Lecture 1

Question 1

Name the techniques used to study cells

Answer 1

1) microscopy (light and electron)

2) biochemical techniques

3) genetic techniques (particularly in yeast)

4) combinations of the above

Question 2

Define light microscopy

Answer 2

Study of cells using visible light and lenses to magnify and resolve cellular structures

Question 3

Define transmission light microscopy

Answer 3

Technique using transmitted light to visualize internal cellular structures

Question 4

Define tissue culture

Answer 4

Growth of cells from a tissue or organism in a controlled artificial environment (lab)

Question 5

Define fluorescence light microscopy

Answer 5

Microscopy using fluorescent dyes to label specific cellular structures for visualization (important for cell biology)

Question 6

Name the history of microscopy

Answer 6

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

Question 7

What is microscopy?

Answer 7

Study of objects too small to be seen by the naked eye using light and electron microscopes

Question 8

What are biochemical techniques?

Answer 8

Methods to study biological processes at the molecular level, e.g., protein purification and analysis

Question 9

What are genetic techniques?

Answer 9

Methods to manipulate and study genes, e.g., gene cloning and sequencing (especially in yeast)

Question 10

What is yeast?

Answer 10

Single-celled fungi often used in genetic research due to their simple genetics

Question 11

What is electron microscopy?

Answer 11

Microscopy using a beam of electrons to visualize the ultrastructure of cells and tissues

Question 12

What are Leeuwenhoek’s animacules?

Answer 12

Microorganisms observed by Leeuwenhoek, including yeast and rotifers

Question 13

What are rotifers?

Answer 13

Small multicellular organisms

Question 14

What did Robert Hooke do?

Answer 14

• 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

Question 15

What is the micron scale?

Answer 15

Scale of measurement in microscopy, equivalent to one millionth of a meter

Question 16

What is the nanometer scale?

Answer 16

Scale of measurement in microscopy, equivalent to one billionth of a meter

Question 17

Which type of microscopy would you use to visualize a bacterium?

Answer 17

• 1-2 microm across
• can tell general shape but not any detail inside w LM -> need to be clever

Question 18

Which type of microscopy would you use to visualize a columnar epithelial cell w microvilli in respiratory epithelium?

Answer 18

• 15 microm > 2/10 microm -> can distinguish details
• most organelles/membrane bound compartments are big enough to distinguish (only see microvilli + nucleus)

Question 19

slides14,15,16,17

Question 20

What microscopes are used in biology labs?

Answer 20

• 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)

Question 21

Name the important components of a conventional transmission microscope (light)

Answer 21

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)

Question 22

What are the important types of light microscopy?

Answer 22

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)

Question 23

How do you study a cell if it is transparent under normal brightfield illumination?
(aka magnify cell without treating it in any way)

Answer 23

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

Question 24

What are the different kinds of samples?

Answer 24

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

Question 25

Compare the different techniques for studying live cells without staining

Answer 25

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

Question 26

What is the advantage of methods to observe unstained live cells?

Answer 26

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

Question 27

Why are tissue culture cells often used in cell biological research?

Answer 27

microscopy of cells in tissue culture is very easy

Question 28

What are the 3 types of tissue culture?

Answer 28

1) organ/explant culture 2) primary cells 3) continuous cell lines

Question 29

Compare the 3 types of tissue culture (slides 31-36)

Answer 29

1) organ/explant culture: keeps most of the physiological conditions from a living organism 2) primary cells 3) continuous cell lines

Question 30

Define contact inhibition

Answer 30

- 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

Question 31

Do cancer cells exhibit contact inhibition?

Answer 31

ex: HeLa cells No, they will continue to grow in tissue culture, piling up on each other they are called "transformed cells"

Question 32

Name the 4 types of tissue culture cells

Answer 32

1) primary cells 2) non-immortalized cell line 3) immortalized cell line 4) transformed cell line

Question 33

Describe primary cells

Answer 33

= same cells obtained from the source, not immortalized or transformed !!transformed only if obtained from a cancer

Question 34

describe non-immortalized cell line

Answer 34

= when primary cells are alowed to reproduce in tissue culture for many generations (not indefinite)

Question 35

describe immortalized cell line

Answer 35

= cell with mutations allowing indefinite growth in tissue culture (ex: allowing telomerase expression)

Question 36

describe transformed cell line

Answer 36

= 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

Question 37

How do you maintain cells in culture?

Answer 37

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

Question 38

Fluorescence microscopy