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Cell Structure and Dynamics > Lecture #1 - Strategies for Cell Biology > Flashcards

Lecture #1 - Strategies for Cell Biology Flashcards

1
Q

Cell membrane

A

Lipid bilayer driven together by hydrophobic effect

Hydrophobic effect - hide the hydropobic fatty acid tails from the outside

Often have integral membrane proteins that cross the bilayer and bound peripheral proteins

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2
Q

Cell Membrane Peripheral proteins

A
  1. Can be bound directly to the lipid head by charge
  2. Can have soluble protein with lipid group that can intercalate into membrane
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3
Q

What surrounds/seperayes organelles

A

Cell is defined by membrane compartments

There are membranes surrounding all organelles that separate the organelles from one another in a cell (Ex. Membrane around golgi + membrane around mitocondria etc.)

EXCEPTION - Biological condensates us liquid phase seperation (condesates are beleived to be functioning organelles)

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4
Q

Non-ionic detergents Vs. Ionic Detergents

A

Non-ionic detergents (triton X) –> disrupts the lipid bilayer (makes micelles)
- Gentle –> does NOT destroy the structure of proteins or interactions with one another

Ionic (SDS) –> destroys the lipid bilayer AND will denature proteins
- Lose protein-protein interactions + lose the structure of proteins

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5
Q

Plant Vs. Animal Cell Cell Compartments

A

Animal cell (left) ; Plant cell (right)
- Plant cell = has cell wall + chlroplorast

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6
Q

How do we know the structure of cels

A

1670 – can see cells BUT the details of cell compartments was found in 1960s

1960s – used electron microscope to see cells in detailed (High resolution of cell structure )

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7
Q

Subcellular Fraction - Use

A

Method for purifying organelles in order to study them (function, localization of protein, reconstitute fave process in vitro)

Process:
Start with whole cell lysate –> Disrupt cell without detergents (membrane and organelles stay intact) + add homogenization buffer for osmotic support (mimic osmotic strength of cytosol) –> break open cell and organelles spill out –> centrifuge at different speeds to collect organelles based on size

Ex - Where is my protein of interest in the cell?

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8
Q

Subcellular Fractionition centrifugation

A

When centrifuge organelles the larger and more dense go down faster

Organelles have different densities = can seperate them

Example – Nuclei go to pellet –> then take the supernatnet and spin again and the mitocondria will go to pellet etc.
- Order – broken cells –> nuceli –> mitocondra/lysosomes/peroxisone sediment –> plasma memebrane and ER sediment –> ribosomes sediment (in cytosol)

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9
Q

In subcellular Fractionatiion organelles are seperated by

A
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10
Q

Velocity gradient Vs. isopycnic

A

Veolicity Gradient = seperates based on size

Isopynic Gradinet = sucrose gradnient (isolates based on density)
- Needed to separate mitochondria from lysosomes and peroxisomes

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11
Q

Co-Immunoprecipitation - Use + Process

A

Use - to know which proteins interact with other proteins in cells + purify protein of interest

Process - –Add AB for a protein of interest to whole cell lysate –> add beads for AB –> IF the protein is bound to other proteins in a complex when you centrifuge the protein of interst bound to the bead you will get the protein of interest and the other proteins that it is bound to

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12
Q

Co-IP for proteins in membrane

A

Need to keep the protein complex in tact

IF the complex is in the membrane then you need to free the proteins from the membrane and add a non-ionic detergent because Ionic would separate the proteins from the binding partners

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13
Q

What do you run for Co-IP

A

Cell with protein and cell without protein (control) –> run a gel to separate the proteins in pellet

Gel:
- See that there is a lot of non-sepcifc binding (have bands in the cell without protein – would ignore those in the ‘cells with protein’ lane)
- See protein of interest (indicated by purple dot) in cell with protein lane + get other bands that are NOT in cell without protein –> THOSE proteins are likely in a complex with the protein of interest

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14
Q

Proximity labeling - Use

A

Label proteins that tanseintley intercat with protein of interest (find binding partners and protein – protein interactions)

