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ANAT 262 > Microscopy I > Flashcards

Microscopy I Flashcards

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1
Q

How do we know what we know about cells

A

Lm and em
Biochemical techniques
Genetic techniques - yeast
Combos of any of these

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2
Q

When lm important

A

Most important until 1950s - bc not much biochem

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3
Q

When EM important

A

Dominant from 1950-70s

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4
Q

WHAT dominated from 80s-now

A

Combo of biochem and yeast genetics - dominated from 1980s - present

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5
Q

What is making comeback

A

Light microscopy
Bc new ability to follow dynamics of proteins in living cells

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6
Q

Antony van Leeuwenhoek

A

1632-1723
Made microscopes - powerful magnifying glass
Little animals - rotifers, multicellular animals but small
Visualized yeaSt

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7
Q

Robert Hooke

A

1635-1703
Invited compound microscope- multiple lenses, easier to make
Contemporary of van Leeuwenhoek but English
Gave name cells to statures he saw in cork and in wood
Spread idea of microscopy

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8
Q

Describe cell = generally

A

Bacterial cell = 1-2microns
Polarized epithelial cell = 15 microns, bigger

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9
Q

Describe micronscale - image

A

Light micropscpae =
Antibody staining
See 15 microns, cell, nucleus has no mcirotubuels, see microtubuels outside nucleus
Many parts of cell = too small, cannot see details
Large cell = 30microns, Rbcs = 4micro
100microns = human hair, so large cell = 1/3 of human hair on avg

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10
Q

Describe nanometers scale

A

Electron microscope - cannot see with lm
Higher resolution = can visualize protein molecules and diff aas
Axons of neurons (diameter = 1 micron), see myelin, more prep for em samples
Microtubule = 24nanometer diameter- diffusion of light makes it look thicker than is
But if 2 objects = less than 250 nanometers = cannot resolve, images blend together
Limited by resultion of light =lm
CANNOT SEE THIS WITH LM

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11
Q

Describe pm of cell- em image

A

Bilayer = 7-8nm typically, depends on where cell is (1 nm = 1/1000 of micron)
Can see the 2 layers with em
Hydrophobia inside,2 fatty acid chains, integral membrane proteins embedded in membrane
Cannot see head groups - but just see 2 layers and space inside
Mid 1950s, better now

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12
Q

What can we see with naked eye

A

1cm-less than 1 mm
1/4 mm or bigger, 250microns (1mm = 1000 microns)

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13
Q

What can we see with lm

A

Large range - 1/4mm to less than 1 micron
Plant cell,animal cell (10-30microns), bacterium
For internal stature - shapes outside

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14
Q

What can we see with em

A

Em = 1/4 mm to o.1nm = atom
Can see v small things, plant cell, animal cell,virus, ribosome, globular protein, small molecules, atom
Get good internal structure

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15
Q

What are issues with lm

A

Resolution limited by diffraction of light - as long as not closer than 0.25 microns/250nm

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16
Q

What are issues with em

A

Energy to sample and cook in process - poor contrast, heavy metal staining = problems
More elaborate sample prep - cannot be done for live cells

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17
Q

Micrometer, nanometer, Angstrom units

A

Um = micrometer = 10^-6m
Nm =nanometer = 10^-9 m
A = Angstrom unit = 10^-10 m

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18
Q

Describe light microscopy

A

In bio labs
Compound microscopes used
Flourecnse microscopes used too
Now common = new kind fluorescence microscope = Confocal microscope

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19
Q

Describe conventional transmission light microscope

A

For histology
To see sample = light goes through it= some absorption or something else that created contrast
Illuminator, mirror = reflects light
Stage position adjustment = light absorption/contrast
Condenser = sends light through it then can see specimens

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20
Q

Name all the types of lm

A

Transmitted light = brightfield, phase contrast
Fluorescence
Confocal = specialized kind fo Fluroesnce micropsopy - deals with large or 3d samples
Super resolution - recent, Much better resolution

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21
Q

Are cells opaque

A

Most cell = transparent
Very thin

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22
Q

How to make cells visible

A

Normal - brightfield* illumination = cells v haard to see
To make visible = staining with dyes (hematoxylin followed by eosin)
Or use special optics

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23
Q

Name special optics

A

GOOD FOR LIVE CELLS
Phase contrast*
Differential interference contrast = dic/nomarksi
Dark field - illuminated from side, look at scattterd light

