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Histology (Emily) > Lecture 1 - Microscopy, Staining, Cell Membrane Organelles > Flashcards

Lecture 1 - Microscopy, Staining, Cell Membrane Organelles Flashcards

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1
Q

Resolution

A

measurable distance b/t 2 points at which they can still be seen as separate

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2
Q

What is the resolution of the human eye?

A

0.2 mm

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3
Q

____ wavelength = better resolution

A

Shorter wavelength = better resolution (light < UV < EM)

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4
Q

Light Microscope

A

light, condenser lens condenses light on specimen, passes through to objective lens, ocular lens enlarges virtual image.

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5
Q

TEM (Transmission Electron Microscopy)

A

same as light except cathode emits electrons through sample (done in vacuum)

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6
Q

SEM (Scanning Electron Microscope)

A

electrons reflect off specimen (coat in metal). Get a 3D view of the specimen, shows what’s on the surface of membrane not what’s inside. Resolution is not as good as TEM.

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7
Q

Atomic Force Microscope (AFM)

A

non-optical, cantilever w/ sharp tip dragged along specimen, laser pointed at tip of cantilever and reflect to photodiode (stationary, the stand moves to hit the it)

  • bumpy images, very high resolution (can see single DNA molecule, nuclear pore etc.)
  • No fixation, can look at living cells; examine dynamic changes
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8
Q

What are the six steps to preparation of samples?

A
  1. Fixation: stop metabolism, cross-link proteins to make them inactive, prevents enzymatic degradation, kills pathogens.
  2. Dehydration: alcohol up to 100%, 70% common; removes fat, mucosa, golgi etc.
  3. Embedding: allows specimen to be thinly sliced
  4. Sectioning
  5. Mounting
  6. Staining
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9
Q

Fixation for FM v. EM

A

LM: Formaldehyde

EM: Glutaraldehyde

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10
Q

Embedding for LM v. EM

A

LM: Paraffin

EM: Epoxy resins

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11
Q

Sectioning LM v. EM

A

LM: Microtome (metal knife)

EM: Ultra-microtome (diamond/glass)

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12
Q

Mounting LM v. EM

A

LM: Glass Slide

EM: Copper grid

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13
Q

Staining LM v. EM

A

LM: Hematoxylin and Eosin

EM: Heavy Metals

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14
Q

What type of dye is Hematoxylin? Types of structures stained?

A

Basic dye (acidophilic stain)

Stains acidic structures BLUE

Stains DNA, ribosomes, nucleus

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15
Q

What type of dye is Eosin? Types of structures?

A

Acidic dye (Basophilic stain)

Stains basic structures PINK

Stains cytoplasmic stuff, proteins, protein-filled vesicles

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16
Q

Acidophilic

A

Loves acid, therefore basic and positively charged (BLUE)

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17
Q

Basophilic

A

loves base, therefore acidic and negatively charged

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18
Q

PAS (Periodic Acid-Shiff Reaction)

A

Carbohydrates stain purple. Used to demonstrated glycogen in cells, mucus (goblet cells), basement membrane, and reticular fibers.

  1. Periodic acid cleaves bond between adjacent carbon atoms to make aldehydes.
  2. Aldehyde groups react with Schiff reagent.
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19
Q

Mallory’s Trichome

A

stains connective tissue (collagen – blue)

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20
Q

Toludine Blue

A

stains mast cells dark blue/purple

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21
Q

Mucicarmine

A

Stains mucus (pink)

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22
Q

Silver Stain

A

Stains for reticular fibers

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23
Q

Immunocytochemistry

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A

Using antibody with specific marker to bind specifically to molecule of interest or specific cell/tissue type (like melanocytes).

Usually used with enzymes.

24
Q

Autoradiography

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A

Radioactive labeled molecules can be injected into animals/tissues and traced to localize the larger molecules in cells and tissues.

