Lecture 1

Question 1

T/F: Compound microscopes have multiple lenses

Answer 1

T

Question 2

Light microscopes allows us to see things on what scale?

Answer 2

Micron

Question 3

What structures are hard to see with light microscopes ?

Answer 3

-Nanometer scale
- Two structures less than 250 nanometers apart

Question 4

What is used to see things on a nanometer scale?

Answer 4

Electron microscopes

Question 5

Nanometer to micron

Answer 5

1 nanometer is 1/1000th of a micron

Question 6

What scale can we see with the naked eye ?

Answer 6

1/4 mm to bigger

Question 7

What scale can be see with the LM?

Answer 7

something larger than 1mm all the way down to something with structures that are separated more than 250 nanometers apart

Question 8

What limits LM?

Answer 8

Resolution of LM is limited by the fraction of light

Question 9

What can be seen in LM?

Answer 9

• Organelles is plant/animal cells
  -Bacterium but not their internal structure

Question 10

What can be seen in EM?

Answer 10

-Internal structure of bacterium
-All the way down to a structure the size of an atom

Question 11

What limits EM?

Answer 11

How long your sample can go without being cooked

Question 12

Which technique EM or LM can be used to image live cells?

Answer 12

Light microscope

Question 13

Transmission Microscope

Answer 13

-Light source(bulb/mirror)
-Condenser
-Ocular lense

Question 14

Transmitted LM

Answer 14

Light is transmitted through the sample of interest and can be absorbed which creates contrast and a visual of the sample

Question 15

Two types of transmitted LM

Answer 15

1. Brightfield
2. Phase Contrast

Question 16

Why is cells being thin a problem for LM?

Answer 16

Cells that are thin and transparent will be hard to see using brightfield illumination since there is no contrast

Question 17

How do we deal with thin cells for LM?

Answer 17

We use stains for dead cells
or we use special optics for live cells

Question 18

Types of special optics?

Answer 18

-Phase contrast
-Differential-interference contrast
-Dark field

Question 19

How can we look at cells within tissues ?

Answer 19

1. Fix them
2. Embed them in paraffin
3. Section them
   This makes them small enough. to visualize. Hard to keep these alive when we section

Question 20

Tissue Culture cells

Answer 20

-Fried egg shape, flat, easy to work with
-Can be fixed or alive

Question 21

Problem with tissue culture cells?

Answer 21

Less representative of human cells since they are grown and don’t come directly from us

Question 22

How do we observe unstained live cells?

Answer 22

Inverted microscope prevents the microscope from getting wet since objetive is below the sample

Question 23

Organ culture?

Answer 23

Keeping the kidney alive by constantly perfusing it with nutrient solution

Question 24

Explant culture?

Answer 24

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

Question 25

Primary cells

Answer 25

Cells that were actually at one point in the organism of interest

Question 26

Continuous cell lines?

Answer 26

When primary cells grow and divide and produce cells that have grown completely in an incubator

Question 27

Mortalized Cell line?

Answer 27

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

Question 28

T/F: Older organisms reach crisis faster?

Answer 28

True

Question 29

Immortalized cell lines

Answer 29

These cell lines express telomeres which prevent chromosome shortening during division and prevent the “crisis” from occurring ex. germ cells

Question 30

Transformed cells

Answer 30

Cells lose contact inhibition and have abnormal mitosis. Ex. Cancer cells, mutated cell lines

Question 31

Contact inhibition

Answer 31

Culture cells will only form a single monolayer on a plate and then will not continue to grow

Question 32

Contact inhibition in cancer cells

Answer 32

Cancer cells/transformed will continue to grow and piling up on each other due to a loss in the ability to regulate growth

Question 33

Artificial Medium used to maintain cells?

Answer 33

-pH 7.4 -Nutrients -Glucose -Serum(growth factors) -Antibiotics -37 degrees Sterile environment

Question 34

Why do we add antibiotics to artificial medium?

Answer 34

Since cells divide every 24 hours bacteria can get in and divide every 20 minutes. Prevents bacteria from taking over

Question 35

Advantages of fluorescent light microscopy

Answer 35

-No background allows you to focus on interest of study

Question 36

How do fluorescent molecules work?

Answer 36

Fluorescein and rhodamine absorb light of high energy and emit light at lower energy Rhodamine(green) emits red

Question 37

T/F: Fluorescent molecules always emit a light of longer wavelength than you were shining on the sample?

Answer 37

True

Question 38

Modern fluorescent microscope?

Answer 38

Epi-fluorecence microscope

Question 39

Autofluorescence

Answer 39

Cells nautral fluorescence

Question 40

Three ways to label proteins for fluorescence?

Answer 40

1. Chemically label the protein outside the cell and then add it into the cell 2. Create an antibody against the protein and then stain the cell with the antibody 3. Fuse the protein of interest with Green Fluorescent Protein (GFP) and express