Microscopy Flashcards
benefits of fluorescence microscopy (general)
can look at chemistry in vivo (molecule movements etc…)
can overcome optical limits (super-resolution techniques)
particle formalism?
Light particle travelling in straight path like bullet
can account for refraction
wave formalism?
light as a wave with Electric and magnetic components (oscillation in these fields)
Phase
basically where in space the peaks and troughs of wave are in the oscillations
polarisation
orientation in space in which the electric and magnetic vectors are oscillating (always orthogonal to each other tho)
amplitude
height of wave peak from mid point of peak/trough
perceiving amplitude
can see brightness of light
perceiving polarisation
can use polarised filters to see the wave’s polarisation
perceiving phase
can’t (light moves too fast to detect nm difference in were peak and troughs are)
interference
light interacting w light
2 peaks or troughs from diff waves line up together
or 1 peak/1 trough
constructive vs destructive interference
in destructive
if same amplitude will cancel each other out and perceive nothing
diffraction
scattering/bending of incedent light via interaction w details of structure of object/sample
requires wave formalism
refraction
change in path of light as result of passing through transparent medium
refractive index
n
how much light slows down in medium
denser medium = higher n
because more electron density
refraction mechanism
light ray in less dense air
hits denser glass
slows down
if comes at interface btwn media at angle that isn’t 90deg (theta1)
changes direction to another angle (theta2)
path bends basically because light reaches a point in the second medium faster on this bent path than if it took straight line there (ie it spends more time in the “faster” medium - path of least resistance i guess)
lenses (basic)
media interface is curved
the path orthogonal to surface (the normal i think its called??) changes along its length.
concave - diverges rays
convex - converges rays
can adjust degree of this by changing lens density/curvature
focal length - f
distance from the lens where parallel rays entering the lens will converge
focal length changes w lens material/curvature
higher curvature - shorter f
object >2f away from lens
miniature image created between f and 2f away from other side of lens
object =2f away from lens
same size image =2f away from other side
between 2f-1f away from lens?
magnified image formed >2f away from lens
look at diagrams in notes if confused - can draw diagram i guess to help
object =1f away from lens
no image formed - all rays entering lens emerge parallel and never converge
object <1f away from lens
rays all diverge - no image
compound microscope basic
2 stages of magnification
-objective lens
-ocular (eyepiece)
focus by moving sample up and down relative to fixed lens
image formation in compound microscope
Rays from sample focused by objective lens onto Ocular lens
Real intermediate image formed on the ocular lens by first set of lenses
ocular lens then diverges the rays from the Real intermediate image
allowing the lens in the eye to refocus them onto the retina forming Real final image
can put camera/detector where the Real intermediate image is formed
trans-illumination microscopes
Upright and Inverted microscopes
shine light Through the sample instead of onto it
inverted microscope used to see cell culture in liquid medium
can put lens underneath the culture plate and therefore get as close as possible w/out lens going into medium
Resolution definition
ability to distinguish two points v close together (eg two single GFP molecules [2nm across])
how do point sources show up in microscopy
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
Huygen’s Principle
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
Diffraction and interference - Double Slit experiment
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
Slit distance importance
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
diffraction patterns from sample importance
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)
Diffraction pattern and lenses
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
Airy Disc origin
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
limits of resolution
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
Numerical aperture
NA = n (refractive index of lens) * sin(theta)[angle of light lens can collect, from diameter/closeness to sample]
Abbé equation
resolution limit = (0.61 * wavelength)/NA
NA of 1.5 achievable
>1.5 v special
how does wavelength affect resolution
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
useful size range for visible light microscopy
the useful range for resolving details for visible light is coincidentally v useful for studying cells (details of 300 micrometres - 300 nanometres)
use of chemical stains
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
Contrast methods uses
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
Phase contrast microscopy - detecting change in phase from light passing through cell
converts invisible phase differences
into visible contrast differences
- light diffracted by sample is found all over diffraction plane, and is considered ~1/4 phase shifted relative to non diffracted light
- light not diffracted by sample (aka 0th order light) is found only in centre of diffraction pattern
- 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
differential interference contrast (DIC)
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
benefits of using fluorescence microscopy
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
examples of things that can be observed w fluorescence microscope
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
energy & wavelength
photon’s energy is inversely proportional to its vibrational frequency
shorter wavelength - higher energy
Basis of fluorescence in a fluorochrome
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
issues within fluorophore
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)
properties of fluorescent molecule
conjugated double bonds within its structure
Stokes shift
emission spectrum always Red-shifted from absorption spectrum
Called Stokes shift
is defined by the chemistry
Absorption vs Emission spectrum
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
different fluorochrome use in microscopy
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
Fluorescent dyes that bind cellular structures
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
Rhodamine-Phalloidin stain
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
Dyes on Ab
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
engineering of new fluorescent dyes
can alter the conjugated structure to alter wavelengths of their absorption/emission spectra
Epifluorescence microscope
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
Epifluorescence filter set
- Excitation filter
only allows shorter wavelength through (wavelength required for activating fluorophore) - 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
GFP properties
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
why is GFP fluorescent
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)
mutated GFP
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
mStaygold
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
what makes microscope image fuzzy
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
methods of removing out of focus light
Optical
-Laser Scanning confocal microscopy
-TIRF
Computational
-Deconvolution
Deconvolution
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
Optical solutions to fuzzy images
Laser Scanning confocal microscopy
TIRF (total internal reflection fluorescence)
Laser Scanning confocal microscopy
-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
improving laser scanning confocal microscopy
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
TIRF
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
uses of TIRF
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
FRAP
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
photoactivatable/photoconvertible proteins proteins
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
FRET
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
Usage of FRET to measure protein interaction/comformation
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
Super resolution microscopy (aka Nanoscopy)
PALM - Photoactivated localisation microscopy; ~20nm
SIM - structured illumination microscopy; ~100nm
STED - Stimulated emission depletion; ~20nm
Use of SIM
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
PALM
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)
