Light Microscopy Lectures Flashcards
What is the history of LM?
- most important technique until 1950s
- EM 1950s->1970s (can visualize at nm scale)
- Now, combination of biochemistry & yeast genetics
- LM is now making a comeback, as it can follow dynamics of proteins in living cells.
What are Antony Van Leeuwenhoek and Robert Hooke known for?
(1) Antony -> developed powerful magnifying glass, microscopic organisms ‘animalcules’
(2) Robert -> Contemporary version, gave the name ‘cells’ to structures he saw in Cork and Wood.
What are the important types of light microscopy?
1) Transmitted light (bright field, phase contrast)
2) Fluorescence
3) Confocal
4) Super-resolution
Why are cells hard to see?
- They are transparent
- With normal brightfield, they are very hard to see. To make visible, use visible dye or special optics (phase contrast).
What kinds of samples are we trying to visualize in LM?
- Tissue cells are usually fixed, embedded in paraffin or plastic, and thinly sectioned.
- Tissue culture cells, often egg-shaped or flat, are easier to work with but not representative of normal tissue. They can be fixed or alive, though live cells are more challenging to handle. Microscopy of tissue culture cells is simple, making them useful for cell biology research.
What are advantages of methods to observe unstained live cells?
1) Prolonged observation of live cells.
2) Can study movements in cell division and of intracellular structures.
3) Can film or record on video tape with camera.
4) For live cells, inverted microscopes often used.
In terms of tissue culture, what is the difference between organ culture and explant culture?
(1) Organ Culture
- Dissect from host (mouse), keep most of the physiological conditions from living organisms, put into organ culture.
(2) Explant Culture
- Dissect from host (mouse), finely chop, place into primary explant culture.
- easier to maintain in culture and still presents 3D organization.
Continuous Cell Lines
- start with primary cell culture.
- trypsin digestion to detach cells.
- establish cell line by culturing cells through successive passages (eg., 1st passage, split at 1:3 ratio)
- These cells have undergone immortalization, either naturally (e.g., cancer-derived cells) or artificially (e.g., via genetic modification or viral transformation). They can divide indefinitely under proper conditions.
Immortalized Cell Lines
- Derived from rodents (common)
- grow only as a monolayer culture due to contact inhibition (cells stop growing when they touch each other)
- mutation (eg., allowing telomerase expression) - allow indefinite growth in tissue culture.
- cells otherwise retain good behaviour.
What is an example of a cell that lacks contact inhibition?
- Cancer cells (HeLa cells).
- will continue to grow in tissue culture, piling up on each other, these are ‘transformed cells’
- Abnormalities/abnormal mitosis.
- cultured cancer cells (transformed cell line)
Non-immortilized cell line
- These cells have a limited lifespan and undergo senescence after a certain number of divisions (Hayflick limit). They maintain many characteristics of normal primary cells.
- Primary cells, allow to reproduce in tissue culture, can be grown for many generations, but not indefinitely.
What are primary cells?
- same cells obtained from source
- not to be immortalized/transformed usually
- but primary cells obtained from cancer cells are usually transformed
How are cells maintained in a culture?
(1) Artificial Medium
- Physiological pH (7.4), carbonate buffer, C02 gas, pH indicator (phenol red)
- nutrients (amino acids, vitamins, salts)
- glucose
- serum (growth factors)
- antibiotics (optional)
(2) Temperature (37C - humidified environment)
(3) Sterile environment.
How does fluorescent microscopy work?
- Fluorescent molecules can absorb light of high energy (short wavelenghts) and emit light at lower energy (long wavelenghts).
- Tissues & Cells are irradiated with a blue-violet or UV light so the emission is in the visible part of the spectrum.
- Appear as bright and colored on a black background.
- Sensitivity of the method is very high.
- Cells have natural autofluoresence but this is not useful.
In FM, how do you label proteins?
- chemically label protein outside cell and add it.
- label Ab against protein and stain cell (cell must be formaldehyde fixed and permeabilized)
- fuse protein of interest with GFP and express.
How are antibodies produced?
- antibodies are immunoglobins, produced by B-lymphocytes and plasma cells of vertebrates.
- most Abs used in immunohistochemistry are on the IgG class of immunoglobins.
- Y shaped molecules, with two ight and two heavy chains.
- 2 antigen binding sites, one at the tip of each arm of Y.
