Lecture 1 Flashcards
Lecture 1: - introduction - transmission light microscopy - tissue culture - fluorescence light microscopy
Name the techniques used to study cells
1) microscopy (light and electron)
2) biochemical techniques
3) genetic techniques (particularly in yeast)
4) combinations of the above
Define light microscopy
Study of cells using visible light and lenses to magnify and resolve cellular structures
Define transmission light microscopy
Technique using transmitted light to visualize internal cellular structures
Define tissue culture
Growth of cells from a tissue or organism in a controlled artificial environment (lab)
Define fluorescence light microscopy
Microscopy using fluorescent dyes to label specific cellular structures for visualization (important for cell biology)
Name the history of microscopy
until the 1950s: light microscopy - to study tissues and organs
1950s-1970s: electron microscopy - to study structures inside the cells
1980s - present: combination of biochemistry and yeast genetics
!light microscopy is coming back bc of its new ability to follow dynamics of proteins in living cells
What is microscopy?
Study of objects too small to be seen by the naked eye using light and electron microscopes
What are biochemical techniques?
Methods to study biological processes at the molecular level, e.g., protein purification and analysis
What are genetic techniques?
Methods to manipulate and study genes, e.g., gene cloning and sequencing (especially in yeast)
What is yeast?
Single-celled fungi often used in genetic research due to their simple genetics
What is electron microscopy?
Microscopy using a beam of electrons to visualize the ultrastructure of cells and tissues
What are Leeuwenhoek’s animacules?
Microorganisms observed by Leeuwenhoek, including yeast and rotifers
What are rotifers?
Small multicellular organisms
What did Robert Hooke do?
- designed different microscopes around the same time that are more similar to today’s microscopes
- coined the term ‘cells’ after observing structures in cork and wood
What is the micron scale?
Scale of measurement in microscopy, equivalent to one millionth of a meter
What is the nanometer scale?
Scale of measurement in microscopy, equivalent to one billionth of a meter
Which type of microscopy would you use to visualize a bacterium?
- 1-2 microm across
- can tell general shape but not any detail inside w LM -> need to be clever
Which type of microscopy would you use to visualize a columnar epithelial cell w microvilli in respiratory epithelium?
- 15 microm > 2/10 microm -> can distinguish details
- most organelles/membrane bound compartments are big enough to distinguish (only see microvilli + nucleus)
slides14,15,16,17
What microscopes are used in biology labs?
- compound microscopes (higher magnification -> more sophisticated versions of regular lab ones)
- fluorescence microscopes (to visualize, locate and track individual molecules, v sensitive -> can detect few molecules)
- confocal microscopes (new type of fluorescence microscope)
Name the important components of a conventional transmission microscope (light)
1) illuminator + mirror – light source
2) condenser – focuses on sample
3) specimen slide – sample
4) objective lens – multiple lenses for diff zooms
5) ocular lens – eyepiece to look through (in research microscopes: direct light to a camera)
What are the important types of light microscopy?
1) brightfield, phase contrast = transmitted light
2) fluorescence
3) confocal = a specialized kind of fluorescence microscopy (covered in lecture 3)
4) super-resolution (recent invention, covered in lecture 3)
How do you study a cell if it is transparent under normal brightfield illumination?
(aka magnify cell without treating it in any way)
i can…
1) stain with dyes: H&E
2) use special optics (for live cells without any staining): phase contrast, etc.
problem: none of these are enough if you want to locate diff molecules in the cell
What are the different kinds of samples?
1) cells in tissues
- usually fixed
- embedded in paraffin/plastic
- sectioned (bc too thick for light microscope)
2) tissue culture cells
- fixed or alive
- grow directly on slide/glass
- flat and fried egg
- easier to work with BUT not normal env to study the behaviour of cells in tissues
3) live vs fixed
- tissue culture cells can be fixed or alive
- intact tissues are difficult to work with alive
- both techniques are good for visualizing proteins
Compare the different techniques for studying live cells without staining
cell growing in tissue culture on cover slip directly
1) bright field
= Standard illumination method for microscopy, suitable for stained or strongly absorbing specimens
- no stain, just put cell under microscope
- transparent cells (no contrast)
- cant differentiate organelles
- can locate cells
- can visualize nucleus
2) phase contrast
= Microscopy technique to enhance the contrast of transparent and colorless specimens
- one of the optical tricks to see nucleus better
- bubbles = see vesicles (endosomes) across cell and track them
- dark on ends = actin cytoskeleton
- cant see other organelles much
What is the advantage of methods to observe unstained live cells?
allow the prolonged observation of live cells
filmed using microcinematography/recorded on videotape with video camera
inverted microscopes are often used for live cells
ex: studying the movements in cell division and of intracellular structures
Why are tissue culture cells often used in cell biological research?
microscopy of cells in tissue culture is very easy
What are the 3 types of tissue culture?
1) organ/explant culture
2) primary cells
3) continuous cell lines
Compare the 3 types of tissue culture (slides 31-36)
1) organ/explant culture: keeps most of the physiological conditions from a living organism
2) primary cells
3) continuous cell lines
Define contact inhibition
- appears in tissue culture cells obtained from primary cultures
- cells form a single monolayer on a plate
- cells will not continue to grow once the space is filled
Do cancer cells exhibit contact inhibition?
ex: HeLa cells
No, they will continue to grow in tissue culture, piling up on each other
they are called “transformed cells”
Name the 4 types of tissue culture cells
1) primary cells
2) non-immortalized cell line
3) immortalized cell line
4) transformed cell line
Describe primary cells
= same cells obtained from the source, not immortalized or transformed
!!transformed only if obtained from a cancer
describe non-immortalized cell line
= when primary cells are alowed to reproduce in tissue culture for many generations (not indefinite)
describe immortalized cell line
= cell with mutations allowing indefinite growth in tissue culture (ex: allowing telomerase expression)
describe transformed cell line
= Cells that have lost contact inhibition and exhibit abnormal mitosis, often cancer cells
can be transformed through mutations or through viruses/introduced DNA to express oncogenes
How do you maintain cells in culture?
1) artificial medium
- physiological pH (7.4): carbonate buffer, CO2 (gas), pH indicatory (phenol red)
- nutrients (aa, vitamins, salts)
- glucose
- serum (growth factors)
- antibiotics (optional)
2) temperature
- 37C through humidified environment
3) sterile environment
Fluorescence microscopy