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15
Q

Proximity labeling - Process

A

Process – Bait protein is fused gentically to enzyme (BirA) that biotynylates nearby proteins when biotin is added –> BirA adds biotin to nearby proteins –> run recation for some time –> stop reaction –> NOW have protein that is covalent bonded to biotin

Once have biotin bound to proteins – use Ionic detergents to separate the proteins –> immunoprecipiate the proteins using biotin –> separate the proteins and identify the proteins that were close to the protein of interest
- Can separate the proteins because the proteins are already taged with the covalent tag

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16
Q

Pulse-Chase labeling - Use

A

Use - Following proteins around a cell (looking to see if the protein is moving) + can see if something is post-translatinoally modified
- Trace DNA or RNA or Protein turnover/movement in cells

Example – labeling proteins –> Protein is first seen in the ER –> THEN the radioactivty is in the golgo –> THEN the portein becomes glycosylated

Overall:
Pulse with radioactive molecule
Chase with unlabeled molecule

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17
Q

Pulse-Chase labeling - Process

A

Process – Add radioactive labeled molecule to the cell (Ex. Add radioactive amino acid if studying protein) –> keep labeled molecule for some period of time and then remove/stop adding –> NOW proteins syntehsized during that period of time is labeled –> immunopurify and use autoradiography tp detect the labeled protein

LIMITATION - Can see levels of a protein increase or decrease but this does not tell you whether its being degraded more or snthesised less

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18
Q

Gold standrad for showing that you understand a biological process

A

Do in vitro reconstitution - be able to make the process happen from purified proteins

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19
Q

In Vitro Reconstitution

A

In Vitro Reconstitution = make the process happen from purified proteins (reconstitute protein action in an isolaed organelle)
- Only been done a few times ; in most cases the process is too complicated (only works if there are not too many proteins involoved)

Ex Na-K ATPase

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20
Q

In vitro reconstitution process

A
  1. Solubilize cell or organelle with non-ionic detergent (keeps Na+-K+ ATPase complex intact)
  2. Purify protein complex
  3. Reconstitute - remove detergent & mix with lipids to form artificial membranes (with Na+-K+ ATPase)
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21
Q

In vitro reconstitution Sucess

A

Examples:
1. Actin Polymerization and filaments formation –> You can push a bead around in test tube by mixing 5 proteins togther
2. Antigen presentation – can get rid of the APC by putting the proteins that are important for antigen presentation into artificial lipid bilayer and have T/B cell interact with that and get formation of immunological synapse
3. Giant lipid bilayers
4. MinD and MinE proteins in bacteria –> Can add MinE and MinD in well shaped line bacteria –> proteins will undergo this oscelation pattern on the glass slide

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22
Q

What biological process has NMOT be reconstutted in vitro (options - Actin polymerization + DNA synethesis + Cell motility + protein synthesis)

A
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23
Q

Work horse of cell biology

A

Imaging

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24
Q

Light Microscopy

A

Can see cells + Can see organelles (Ex. Mitocondria) + can see structures/nucleus
- Can see ribsomes under favorbale conditions (hard to see because they are below 0.2 micrometers)

LIMITATION - only good until 200 nm (0.2 micrometeres)

Image - shows the size of things you can image

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25
Q

Microscope history

A

1630 – first microscope (1 lense)
1645 – Made compound microscope (uses 2 lenses) –> 2 lenses allowed for an increase in magnification to be able to see better
1670 – Robert Hook looked at Cork –> called the units in cork ‘cells’ = dawn of cell biology
1980 – New microscopes –> Now have 3 objectives and can be higher power BUT still not that different
- In 1980 started having CV cameras to record image and put the image on a computer –> AFTER THIS things stared to evolove more
2010 – imaging takes off –> Have tables with optics and computational analysis

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26
Q

How does light microscope work

A

Light microscopes - look at the absorbance of light by the cell

Does not look good in unstained cell (just looking at the light absorbed by an unstained cell does not look good) –> MEANS light microscopy is often used for histology (USED ON FIXED CELLS)
- Used to stain for organelles in different colors

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27
Q

Phase contrast microscopy

A

How does it work - Looks at index of refraction in different parts of the cell

Use - Look at live cells

Image - shows different wave patterns in stained vs. Unstained cell

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28
Q

Difference interference contrast (DIC)