- parts of Ag that bind to Ag-binding site are epitopes or antigenic determinants.
- Abs can be generated against most macromolecules, not generated against the full protein but against part of the peptide sequence of that protein, using a synthetic peptide.
- also possible to generate Abs against small molecules such as amino acids and monoamines if they are conjugated to carrier protein.
How are Abs generated for Immunocytochemistry?
(1) Immunisation
- animals injected at specific intervals with a suspension of the antigen, conjugated or not to a carrier.
- at end of the immunisation, blood is removed from the animal and after clotting, serum separated and tested by immunocytochemistry.
When you have a new Ab, what steps do you take to make sure it is characterized correctly?
(1) Test Ab to make sure it works correctly (only targets the intended antigen) -> After using Ab for staining, check its specificity.
(2) To confirm Ab specificity, mix Ab with Antigen before applying to tissue. If Ab binds to Ag, no staining should appear (If Ab and Ag are bound, can’t bind to tissue)
(3) Check for cross-reactivity.
If Ab is good, animal needs regular injections of the antigen to keep producing the Ab*
What is the difference between Polyclonal and monoclonal Abs?
(1) Polyclonal (antiserum)
- Produced by multiple clones of lymphocytes/plasma cells in an animal.
- Recognize multiple epitopes on the same antigen.
(2) Monoclonal
- Produced by a hybrid myeloma cell line (fusion of myeloma cells with lymphocytes from an immunized animal).
- Recognize only one epitope on the antigen.
- Can grow indefinitely in culture.
- Only from rats & mice.
Myeloma cell lines available for production of monoclonal Abs are azaguanine-resistant. What medium can they not survive in?
HAT Medium
How are monoclonal antibodies produced?
(1) Immunization
- Rats/mice are immunized with the target antigen to generate polyclonal antibodies.
(2) Serum Testing
- Blood is collected, and antibody response is tested using immunocytochemistry.
Animals producing the best antibodies are selected for further immunization and cell fusion.
(3) Cell Fusion
- At the end of immunization, lymphocytes from the animal are fused with myeloma cells to create hybridomas.
HAT medium eliminates unfused myeloma cells, allowing only fused hybridomas to survive.
(4) Screening & Cloning
- Hybridomas are cultured in wells, and antibody production is tested in the spent culture supernatant.
Initially, each well may contain multiple hybridoma clones.
Cloning is done by plating hybridoma cells at one cell per well.
- The best clones are selected, expanded, and re-cloned to ensure a pure monoclonal antibody-producing hybridoma.
What are the advantages of Monoclonal Antibodies?
(1) High yield, low cost once generated.
(2) Hybridomas are immortal, unlike animals producing polyclonal antibodies.
(3) Highly specific staining with minimal background.
(4) No need for affinity purification, unlike polyclonal antibodies.
What are the two different types of IF?
(1) Direct
- Fluorescente Ab (labeled) -> Ag
(2) Indirect
- Add fluorescent anti-antibody on the antibody (unlabeled) to the antigen.
What are the key steps in IF?
(1) You have a live cell, can’t cross membrane.
(2) Formaldehyde fix -> kills cell (preserves their structure)
(3) Permeabilize cell (with detergent, now they can enter membrane)
(4) Add primary Ab.
(5) Add secondary Ab.
What are some important variations on IF?
(1) Can also fix/permeabilize with methanol.
(2) If you skip permeabilization step, can only stain things on the surface of cells (not tissue)
(3) You can use small amount of glutaraldehyde for stronger fixation (preserves them better), but it is fluorescent itself, can interfere with staining.
(4) Tissues are sectioned before staining (unlike culture cells)
What are the advantages and disadvantages of GFP?
(+) of GFP
- can be used in living/fixed cells
- if DNA is introduced into cell, GFP-tagged protein is produced by cell and already fluorescent.
- good for live imaging.
- used with other colours of fluorescent proteins or in combo with IF.
(-) of GFP
- sometimes GFP fusion proteins does not fold properly/misbehaves
- protein may be present in cell in higher than physiological amounts
- endogenous protein in cell is not well visualized
- expression of GFP proteins in whole animals is much more difficult than in tissue cultured cells.
How are GFP proteins constructed?
- plasmid grown in bacteria, purify plasmid, transfect mammalian cell, GFP-protein fusion, expressed after 24 hours, examine living cells on heated microscope stage.
