Microscopy Flashcards

Question

Resolution definition

Answer 1

ability to distinguish two points v close together (eg two single GFP molecules [2nm across])

Answer 2

not as clean points but as smeared out discs Airy discs can make it hard to resolve two v close together points until they get far apart enough to distinguish the discs this hard limits the resolution of basic light microscopy result of wave properties

Answer 3

every point on a propagating wavefront can serve as source of secondary wavelets that are emitted radially from this source this is what causes waves to show up in the geometric shadow of an aperture (where without wave formalism would just expect dot of light from aperture

Answer 4

infinitely thin slit acts as single point source of radially emitted waves (from Huygen's principle diffraction stuff) one slit first (S0) two slits next to each other (S1, S2) then hit by radially emitted waves from first slit each of these slits act as point sources now place surface somewhere in front of slits get max and min points of light where constructive and destructive interference from peaks and troughs from radial waves coming from S1 line up with the same from S2 the interference occurs at different angles depending on distance of slit closer = higher angle = information on diffraction pattern is further out from pattern's centre

Answer 5

closer slits peaks line up in row at higher angle means that the max point bright spot on the diffraction pattern on screen shows up further from the optical axis

Answer 6

diffraction pattern from sample carries information about the point sources within the sample (eg diffraction from light hitting point of sample, self illuminating fluorophore...) can give info about distance between point sources (eg x-ray crystallography)

Answer 7

refraction from lens can direct light rays scattered (diffracted) by a sample no lens = diffraction pattern lens = can produce image interference from closer together points appears at higher angle from optical plane if your lens cannot collect the high angle information from the diffraction pattern then will lose info from the closest together points in the sample RESOLUTION is reduced less detail is resolved ie adding an aperture (to block higher angle light) or using smaller lens fail to collect higher angle info less detail is resolved

Answer 8

result from incomplete interference of diffracted light at the image plane due to the highest-angle diffracted light not entering the optical system therefore not contributing to the final image therefore cannot contribute to interference that produces the final image

Answer 9

no lens has perfect resolution resolution depends on angle (theta) of cone of light that the objective lens can collect from the sample this angle depends on the lens diameter and distance from sample to lens lens would have to be infinitely wide/close to collect all info resoliution also affected by wavelength of light

Answer 10

NA = n (refractive index of lens) * sin(theta)[angle of light lens can collect, from diameter/closeness to sample]

Answer 11

resolution limit = (0.61 * wavelength)/NA NA of 1.5 achievable >1.5 v special

Answer 12

spots in the diffraction pattern contain info about distance btwn points on objec ie the diffracted waves carry this info shorter distance = info on diffraction pattern is farther from origin both spacing of the waves AND the wavelength relative to each other matters if wavelength much < spacing portion of diffracted light containing relevant spacing info occurs at smaller angle so the ability of the objective to collect this info is improved with shorter wavelengths ->image is sharper/better resolved hence means that when details in object are vv close together wavelength needs to be vv small to generate useful info about those details -why x-rays are used in protein crystallography

Answer 13

the useful range for resolving details for visible light is coincidentally v useful for studying cells (details of 300 micrometres - 300 nanometres)

Answer 14

diff chemical stains bind structures w diff properties can be useful for generally classifying cells BUT need to fix them first so not so useful for studying live cells

Answer 15

uses diffraction and interference to generate contrast between structures (unlike stains which absorb diff amounts of energy from diff wavelength of light to generate contrast) cells are mostly transparent light passing through them is slowed down (aspect of refraction) this alters the phase of light that passes through the cell compared to light that didn't

Answer 16

converts invisible phase differences into visible contrast differences 1. light diffracted by sample is found all over diffraction plane, and is considered ~1/4 phase shifted relative to non diffracted light 2. light not diffracted by sample (aka 0th order light) is found only in centre of diffraction pattern 3. special piece of glass - Phase plate - at diffraction plane can alter phase of MOST of diffracted light without altering phase of non diffracted light (designed to not affect light in centre of diffraction plane) causes overall 1/2 phase shift of diffracted light relative to non diffracted causing destructive interference between them causing great contrast in image

Answer 17

aka Nomarski optics more complicated theoretically but still easy to use measures relative phase difference instead of absolute phase difference differences greatest at edges, gives: - false 3D effect -well defined edges in image

Answer 18

vs transmitted light microscopy (only info on physical properties) fluorescence can give info on biochemical properties can target diff organelles/proteins can see when certain things are active w reporters eg

Answer 19

localisation/behaviour of cell components turnover of molecules at subcellular sites (via FRAP) protein-protein interactions and proximity (via FRET, and FLIM) diffusion of individual molecules behaviour of single molecules (TIRF) spacial/temporal ion conc changes cell signalling activity (eg of kinases) endo/exocytosis, membrane trafficking organelle activity