A

How does it work - looks at gradient of index of refraction (Looks at how index changes across different areas )

Use - Looks at living cell + Gives 3D image

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29
Q

Limitation of Phase contrast + DIC

A

Limitation of DIC and phase contrast = often want to look at molecules within cels NOT cells themselves –> where fluorescence comes in

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30
Q

Fluorescence

A

Fluorescence – when somethings is excited with a wL of light and it emits a different color

How does it work - Molecule can absrob wL of color –> once absorb color the electronic structures are pushed up to excited state –> then the electronic structures have decay from excitation –> big decay to ground state causes emission of light

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31
Q

Fluorescent exitation vs. emission wL

A

Exitation = always higher energy than emission because of decay

MEANS WL of excitation is shorter (ex. 450) and frequencey is higher
- wL of emission in this case = 550

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32
Q

Stock shift

A

Stock shift – Shift to longer wL for emission (Ex. Absribes in blue range and then emits in green/ywllow range)
- ALL flourescent molelecules have shift to longer wL for emission

All fluorescent molecules use this principle in different colors
- Example – FITC = exicted by blue light and emits green light VS. DAPI is excited by UV light and emits violet light

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33
Q

Upright Floruesnece microscope - Heart if the microscope

A

Heart of microscope = Dicromatic mirror

Dichrmatic mirror (beam splitting mirror) –> coated glass that reflects the blue light it was excited with

Ex – IF the molecule is excited with blue light and emits green light –> Glass is place diagnolaly so the blue light is refected onto specifimen –> IF there is floruaphore in the specimen that is excited by the blue light THEN it will emit a green light –> The mirror use will be transparent to let the green light through = can see the emission of the green light
- END - Mirror won’t let any blue light to transmit ; will only let the green light transit

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34
Q

What is needed for each flouraphore in Upright Floruesnece microscope

A

Need a different mirror for each flouraphore

Black cubes = go into microscopes and adapts the microscope for each floruaphore
- Have barrier filters + the mirror in the middle of the cube –> all 3 are in 1 cube

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35
Q

Additional components in Upright Florescent microscope

A

Barrier filters

  1. Excitation filter - If have blue +white + green + red light and want to bring the blue light in THEN use barrier filter to only allow blue light through
  2. Emission filter (cleanup filter) – only lets green through

NOW use lasers to clean up light emission/excitation = don’t need barrier filter

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36
Q

Example image from Upright Florescent microscope

A

Image has multiple colors BUT in reality this is NOT done with color cameras

Use 1 filter for flourphore for tubules –> get 1 black and white inage + use 1 filter for floraphore for DNA and get balck and wite picture + use 1 floraphore for histones and get balck and white picture –> END get 3 black and white pictures from the 3 filter cubes –> THEN false color the images and overlay them

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37
Q

Confocal Flourescent Microscope Vs. Normal microscopes

A

Normal imaging – get light from all planes of cell (all layers of cell)

Confocal – bring light in through a small pinhole to focus the light on one plane in cell THEN when you look at emission signal you have another pinhole that blocks everything going to the detector
- Emitted florusnce from an in focus point is focused at the pin hole and will reach the detector BUT a light from out-of-focus point is out of focus of pinhole and is excluded from the detector

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38
Q

What is seen from confocal

A

ONLY see slice of cell (NOT the whole cell)
- Tyoically see 1 micron slice ; cell is 20 microns in height = need 20 slices

Can adjust where the pinhole is – can move slice through –> Can make 3D reconstruction of cell

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39
Q

Confocal - Overall

A

Scans the sample with narrow excitation beam and collects emitted light through a pinhole so that only light from a single focal plane is collected

Use - Creates sharper image from single plane + 3-D images by producing a “Z-stack” of planes at increasing distances from the cover glass substrate

40
Q

Example Confocal

A

Top = epifloreunce microscope ; bottom = confocal (much sharper image)

41
Q

Deconvolution Microscopy

A

Uses a Z-stack of images with a standard epifluorescence microscope and uses computer simulation to subtract the out-of-focus light in each plane and constructs a 3D image

