Lecture #1 - Strategies for Cell Biology Flashcards

Question

Microscope history

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

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

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

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

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

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

Answer 24

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

Answer 25

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

Answer 26

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

Answer 27

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

Answer 28

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

Answer 29

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

Answer 30

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

Answer 31

Issue – NOT as good as the original chemical flouraphores Solution - using HALO tag

Answer 32

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

Answer 33

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

Answer 34

Need the background to be dark to see these events

Answer 35

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

Answer 36

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

Answer 37

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

Answer 38

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

Answer 39

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

Answer 40

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

Answer 41

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

Answer 42

Biosensor = way to measure something happening in living cell without breaking the cell Example - Fret has been used to build Biosensors

Answer 43

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

Answer 44

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

Answer 45

Looking at function of protein in living cells using the biosensor Example - Looking at binding interaction

Answer 46

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%)

Answer 47

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)

Answer 48

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

Answer 49

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

Answer 50

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

Answer 51

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

Answer 52

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

Answer 53

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

Answer 54

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

Answer 55

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

Answer 56

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

Answer 57

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

Answer 58

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

Answer 59

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)

Answer 60

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

Answer 61

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

Answer 62

Murray pattern – two patterns when overlap make a different pattern

Answer 63

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

Answer 64

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)

Answer 65

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

Answer 66

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

Answer 67

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

Answer 68

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

Answer 69

See membarnes and get structres