transmitting electrons through the specimen
TEM vs Light Microscope:
Illumination
TEM uses electrons, the light microscope uses light
Sections
TEM has sections 50-90 nanometers thin, the light microscope has sections 10-25 micrometers thin
Lenses
TEM has electromagnetic lenses, the light microscope has glass lenses
Imaging
TEM uses a screen, the light microscope uses your eyes
Has many different techniques
Plastic Thin Sectioning
Process:
Fixation
Tissue is fixated using glutaraldehyde to cross link the proteins and OsO4 to cross link phospholipids
Dehydration
Infiltration
Uses epoxy plastic
Ultra microtone (usually a diamond knife) is used to cut samples ultra thin
Staining
Electron microscopes can only detect staining if the stains are from heavy metals
Lead is used for staining the membrane and uranium is used for counter staining
Negative staining can also be used
When everything but the sample is stained
Freeze Fracture
Process:
The cell is frozen
The cell is cracked open with a knife to remove the outer membrane
Platinum and carbon cover the cell to make a mold of the interior
Ultrastructural Immunocytochemistry
The same idea as immunocytochemistry but instead of dyes, gold particles are used on antibodies
Gold is relatively cheaper and comes in many sizes
Shows up on TEM as little black dots
The double label protocol uses gold particles of two different sizes can be used to see it better when it shows as a black dot
Ultrastructural Autoradiography
An ultra thin sample is placed in silver halide solution
A radioisotope is also placed
The slide is fixed and developed
Silver not bound to the radioisotope is washed off
Silver that remains is detected on TEM
The High Voltage Electron Microscope (HVEM) has a better limit of resolution than TEM because its accelerating voltage is, 1,000 kV, not 100 kV as in a typical TEM
Best used for imaging thick sections
Vivascope
Not technically a microscope
Uses confocal imaging via a handheld device for skin biopsies
Used by dermatologists
Helps determine the point of care
Non-invasive
Laser Capture Microdissection (LCM) Microscope
Uses a laser to cut off smaller samples from a larger one
Sample sizes that can get cut off range from 7.5 micrometers to 30 micrometers
Can be individual cells or a a group of cells
Atomic Force Microscope
A scanned proximity microscope, does not have lenses
Used for surface analyses
Measures and analyzes many forces
Scanning Tunneling Microscope
Looks at a surface atom by atom
Ultra high resolution
Does not use electron beams or light
A scanned proximity probe microscope
Two Photon Microscope
Designed for deeper tissue imaging like brain tissue
Less phototoxicity, damage to a sample caused by light compared to other microscopes
Is preferred over confocal microscopy