42
Q

Light Sheet Microscopy

A

Overall - light comes from the side illuminating only 1 focal plane

Special lense brings as a sheet of light across into the cell –> Only excites the fluorophore in the slice where the sheet is going through the cell

Use - Get 3D image of cell (done by moving sheet up and down through cell –> put together information from the slices)

Image - see 3D view of cell

43
Q

Total Internal Reflection Florescence microscopy

A

Based on idea of fiber optics

Overall - Excitation beam enters along the edge of the objective barrel and bends sharply when it hits the lens –> Evanescent field penetrates cell and excites fluorophores on the basal surface

44
Q

Internal reflection

A

Internal reflection - When light moves from one index of refraction to another the light is bent

45
Q

Total Internal Reflection Florescence - Internal Refection

A

Normally – the light is brought in the center of the lense BUT INSTEAD (In TIRF) if you move to the side the light comes towards the surfae of the glass at a glancing angle –> the light is internally reflected and NONE of the light goes to where the cell is BUT there is a small amount of energy that moves beyond interphase at critical angle (occurs in the Evacent feild) –> field penetrates 100-300 nm
- If a fluorophore is in the evancent field you can excite the fluorophore

46
Q

Total Internal Reflection Florescence - Use

A

Use - useful for viewing parts of the cell that are close to the basal surface (ex. plasma memebrane) + can visualize the behavior of single molecules in cells (see flourence from 1 molecule)

Example - Cell surface receptor on the membrane (close to where the cell touches the glass) THEN you can light up the floraphores BUT you won’t see flouraphores in the rest of the cell which create a DARK background – can see things you wouldn’t see before

47
Q

Total Internal Reflection Florescence - Sensitivity

A

TIRF = Creates a dark background to be able to better see things

See the small number of fluorophores that are exited by the wave and NOTHING else (see no other fluorophores in the cell) = becomes more sensitive

Image - Epiflrousnece shows 1 thing BUT where the cell is actually close to the surface is a much smaller part of the cell

48
Q

Multiphoton Floruesnce microscopy - Use

A

Use – Looking at thicker specimens (Ex. nuerons deep in rat cortex or embryo or polytene chromosomes)
- Can see seep things in tissue

49
Q

Issue with imaging thicker specimens

A

Normally - Issue in thicker specimens = the excitation beam is scattered in tissue = don’t get a lot of excitation signal in thick specimens

Scatering = proportional to the 1/4th power of wL –> If the wL is longer then scattering is less
- Blue light = shorter wL = more scatter VS. Red light = longer wL = less scattering

50
Q

Solution in Multiphoton Floruesnce microscopy - Use

A

Can couple the photones and get exictation of the floruaphore with 2 photons of red light by focusing to 1 spot = get less scattering

Normally have blue excitation/green emission (would have more scattering) INSTEAD can be exited by red light as long as 2 photons come at the same time

END - using a longer wL (ex. Red) to exite the floruaphore so it will be scattered less and can penetatte farther into the tissue

51
Q

Past flouraphores Vs. Green Fluorescent protein

A

PAST florraphores = all organic moelcules –> Done by covalentley attaching moelcules to proteins and then injecting proteins into cells (hard to do)

Solution - GFP

52
Q

GFP

A

Bioluminescent molecule –> when the protein is made it is automatically flourescent (don’t add anything to it)

Can mutated GFP through directed evolution –> made different colrors by slightly mutating the Amino Acids suroudning the floraphore

53
Q

Post GFP discoveries

A

People started to look at other naturally florescent biological systems –> found RFPs

Now have many RFPs - All made by directed evolution (have different colors with different absorbances and different stabilties)

ALSO NOW have IR ones

54
Q

GFP - Use

A

Use – Can fuse GFP/RFP etc to protein of interest and now you can follow the protein of interest around a cell because GFP is fused to it

IF have protein of interest you want to follow – you can hook GFP or you could hook HALO

55
Q

GFP issue

A

Issue – NOT as good as the original chemical flouraphores

Solution - using HALO tag

56
Q

HALO Tag

A

HALO = enzyme that has a ligand –> when the ligand binds it makes a colvalent bond in the binding pocket of the enzymes and acts as a suicide inhibitor