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24
Q

Name types of samples

A

Cells in tissues
Tissue culture cells
Live vs fixed cells

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25
Describe cells in tissues
Usually fixed, embedded in paraffin wax or plastic or freezed = usually for thicker samples
26
Describe tissue culture cells
Usually shaped like fried eggs - very flat Much easier to work than tissueswith but usually not normal - less representative of body
27
Describe live vs fixed cells
Tissue culture cells = can be fixed or alive - actual intact tissues hard to work with alive (formaldehyde) - cannot section and must keep alive but sometimes still done
28
Which cells are easier to deal with
Tissue culture cells
29
Describe resp epi - lm
Get cells in correct 3d representation Cannot do this with tissue culture - bc lose 3d relationships Cells in tissues
30
Describe bright field
No staining Can see lysosomes, nucleus’s endosomes
31
Describe phase contrast
No staining Works for live cells
32
Describe dark field
Lysosomes around nucleus Packed with protein - scatters light, so v visible with dark field
33
Advantages of methods to observe unstained live cells
Allow prolonged observation of live cells Movements in cell division and of intracellular structures can be studied Particularly if filmed - microcinematography - recorded on videotape or camera
34
How are live cells microscoped
Inverted microscopes used = Objective - comes from below, coverslip in bottom of dish - keep dry from media
35
What is used for cell biological research
Tissue culture cells Bc = microscopy of cells in tissue culture = v easy
36
What is tissue culture
Cells grown outside organism
37
Describe organ culture
Done sometimes Perfume with solution, tricky tho Keep most of physiological conditions from a. Living organism
38
Describe explanations culture
Host - dissect - fine chop organ, and do primary explant culture- lay on coverslip on media solution - can be grown Easier to maintain in culture - and still presents 3d cell organization
39
Describe primary cells
Use protease = trypsin to digest and separate tissues = grow and maintain on dish, new cells = grow up in incubator
40
Describe continuous cell lines
Primary cell culture - must be able to go through mitosis Digress connections with substrates 1st passage - 1:3 split ratio - vary depends on cell type - can keep going, could be 1:20 or more = can repeat this process But after 30-40 times = will have crisis, telomeres shorten
41
Describe immortalized cell lines
No crisis, no death
42
How do cells grow
Grow only in monolayer culture due to contact inhibition properties
43
What exhibits contact inhibition
Tissue culture cells obtained from primary cultures = will eexhbit = form single monolayer on a plate - will not continue to grow once space filled
44
What lacks contact inhibition
Cancer cells (hela cells) lack it = will continue to grow In tissue culture and pile up on each other = transformed cells Oncogenes - lost ability to regulate growth, can also transform in culture with virsues
45
Define primary cells
Same cells obtained from source Not immortalized or transformerd (But primary cells obtained from a cancer are usually transformed cells)
46
Define non immortalized cell line
Primary cells = reproduce in tissue culture Can be grown for many generations in tissue culture but not indefinitely
47
Define immortalized cell line
Mutations = like Telomerase expressed = allow for indefinite growth in tissue culture Cells otherwise retain good behaviour = contact inhibition
48
Define transformed cell line
Cells lose contact inhibition and have other abnormalities - like abnormal mitosis Equivalent to cancer cells, almost always immortalized Easy to grow- used in labs a lot
49
How can cell lines become transformed
Other cell lines can become transformed = through mutations or through use of viruses or introduced dna to express oncogenes
50
Name the 3 things needed for maintaining cells in culture
Artificial medium Temperature control Sterile environment - Prevent external contamination
51
Describe artificial medium
Physiological ph = 7.4 Nutrients- aas, vitamins, salts Glucose Serum - growth factors Antibiotics - option
52
What is needed for physiological ph
Carbonate buffer, co2 gas, ph indicator (phenol red - turns yellow bc cell produces waste)
53
Describe why we put antibiotics in cell culture
Cells divide every 24 hrs - but bacteria divides very fast - so put antibiotics in case external contamination
54
Describe temp of cell culture
37 degrees Celsius Humified environment
55
Describe fluorescent molecules - fluorescence microscopy
Shine one colour and get another off it Fluorescent molecules - like fluoresciene (green) or rhodamine (red)- excited by green light, absorb light of high energy (short wavelength, blue = flouresces green) and emits light at lower energy - longer wavelength
56
Describe fluorescent microscopy - method
Tissue and cells are irritated with a blue violet or ultraviolet light so that emission is in visible part of spectrum - could also use infrared waves
57
Describe fluorescent microscopy - results
Fluorescent structures appear as bright an coloured on black background
58
Describe fluorescent microscopy - sensitivity
Sensitivity of method very high
59
Describe early fluorescence microscopes
Not her great See something fluoresce Gas/liquid excitation filter = blocks uv rays - need filter to get ride of excitation light - must be efficient, and selective but hard to fine one Carbon arc lam = provides uv rays
60
Describe epi florescence microscope
Blue light to excite - selective filter Beam splitting mirror = reflects blue and transmitts green Then blue hits sample, objective lens - fluorescence excitation from objective lense and also the condenser 2nd filter barrier - then green light goes through to eyeball
61
Describe locating modules with fluorescence - gen
Cells have some slight natural flureosnce - auto fluorescence - not useful most of Tim e exception = chlorophyll Need techniques to label proteins of interest with Florenscnet molecule
62
Name the 3 ways to label proteins
1- Chemically label protein outside cell and add it - doesn’t always work 2- Label antibody against protein and stain cell - but cell must be formaldehyde fixed and permeabilized 3- Fuse protein of interest with gfp and expressed (recomb gene in living cell)
63
Describe micro injection
Immuno + gfp used Controlled = used to potion needle Foot pedal = produce gas pressure behind needle and once on cell = can inject, 50% chance of killing cell

ANAT 262 (24 decks)

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