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.