Attatch HALO to protein of interest + can take the old flouraphores and attatch them to the halo ligand –> incubate the cell with the ligand which binds to halo (halo is bound to protein of interest)

Very specific because the ligand has no other place to recat = gives specific labeling of your protein with a better floraphore than GFP

57
Q
A

Two photon – can see down 200 um in tissue (need 2 photon to have less scattering)

Confocal and epiflouresnce is only good until 30 microns VS. 2 photon can go 200/300 microns into tissue

58
Q
A

Need the background to be dark to see these events

59
Q

Fluorescence Recovery After Photobleaching (FRAP) - Use

A

Overall - measures molecular diffusion in cells (Ex. diffusion coefficient of receptor in plane of membrane) + can see where the molecule came from (get diffusion + shuttling)

Process - Fluorescent molecules within a cell are imaged then a section of the cell is photo-bleached with a strong laser –> that area will lack flourenscence –> Overtime, the molecules from the surrounding area move into the bleached zone and the fluorescence is recovered
- The kinetics of the recovery indicate single or multiple species involved in the recovery

60
Q

Basis of FRAP

A

IF you shine a bright at too high of an excitation length at a fluorophore then the fluorophore will make reactive oxygen species and destroy themselves (once they are destroyed, they are never be florescent again)

WHY does flouresnece come back in FRAP - Fluorescence comes back NOT because the molecule recovered BUT because other molecules form different regions diffused into the area –> MEANS you can get the diffusion coefficient of the molecule

61
Q

How does FRAP show where the molecule came from

A

Can see where the molecules came from because can see which part of the cell is getting dimmer (ex. Can see if the molecule came from the cytoplasm or the nucleas)

Example - Image –> molecule is coming from the nuclease and the cytoplasm because they are both getting dimmer as the bleached area is filled in

62
Q

2nd method for FRAP

A

Can have protein that be activated instead (normally the protein is green but when add light the protein turns red) –> in the photobleached area you get rid of green flroreusnce and get red THEN the red goes away and green comes in –> get double view of diffusion coefficient
- Instead of area being white – it would be red

63
Q

Example FRAP

A

Example - Surface receptor on cell

Cell has been treated with something that blocks Actin –> cells rounded and not moving –> bleach fluorophore (GFP) in the edge of cell –> see after some time fluorescence comes back in because the recesptors coming from the edges (diffusing in plane of memebrane) = can tell you diffusion coefficient of the receptor

64
Q

Fluorescence Resonsence Energy Transfer (FRET)

A

Use - See if 2 molecules are in close proximity in living cells (intramolecular interactions)
- Molecules need to be within 60A to each other

Example - see if 2 molecules are both in mitocondria

65
Q

Fluorescence Resonsence Energy Transfer (FRET) Floraphors

A

Donor fluorophore (1) that in an excited state may transfer its excitation energy to a nearby second acceptor fluorophore (2)
- Emission of donor os close to excitation of the other (Ex. – CFP emits at Cyan wL and YFP is excited by Cyan wL )

Donor fluorophore (CFP) is on one protein and the acceptor fluorophore (YFP) on another –> there will only be fluorescence of YFP if energy from the excited CFP fluorophore is nearby –> MEANS if you detect YFP fluorescence it would imply interaction of the two proteins with different fluorophores attached
- If the proteins are far apart then you would only get Cyan BUT if the proteins are close then you get YFP because the energy is transfered from CFP to YFP

66
Q

Biosensor

A

Biosensor = way to measure something happening in living cell without breaking the cell

Example - Fret has been used to build Biosensors

67
Q

FRET biosensor

A

Example – Using a helix that intercalates into plasma membrane and helix is sensitive to voltage and YFP and CFP are attatched to the helix

Start CFP and YFP are far away –> When cell has an action potential here is a change in the voltage of membrane –> helix changes confirmation at different membrane potentials –> change in voltage chnages which chnages the helix confirmation which brings CFP and YFP together –> get different emission which you can read out as signal