Answer 20

photon's energy is inversely proportional to its vibrational frequency shorter wavelength - higher energy

Answer 21

electron in ground state because energy is quantised - if electron absorbs photon w just right energy -can jump it to excited state radiates energy as heat and molecular vibration, moves slightly lower then drastic jump back to ground state and emits remaining energy as photon since some energy lost since absorption energy of photon emitted is lower than when absorbed ie it is a longer wavelength than absorbed photon process takes ~couple nanoseconds

Answer 22

electrons in same state cannot have same spin (pauli exclusion principle) two in ground 1 up spin 1 down spin one goes into excited state one remains in ground one electron can FLIP its spin blocks excited electron from returning to ground keeps it in Excited Triplet State -More reactive - can lead to photobleaching through reaction (mainly w oxygen) -or phosphorescence can occur (slower)

Answer 23

conjugated double bonds within its structure

Answer 24

emission spectrum always Red-shifted from absorption spectrum Called Stokes shift is defined by the chemistry

Answer 25

absorption spectrum doesn't have to be single peak but the stokes shift is from the PEAK of the absorption not from the specific absorbed wavelength from absorption spectrum so emitted wavelength is independent of the incoming absorbed wavelength changing the wavelength absorbed by a specific fluorophore doesnt change wavelength just changes the frequency of the fluorescence

Answer 26

Fluorescent dyes that bind directly certain cellular structures well characterised Fluorescent dyes chemically conjugated to target molecule (eg Ab, small celluar molecule, proteins) Fluorescent proteins that can be fused genetically to gene of interest

Answer 27

FM4-64 Hydrophobic enriches in membranes can be used to study endocytosis (will end up in endosome) DAPI binds v specific to minor groove of DNA molecule v good v specific can use diff dyes w diff spectral properties to co-stain diff things and see relative localisation

Answer 28

Phalloidin toxin binds and depolymerises actin tetra-methyl Rhodamine conjugate to phalloidin via free amino group -can also use tetra-methyl Rhodamine to label other proteins but need to be careful as it can bind to any lysine and kill protein function

Answer 29

chemically conjugate onto Fc portion of Ab usually onto a secondary Ab that binds Fc portion of Primary Ab specifically targeted to protein of interest can use diff tags for primary Ab eg Haemaggluttanin (HA) genetically fuse HA peptide to gene of interest can now use HA targeted Primary Ab to localise to protein of interest w/out needing to create new specific Ab to protein of interest

Answer 30

can alter the conjugated structure to alter wavelengths of their absorption/emission spectra

Answer 31

instead of transilluminating sample (like in transmitted illumination microscopy) separate lamp on top of sample light reflected onto sample by dichromic mirror fluorescence activated in sample - radiates in all directions fluorescence that goes up it collected in objective and sent to eyepiece dichromic mirror only lets light of the fluorescence wavelength pass through allows detection of weak fluorescence signals

Answer 32

1. Excitation filter only allows shorter wavelength through (wavelength required for activating fluorophore) 2. Dichromic mirror acts as mirror to the shorter wavelength reflects light from illumination lamp onto sample longer fluoresced wavelength can pass through this mirror this is due to diff coats of metal oxides 3.Emission filter allows light of emission spectrum to pass through selects further the light allowed to eyepiece from what passed the mirror tnis + the mirror allows strong selectivity and extinction of unwanted non-fluoresced light as the fluoresced light is a v small proportion of light inside the microscope filter set can allow almost all extinction to almost all transmission in just tiny range of wavelengths

Answer 33

fluorescent all on its own doesn't need help can genetically fuse to proteins by short flexible linker in vivo avoids fixation artifacts can use to study dynamics but is prone to photobleaching and attaching protein of its size to another may disrupt folding if not careful

Answer 34

beta barrel alpha helix in middle that contains the fluorochrome autocatalytic post translational modification on fluorochrome: -Tyr66 lost a hydrogen - noy dehydrotyrosine -done naturally post translation -allows DOUBLE BOND formed between amino group and carbonyl carbon on the dehydroTyr66 Gly67 next in seq new bond between this and dehydroTyr66 cyclises main chain causing fluorescence (i guess from conjugated system formed)

Answer 35

altering shape altes conjugated system ig can get lots of diff colours useful for co-staining ig though these variants are prone to photobleaching too

Answer 36

purified from Cytaeis uchidae jellyfish can form dimers tho - which may inadvertently aberrantly dimerise protein of interest so need to artificially make monomeric also for some reason doesn't work well in yeast/fungal cells i guess that's what the m stands for

Answer 37

out of focus light light from one point source on sample is focused on detector BUT light from another point source in the 3D structure of sample is not in right place relative to lens so is out of focus this gives fuzziness in image because light is a wave and diffraction stuff - even a 200nm dot becomes fuzzy disc

