1st week (Lecture 1-4)

Question 1

What are intensity profiles

Answer 1

method to turn fluorescent microscopic images into graphs

Question 2

How to make intensity profiles

Answer 2

1. Targeted parts of the cells fluoresce differently by using compounds that bind to specific structures.
2. Line is then drawn to bisect cell
3. Graph intensity of colours as a function of location (along the line)

Question 3

Types of light microscopy

Answer 3

Brightfield, Phase Contrast, Differential interference contrast (DIC), Widefield Fluorescence, Confocal fluorescence

Question 4

Brightfield Microscopy

Answer 4

visible light passes through specimen
BRIGHT background
usually needs stains to make cells visible

Question 5

Phase Contrast Microscopy

Answer 5

visible light passes through specimen
more complex lenses, thus better contrast than brightfield
halo around cells

Question 6

Differential interference contrast (DIC)

Answer 6

better lenses than Phase contrast = better contrast
shadow around cells

Question 7

Widefield Fluorescence microscopy

Answer 7

short wavelength hits specimen and longer wavelength reflects off (high to low energy)
needs fluorescent molecules i.e DAPI, fluorescein

Question 8

Composite images

Answer 8

monochromatic images captured, given colour, and superimposed
either additive or subtractive (think of psych)

Question 9

Confocal fluorescence microscopy

Answer 9

lasers reflects off specimen
shows 2D plane within 3D specimen (like a cross section)
more detail than widefield but more expensive

Question 10

Tomography

Answer 10

multiple confocal fluorescence images assembled to make a 3D model

Question 11

Preparing slides

Answer 11

1. Getting specimen onto slide
2. Making things visible

Question 12

Getting things on slide

Answer 12

Either the whole cell if small enough or a thin section of a cell/tissue cut with a microtome

Question 13

Ultrathin Sectioning

Answer 13

Done with Microtomes

Question 14

Coloured Stains

Answer 14

2 properties: affinity for target & absorbing light
i.e hematoxylin (blood & RNA), Eosin (proteins)

Question 15

Fluorescence Stains

Answer 15

2 properties: affinity for target & fluoresces light
i.e. DAPI makes nuclei fluoresce blue

Question 16

Fluorescent Probes

Answer 16

2 different molecules joined covalently
1 with affinity for target and 1 fluoresces light
i.e antibodies with Alexa Fluor 488

Question 17

Light vs Electron Microscope

Answer 17

Light: 1000 X, visible or UV light, glass/quartz lens, detected by eye/camera
Electron: 20 000X (SEM) 100 000X (TEM), beam of electrons, electromagnet lens, detected by camera

Question 18

Types of Electron Microscopy

Answer 18

Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM)

Question 19

Transmission Electron Microscopy (TEM)

Answer 19

Beam of electrons PASSES THROUGH specimen, 2D image

Question 20

Scanning Electron Microscopy (SEM)

Answer 20

Beam of electrons BOUNCES off specimen, 3D image

Question 21

Preparing specimens for SEM

Answer 21

• cover specimen (sputter coating) with heavy metal atoms so electrons can bounce off i.e coating with gold

Question 22

Preparing specimens for TEM

Answer 22

1. Ultrathin sectioning + metal atom staining (very common)
2. TEM tomography (several Ultrathin sections assembled to make a 3D model)
3. Ultrathin sectioning + (metal atom staining + gold-labelled antibodies covalently attached)
4. Negative staining
5. Shadowing (metal atoms sprayed at an angle)
6. Freeze-fracturing + shadowing

Question 23

Negative Staining (For TEM)

Answer 23

• done when specimen is VERY small
• stain area surrounding specimen
• specimen highlighted against a background of metal atoms

Question 24

Freeze-fracturing + shadowing

Answer 24

• see inside of membranes (top or bottom part)
• specimens are frozen in “freon”
• cuts through membranes
• one of the most common

Question 25

What's part of the Nucleus

Answer 25

Nuclear Envelope, Nucleolus & Chromatin

Question 26

What's part of the Cytoskeleton

Answer 26

Microfilaments (Actin Filaments) , Intermediate filaments, microtubules

Question 27

Cell Theory

Answer 27

Organisms made of one or more cells Cell is the basic unit of organisms cells arise from other cells

Question 28

Cell Specialization

Answer 28

i.e flagella (sperm), microvilli (intestine), chloroplasts

Question 29

Unicellular Eukaryotes

Answer 29

Paramecium, Green Algae, Yeast

Question 30

Multicellular Eukaryotes

Answer 30

Animals: Collagen (protein) as connecting materials Plants: Cellulose (polysaccharide) as connecting mat.

Question 31

5 categories of Human Cells

Answer 31

Epithelial tissue (i.e covering for body surfaces) nervous tissue (i.e motor neurons) muscle tissue (i.e skeletal muscle cells) connective tissue (i.e osteoblasts) blood cells (i.e red blood cells)

Question 32

Small intestine

Answer 32

absorb nutrients (i.e. amino acids, protein, etc.) Duodenum, Jejunum, Ileum respectively

Question 33

Large intestine

Answer 33

recover water before waste is removed

Question 34

Things that increase Nutrient Absorption in the Small Intestine

Answer 34

The organ is long and very coiled up it has folds in the inner membrane Villi & Microvilli (all increase Surface Area)

Question 35

Types of intestinal cells

Answer 35

Absorptive enterocytes ~90% Goblet Cells ~10% Paneth Cells <1% Enteroendocrine cells (EECs) <1%

Question 36

What are Microvilli filled with

Answer 36

Actin Filaments

Question 37

Absorptive enterocytes

Answer 37

make up ~90% of intestinal cells. import nutrients

Question 38

Goblet Cells (secretory cell)

Answer 38

make up ~10% of intestinal cells export protective mucus into gut via secretory vesicles performs exocytosis

Question 39

Paneth Cells (secretory cell)

Answer 39

make up <1% of intestinal cells export (exocytosis) anti-bacterial proteins into gut "P"aneth = "P"rotein

Question 40

Enteroendocrine cells (EECs)

Answer 40

make up <1% of intestinal cells export hormones into blood "entero" = intestine "endocrine" = secrete into blood

Question 41

Stem cells

Answer 41

either reproduce (not by division) or mature into one of the intestinal cells located at the base of villi and matures upwards

Question 42

How do stem cells mature

Answer 42

1. Reproduce (Self-Renewal) 2. AE or one of the other 3 secretory cells (EECs, Goblet, Paneth Cells) 3. EECs or one of the other 2 (Goblet or Paneth Cells) 5. Goblet Cells or Paneth Cells