68
Q

FRET biosensor Example

A
  1. Looking at memebrane potential in muscle cells in heart of living zebra fish –> 1hen heart beats - can see signal in atria THEN in the ventricles (benig read by FRET sensor)
  2. Cdc42 GTPase proteins
    - When protein is active it binds to binding protein –> protein can be bound or not bound
    - Wehn GTPase is active = changes the distance between YFP and CFP –> can read out as biosensor
69
Q

Use of FRET biosensor

A

Looking at function of protein in living cells using the biosensor

Example - Looking at binding interaction

70
Q

Issue in FRET biosensor

A

FRET signal is hard to use because the FRET signal is not huge

If have CFP and YFP apart or together the difference in signal is 20% (difference between apart and together is 20%)

71
Q

Translocation Based Biosensor

A

Goal - want to know where the lipid is being made

Put a protein into a cell (protein is not normally there and is labeled with GFP) –> protein binds to lipids (ex. protein binds to PIP3)

IF the lipid is made then the protein moves to the leading edge of the cell (cells moves towards the chemoattractant) –> As cells move towards the chemoatractant the lipid is being produced at the leading edge of the cell (know where lipid is made)

If break open cells you would see some amount of lipid BUT you wouldn’t know where it is –> Solution – use translocation based biosensor (can see localization of lipid in living cell)

72
Q

Synthetic biology – Induced recruitment

A

Overall – Make things happen in cell –> cause a new phenotype to happen by moving proteins around the cell

73
Q

Synthetic biology – Induced recruitment (Process)

A

Basis – done using protein pair (heterodimer) that is normally apart BUT if you add Rapamycin (RAPA) then the proteins will bind irreversibly and will bind quickly

Fuse to a protein to anchor it somewhere (fuse protein to anchor) + have protein of interest on effector –> when add Rapamycin the two come together
- Move effector at will around the cell and see what happens

MY INTERPRETATION - fuse protein to anchor and then also fuse protein to effefctor and anchor the anchor where you wnat and then have the protein of interste bind to where the anchor is and move the protein of interest around the cell

74
Q

Synthetic biology – Induced recruitment - Use

A

Proof of principle experiment

Effector that you fuse the protein of interest is bound to is GFP (not doing anything just moving molecule around the cell)
- Can move molecules around the cell quickly
- IF molecule has some activity – can see what happens when you move the molecule to the mitochondria

Example – moving molecules to ER then golgi then plasma membrane

75
Q

Synthetic Biology constructs to manipulate cells

A

Example – Inject Cdc42, Rho, or Rac proteins –> move the proteins to the plasma membrane by adding Rapamycin

Can recapitulate what normally happens when you add these proteins (ex. Still makes ruffles or stress fibers or fibroblosts)

Called Actuators – make things happen in a cell

76
Q

Synthetic Biology constructs to manipulate cells - Issue

A

Issue with RAPA = irreversible and global (everywhere in the cell) ; one it is there you can’t move it around

Solution = make a light activated system
- Top right - inhibitor of Rac was caused to be released due to light –> NOW can direct activation (light activation?) to part of cell and can get the phenotype on that part of the cell and get the cell to move in that direction
- Can’t do with RAPA because RAPA would light up whole thing at once
- IlId or Cry2 – light activated actuary systems

77
Q

Single molecule imaging

A

To do single molecule imaging = using Total Internal Reflection Flourescnce microscopy (gets background down)
- Can look at 1 molecule in TIRF because background

78
Q

Single molecule imaging - Use

A

Use – Track molecule (single molecule tracking) + get diffusion coefficient + get shuttling rate (Ex. see ligand binding or molecule moving on/off membrane (can measure shuttling rate)

Image – Looking at receptor –> Take confocal slice through cell and do FRAP on part
- Receptors are found at the bottom of the cell (image looking at the bottom of the cell using TIRM)
- NOT Actually looking at the receptor instead we are actually looking at ligand that binds to receptor –> each spot is flourence from 1 Molecule

Have 10,000 receptors at bottom of cell BUT the reason we can look is because the molecule added is only labeling some receptors at a given time

