Lecture 1 Flashcards
T/F: Compound microscopes have multiple lenses
T
Light microscopes allows us to see things on what scale?
Micron
What structures are hard to see with light microscopes ?
-Nanometer scale
- Two structures less than 250 nanometers apart
What is used to see things on a nanometer scale?
Electron microscopes
Nanometer to micron
1 nanometer is 1/1000th of a micron
What scale can we see with the naked eye ?
1/4 mm to bigger
What scale can be see with the LM?
something larger than 1mm all the way down to something with structures that are separated more than 250 nanometers apart
What limits LM?
Resolution of LM is limited by the fraction of light
What can be seen in LM?
- Organelles is plant/animal cells
-Bacterium but not their internal structure
What can be seen in EM?
-Internal structure of bacterium
-All the way down to a structure the size of an atom
What limits EM?
How long your sample can go without being cooked
Which technique EM or LM can be used to image live cells?
Light microscope
Transmission Microscope
-Light source(bulb/mirror)
-Condenser
-Ocular lense
Transmitted LM
Light is transmitted through the sample of interest and can be absorbed which creates contrast and a visual of the sample
Two types of transmitted LM
- Brightfield
- Phase Contrast
Why is cells being thin a problem for LM?
Cells that are thin and transparent will be hard to see using brightfield illumination since there is no contrast
How do we deal with thin cells for LM?
We use stains for dead cells
or we use special optics for live cells
Types of special optics?
-Phase contrast
-Differential-interference contrast
-Dark field
How can we look at cells within tissues ?
- Fix them
- Embed them in paraffin
- Section them
This makes them small enough. to visualize. Hard to keep these alive when we section
Tissue Culture cells
-Fried egg shape, flat, easy to work with
-Can be fixed or alive
Problem with tissue culture cells?
Less representative of human cells since they are grown and don’t come directly from us
How do we observe unstained live cells?
Inverted microscope prevents the microscope from getting wet since objetive is below the sample
Organ culture?
Keeping the kidney alive by constantly perfusing it with nutrient solution
Explant culture?
Chop the kidney into smaller pieces and place the pieces in media that nourishes them and allows them to grow. If we add protease we can separate each cell so that we get individual cell and then we can grow these on a dish
Primary cells
Cells that were actually at one point in the organism of interest
Continuous cell lines?
When primary cells grow and divide and produce cells that have grown completely in an incubator
Mortalized Cell line?
After 40-60 rounds of makinga continuous cell cultrue the cells will reach a crisis and die because the telomeres will have shortened too much
T/F: Older organisms reach crisis faster?
True
Immortalized cell lines
These cell lines express telomeres which prevent chromosome shortening during division and prevent the “crisis” from occurring
ex. germ cells
Transformed cells
Cells lose contact inhibition and have abnormal mitosis.
Ex. Cancer cells, mutated cell lines
Contact inhibition
Culture cells will only form a single monolayer on a plate and then will not continue to grow
Contact inhibition in cancer cells
Cancer cells/transformed will continue to grow and piling up on each other due to a loss in the ability to regulate growth
Artificial Medium used to maintain cells?
-pH 7.4
-Nutrients
-Glucose
-Serum(growth factors)
-Antibiotics
-37 degrees
Sterile environment
Why do we add antibiotics to artificial medium?
Since cells divide every 24 hours bacteria can get in and divide every 20 minutes. Prevents bacteria from taking over
Advantages of fluorescent light microscopy
-No background allows you to focus on interest of study
How do fluorescent molecules work?
Fluorescein and rhodamine absorb light of high energy and emit light at lower energy
Rhodamine(green) emits red
T/F: Fluorescent molecules always emit a light of longer wavelength than you were shining on the sample?
True
Modern fluorescent microscope?
Epi-fluorecence microscope
Autofluorescence
Cells nautral fluorescence
Three ways to label proteins for fluorescence?
- Chemically label the protein outside the cell and then add it into the cell
- Create an antibody against the protein and then stain the cell with the antibody
- Fuse the protein of interest with Green Fluorescent Protein (GFP) and express