- because GFP is created on bacterial plasmid, it is much easier to use in tissue culture cells than in animals.
What is Digital Wide-Field Microscopy?
- Digital camera is attached to fluorescence microscope.
- Camera is very sensitive, often cooled to reduce noise, this causes fluorescence to be dim.
- Fluorescence in images can be accurately quantitated / images are similar to eye piece.
- Digital images made up of pixels, light intensity represented by number for each pixel in an image.
Why is confocal microscopy better for thick samples?
(1) Light Microscopy (LM)
- Narrow depth of field, especially with high-resolution lenses.
- Out-of-focus light blurs images, reducing sharpness in thick samples.
- Works well for thin samples, but 3D structure is lost when sectioned.
(2) Confocal Microscopy
- Uses lasers, fluorescence optics, and computers to block out-of-focus light.
- Detector captures only in-focus light, improving clarity.
- Scans point by point with a laser, controlled by a computer.
- A type of fluorescence microscope with specialized optics for better imaging.
What are biological uses of confocal microscopy?
- 3D or single slices from fixed samples (thick!!) or from living cells.
- time-lapse in living cells (dynamics of organelles or transport intermediates )
- photo-bleached techniques.
- quantitative information.
What is photobleaching?
- problem with fluorescence microscopy. once excited, a fluorophore remains excited for 1-10 ns, before re-emitting, while excited it can react with molecular oxygen, irreversibly destroying fluorophore.
- when a high energy of light (laser) is applied to a defined region, such as fluorescent molecule continuously in excited state, bleaching can be introduced in a controlled manner, sometimes useful.
FRAP (Fluorescence Recovery After Photobleaching) - What does FRAP Measure? How does FRAP work? What can FRAP be used for?
(1) Movement of fluorescent molecules inside cells (mobile vs. stationary).
(2)
- Region of the cell is photobleached using a laser (confocal microscope).
- Fluorescent molecules diffuse in, refilling the bleached area.
- Diffusion rate is measured as the diffusion coefficient.
(3)
- Detects diffusion of molecules (e.g., in plasma membrane, ER).
- Tracks protein movement between organelles (bleach one, watch it refill).
- Shows diffusion within membranes does not require energy.
Resolution
- Limit of resolution (d) is the smallest distance between two points that can still be distinguished.
R= (0.61×λ)/nsin(θ)
What is the difference between farfield and nearfield in microscopy?
(1) Farfield (h»_space; λ)
- Light is far from the sample.
- Limited by diffraction: the smaller the wavelength, the better the resolution, but still restricted by diffraction limits.
(2) Nearfield (h «_space;λ)
- Light is close to the sample.
- Diffraction is less of a problem, allowing for higher resolution and better imaging of small details.
- Uses optical fibre with very thin tin (30nm), moves slowly over sample.
- super-resolution!
- better for studying materials than live biological samples.
h = the distance between sample and microscopes detection points, in relation to the wavelength used.
high energy = small wavelength = high spatial resolution.
What is PALM (super-high resolution technique)?
- Photo activated Localization Microscopy.
- special photo-activated GFPs (small portion can be ‘turned on’ by near-UV light, imaged & bleached).
- others can be turned off after, process repeated 1000 times.
What is STORM (Stochastic Optical Reconstruction Microscopy)?
- sample put in buffer, most fluorescence dye molecules off (dark state)
- only few molecules stay active and are far enough to be seen individually using strong light and a high quality camera (randomly flicker between dark/active state)
- take images, when enough data can pinpoint exact location of molecules, final image is built by combining all these localized molecule positions.
What are PALM/STORM limitations?
- not compatible with confocal as confocal is not as good with dim images.
- best with thin samples, so faint signal is not swamped with autofluoresence. use of TRIF can help, but limits observations to the bottom of the samples.
- very difficult with living cells (not impossible)
- can replace immunogold, but requires imaging the same sample with EM and STORM/PALM, time-consuming and complicated. Process less practical.
What is TIRF Microscopy? (total internal reflection)
- wide-field fluoresence microscopy with illumination at very shallow angle.
- light reflected from cover-slip surface, with very shallow penetration into the sample (evanescent wave)
- only portion of sample very close to coverslip can be observed.
- very low background, as much of the sample is not visible.
- favourable conditions for observing faint fluorescence from single molecules.
- confocal also eliminates background well but light is lost much more due to complicated optics.