79
Q

How do we look at 10,000 receptors in TIRF

A

Have 10,000 receptors at bottom of cell BUT the reason we can look is because the molecule added is only labeling some receptors at a given time

Because we can see the single molecules we can make video of the binding of ligand to receptor in cell –> things (ligands?) come on and off receptors and when the molecule (ligand?) is on receteptor they diffuse around on the plane of the membrane
- Don’t see molecules in medium because in any frame of the video the molecule moves too far BUT if the molecule is bound to the receptor then they stay in the image for enough frames and you can see it

END - video of ligand binding to receptor and when the ligand is on receptors get some of diffusion coefficient of receptor

80
Q

Single molecule imaging - Issue

A

Issue - only see a few molecules at a time

IF used a higher concertation of ligand THEN the whole image would be white
- Even if have 10,000 molecules that are separated from one another they would look together because each dot is 200 nm and they would overlap

81
Q

Superreslution microscopes

A

PALM + SIM

Resolution limit (0.2) has been broken by super resolution microscopy

Superresolution uses photoactivated GFP –> when shine light GFP turns red BUT If add an even brighter light then it can be photobleaches

82
Q

Issue with resolution of typical microscope

A

Issue with resolution of typical microscope = spots are 200 nm and IF have too many spots then they start to overal

83
Q

Photoactivated localization Microscopy (PALM)

A

Process - Localize GFP molecule –> give low amount of light –> activate a few of the 10,000 receptors THEN find the center of the 200 nm spots ad add dot on computer –> then bleach the spots –> give a new activation signal to activate more receptors –> localize the newly activated receptors –> add new dots on computer –> bleach the dots –> actiavte more receptors –> localize the receptors and add to computer –> keep cycling through and get image (green dots on slide to black dots)

84
Q

PALM - limitation + Use

A

Limitation = how well you localize the center of the spot + takes a long time because need to go through cycles and build image = need slow moving cell or fixed cells
- Need on/off localization because need to localize a few molecules at a time
- PALM = lose resolution

Use – improves the resolution from 200nm to 20nm

85
Q

Structured illumination Microscopy (SIM)

A

Use - Looking at more dynamic behavior in cell
- Get higher resolution of structures in cels
- Can be done in living cells

86
Q

Murray pattern

A

Murray pattern – two patterns when overlap make a different pattern

87
Q

SIM - How does it work

A

Have cell which has a small pattern AND have stcuture of light (illumination pattern) –> when put the structure of light on the pattern on cell you get Moire pattern –> can image the Murray pattern because it is larger
- Know the structure of light being added to the cell but don’t know the pattern in the cell
- Because you know the structure of the light you put on you can back calculate the pattern in the cell that would give that murray pattern for the pattern that you put on

88
Q

SIM - limitation

A

Not AS enhancing as PALM
- PALM = goes to 20 nm vs. SIM goes to 60 nm

BUT can do SIM in a living cell (can make high resolution movie)

89
Q

Electron Microscopy

A

Overall - Much higher resolution than light/fluorescence microscopy + see indovidual molecules + see structure of complexes and moleciles
- Resolving structures < 300nm (can see atomic level)

Light micrscope = uses lense VS electron microscopy uses electrons that are focused by magnets

90
Q

Electron Microscopy Issue

A

Issue – electrons need to fly through vacuum

Because need vacuum –> specimen must be stable to vacuum and stable to electrons hitting it

Limitation – can’t look at living system

91
Q

Preparing samples for Electron Microscopy

A
  1. Fix with cross linkers
  2. Stain with heavy metals
    • Heavy metals defect the electrons
  3. Dehydrate with ethonol
  4. Embed in resin to give more stabilization
  5. Section and put on copper grid

OVERAL – shows FAR from a live cell BUT it is super high resolution

92
Q

Application of electron micrscopy

A

Application of electron micrscopy = cryoEM

CryoEM = freezes the system rapidly –> Rapid freezing improved images
- Before adding cross linkers you freeze the cells which preserves the structures –> THEN you haw and add rtoss likers and heavy metals = get imporved image

93
Q

Image from EM

A

See membarnes and get structres

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Cell Structure and Dynamics (8 decks)

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