25
Enzyme Histochemistry
Using capture agent to precipitate reaction product of the enzyme, not the enzyme itself. e.g. lysosome was identified by precipitating lead ions derived from acid phosphatase activity.
26
Hybridization Techniques
Localizing mRNA or DNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe. Ex. FISH (gene testing by probing chromosomes)
27
Plasma Membrane
- asymmetric, dynamic fluid structure of amphipathic lipids, cholestorol, proteins, glycoproteins and glycolipids - held together by non-covalent interaction between lipids. - more proteins on inner P-face (covered more in cell membrane!)
28
Lipid Rafts
Multiple signaling platform; enriched in glycosphingomyelins (stick out above surface w/ long lipid tails) & cholesterol, thus more dense area of glycogen & protein on “ocean” of lipids
29
Integral Membrane Proteins
Imbedded in membrane (pumps, receptors, channels, enzymes, structural, linkers)
30
Peripheral Membrane Proteins
Not attached to membrane, but attached to membrane proteins. E.g. glycocalyx and microvilli
31
Rough Endoplasmic Reticulum (rER)
- protein synthesis w/ ribosomes for secretion, cell membrane, and internal needs (acidic = basophilic! BLUE) - volume occupied by rER – best LM indication of cell activity; if large, cell is producing lots of protein
32
Smooth Endoplasmic Reticulum
Synthesize steroid hormones, degrade and detox alongside lysosomes and peroxisomes (alcohol abuse shows a lot of sER in liver cells), glycogen metabolism in liver cells, and regulate Ca2+ release into cytoplasm for muscle contraction.
33
Golgi Apparatus
- cisternae layers, vesicles bud from rER and go to **cis-face** (first layer) of Golgi - **trans-face** towards cell membrane - complete synthesis of lipids and proteins through post-translational modification: trim off singal peptides from sequence, hydroxylation of a.a. residues, and glycosylation to make polysaccharides **- sorts/packages proteins into secretory vesicles for export** - Often washed away during stain process b/c contains lots of lipids = negative staining of where golgi used to be!
34
What are the two types of pathways for protein secretion?
1. Regulated 2. Constitutive
35
What is regulated protein secretion?
Some sort of regulatory event triggers secretion. Ex. Clathrin-coated vesicles accumulate and wait in cytoplasm, influx of Ca2+ regulates secretion. If regulated secretion is common in cell, see presence of clathrin-coated vesicles (very pink stored protein).
36
What is constitutive protein secretion?
Immediate/constant secretion of synthesize proteins, ie. collagen in fibroblasts, plasma cell antibodies. Not all have regulated secretion.
37
Endosomes and Transport Vesicles - How are they formed?
Formed folling budding from plasma membrane or Golgi, coated by clathrin. 1. cargo protein reaches cargo receptor at plasma membrane 2. *adaptin* binds to receptor allowing clathrin to interact with receptor 3. *dynamin* cuts the vesicle from the membrane 4. vesicle uncoats and goes to specified location in cell
38
Early Endosome
Sorting of vesicles, those not sent to other places go to late endosome
39
Late Endosome
Acidifying, become lysosomes. Found near Golgi and nucleus.
40
Lysosomes
- Formed from late endosome by delivery of hydrolytic enzymes. - hydrolysis of molecules & organelles – how cell gets nutrients; recycling of amino acids/monosaccharides; destroy foreign organisms; bone reabsorption - has unique membrane that is resistant to hydrolytic enzymes à membrane contains cholesterol and lipids that are highly glycosylated. pH decreases as the lysosomes mature (more acidic)
41
What role does Mannose-6-phosphate play in lysosomes?
M6P directs enzymes to late endosomes.
42
What is I-Cell disease?
No M6P, lots of vescicles with lysosomal enzymes! Eventually unsustainable.
43
Mitochondria
- mobile power generators (ATP) with highest ATP synthase and protein pump concentrations in inner mitochondrial membrane; very distincted membrane covered “bean” shape and cristae. - own genome and complete system of protein synthesis - can initiate apoptosis via release of cytochrome C from intermembrane space = caspases cascade
44
Nucleus - LM and EM features
LM: Largest organelle, distinct dark color EM: Double membrane, dark nucleolus, nuclear pores, dark heterochrom/light euchromatin
45
Nucleolus - LM and EM Features
LM: Not visible unless in living cells EM: Very dense and granular within nucleus
46
Plasma membrane - LM and EM Features
LM: Not visible; can see outline but not the bilayer structure; PAS staining EM: Inner and outer electron dense layers separated by electron lucent layer
47
rER - LM and EM Features
LM: A basophilic region of cytoplasm EM: Flattened sheets and sacs with ribosomes!
48
sER - LM and EM Features
LM: Not visible EM: Flattened sheets
49
Golgi - LM and EM Features
LM: Negative staining; washed away EM: Flattened membrane sheets next to nucleus
50
Mitochondria - LM and EM Features
LM: Not visible EM: Double membrane, cristae
51
Endosome - LM and EM Features
LM: Not visible EM: Tubulovesicular with subdivided lumen
52
Lysosome - LM and EM Features
LM: Only with histochemical staining EM: Membrane bound vesicle, electron dense
53
Peroxisome - LM and EM Features
LM: Only with histochemical staining EM: Membrane bound, electron dense, crystalloid inclusions
54
Cytoskeleton - LM and EM Features
LM: Not visible unless in large fibrils EM: Long linear staining pattern with each type
55
Ribosomes - LM and EM Features
LM: Not visible EM: Small dark dots, often by rER
56
Lipid Droplets - LM and EM Features
LM: Washed out holes, peripheral nuclei EM: Nonmembranous, void

Histology (Emily) (8 decks)

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