Answer 38

Optical -Laser Scanning confocal microscopy -TIRF Computational -Deconvolution

Answer 39

Possible through iteration as you change distance of lens from sample eg (z axis) light from a single point becomes either more or less out of focus signal becomes weaker but area larger can experimentally obtain this change in intensity and spreading as POINT SPREAD FUNCTION if know original image, can calculate microscopic image via point spread v straight forward (like x^3) But in reality want to get original from microscope image - Deconvolution much more complex to convert directly (like cube root of x) -can do through ITERATION -eg making a guess of what original image is -then convert this guess of original to a microscope image and evaluate how close they are -use this info to make another guess at real image -then convert this to microscope image and see how close -repeat until you can approximate the original image >disadvantages: -takes long time to compute -may introduce artifacts

Answer 40

Laser Scanning confocal microscopy TIRF (total internal reflection fluorescence)

Answer 41

-Use laser to activate fluorescence in only one tiny dot on slide -use pinhole between lens and detector to cut out the out of focus light reaching detector -allows focusing on small points of sample at same time while cutting out the out of focus light, letting through mostly just focused light from that small region can then move laser to scan the sample and assemble into image can change focus and repeat process to ensure all points scanned are in focus disadvantages: -slow -not v sensitive (pinhole cutting out a lot of the light) -laser can quickly bleach fluorophores these make it not well suited for live imaging of GFP tagged protein in live cell

Answer 42

Spinning disc laser scanning confocal microscopy multiple laser beams/pinholes in spinning disc laser light comes down through pinhole fluoresced light comes back through same pinhole improves the resolution time enough to be suitable for live imaging

Answer 43

total internal reflection fluorescence microscopy -cell on glass cover slip -hit sample with light from shallower than critical angle -causes all light to reflect -but due to some quantum stuff (not important) some light energy sneaks to other side of glass -this decays within 200nm -so only fluorophores 200nm from slide are activated -so few fluorophores are activated that it means that there is very little out of focus light hits the detector

Answer 44

TIRF can be used to detect single molecules eg kinesin moving across MT can combine TIRF microscopy and normal fluorescence microscopy to get a composite image of small details and larger structures

Answer 45

Fluorescence recovery after photobleaching can use to measure molecular dynamics cell expressed GFP tagged protein can see structure made with this structure not apparently moving - looks static but can see if dynamic photobleach spot on structure can then measure the recovery time of the fluorescence in that spot (as the photobleached molecules move out and fresh ones replace them from soluble phase eg) faster recovery = more dynamic (no recovery = completely static) can measure half recovery time (similar concept to half life) can use this as measure of how quickly components in a structure turn over

Answer 46

can either activate fluorescence or change the colour of fluorescence from stimulus can use to detect protein flow out of a structure (kind of kind of like a pulse chase i guess but w fluorescence activation dont take this comparison too serious) can see dynamicity of components in usually static appearing structure

Answer 47

Fluorescence resonance energy transfer measures direct proximity of two fluorophores one fluorescent molecule - eg green hit with blue light - activates it to fluoresce green light but this blue light cannot activate the ref fluorescence BUT if the activated green fluorophore is very very close - practically bound - to red fluorophore - can transfer energy to red and allow it to fluoresce with just blue light shone

Answer 48

protein interaction attach one fluorophore to one protein - and the other to another no interaction - green light when activated with blue interaction - get red light protein conformation similar concept but with two fluorophores on parts of protein that are far in one conformation and close in another can look at this in a live cell eg can use this to measure kinase activity via measuring phosphorylation level protein has phosphorylation site and another site which binds this phosphorylation site phosphorylation causes these regions to bind bringing the fluorophores together can use this construct to measure kianse activity in cell eg aurora B kinase

Answer 49

PALM - Photoactivated localisation microscopy; ~20nm SIM - structured illumination microscopy; ~100nm STED - Stimulated emission depletion; ~20nm

Answer 50

lamin nuclear pore proteins some NPC protein inside envelope some outside difference of about 50nm hard to distinguish the difference in normal microscopy SIM can differentiate the 50nm spatial difference - can actually tell if nearer inside or outside of membrane

Answer 51

Photoactivated localisation microscopy - use photoactivatable fluorescent molecule -activate just one at a time using low light (statistical thing where fewer photons -> less likely that a fluorophore is hit in right way to activate fluorescence) -under microscope - because of resolution limit, see fluorophore molecule as a fuzzy disc, not distinct point -but because only one is there - can assume centre of disc as where molecule actually is -fades -another one activates, can do same for this disc -repeating this allows resolving of an actual image if had higher intensity light - many fluorophores active - very nearby discs form blobs - hard to pinpoint the centres (ie where the molecules actually are)