biology 177: principles of modern microscopy lecture 13: super-resolution microscopy: part i
TRANSCRIPT
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Biology 177: Principles of
Modern MicroscopyLecture 13:
Super-resolution microscopy: Part I
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Lecture 13: Fluorescent labeling, multi-sprectral imaging and FRET
• Review of previous lecture• FRET• FLIM• Super resolution microscopy
• NSOM
• Scanning probe microscopy
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Summary of spectral unmixing
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Förster Resonance Energy Transfer (FRET)
•Great method for the detection of:1. Protein-protein interactions2. Enzymatic activity3. Small molecules inside a cell
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FRET:
Resonance Energy Transfer (non-radiative)
The Good: FRET as a molecular yardstick
Transfer of energy from one dye to anotherDepends on: Spectral overlap Distance Alignment
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FRET: Optimize spectral overlap Optimize k2 -- alignment of dipoles Minimize direct excitement of the acceptor
(extra challenge for filter design)
donor acceptor
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4nsec
0.8 emitted
Non-radiative transfer
-xx-Less
-xx-Less
FRET Diagram
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The Förster Equations.
r is the center-to-center distance (in cm) between the donor and acceptortD is the fluorescence lifetime of the donor in the absence of FRET
k2 is the dipole-dipole orientation factor, QD is the quantum yield of the donor in the absence of the acceptor
is the refractive index of the intervening medium,FD (l) is the fluorescence emission intensity at a given wavelength l (in cm)eA (l) is the extinction coefficient of the acceptor (in cm -1 M -1).
The orientation factor k2 can vary between 0 and 4, buttypically k2 = 2/3 for randomly oriented molecules (Stryer, 1978).
When r = R0, the efficiency of FRET is 50%(fluorescein-tetramethylrhodamine pair is 55 Å)
KT = (1/τD) • [R0/r]6
R0 = 2.11 × 10-2 • [κ2 • J(λ) • η-4 • QD]1/6
J (λ) eA
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More about FRET (Förster Resonance Energy Transfer)
Isolated donor
Effective between 10-100 Å onlyEmission and excitation spectrum must significantly overlapNote: donor transfers non-radiatively to the acceptor
Donor distance too great
Donor distance correct
From J. Paul Robinson, Purdue University
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Optimizing FRET: Designs of new FRET pairs
• Difficult to find two FRET pairs that can use in same cell• Used as Caspase 3 biosensors and for ratiometric imaging
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Properties of fluorescent protein variants
Shaner et al, Nature Biotechnology, 2004
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Optimizing FRET: Designs of new FRET pairs
• mAmetrine developed by directed protein evolution from violet excitable GFP variant
• Bright, extinction coefficient = 44,800 M-1
cm-1
• Quantum yield = 0.58• But bleaches, 42% of
mCitrine time and 1.7% of tdTomato
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4nsec
1. The acceptor excited directly by the exciting light• “FRET” signal with no exchange• Increased background• Decreases effective range for FRET assay
Problems with FRET
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2. Hard to really serve as a molecular yardstick*• Orientation seldom known
assume k2 = 2/3 (random assortment)
• Exchange depends on environment of dipoles
• Amount of FRET varies with the lifetime of the donor fluorophore
* r = R0, the efficiency of FRET is 50%(fluorescein-tetramethylrhodamine pair is 55 Å)
Problems with FRET
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4nsec
Longer lifetime of the donor gives longer time to permit the energy transfer (more for longer)
Added Bonus: Allows lifetime detection to reject direct excitement of the acceptor (FRET=late)
Amount of FRET varies with the lifetime of the donor fluorophore
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Fluorescence Lifetime Imaging Microscopy (FLIM)
• Measure spatial distribution of differences in the timing of fluorescence excitation of fluorophores
• Combines microscopy with fluorescence spectroscopy
• Fluorescent lifetimes very short (ns) so need fast excitation and/or fast detectors
• Requirements for FLIM instruments1. Excitation light intensity modulated or pulsed2. Emitted fluorescence measured time resolved
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Fluorescence Lifetime Imaging Microscopy (FLIM)
• Two methods for FLIM1. Frequency-domain
1. Intensity of excitation light continuously modulated2. For emission measure phase shift & decrease in modulation
2. Time-domain1. Pulsed excitation that is faster than fluorescence lifetime2. Emission measurement is time-resolved
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FRET and FLIM
• Donor fluorescence lifetime during FRET reduced compared to control donor fluorescence lifetime
• During FRET, donor fluorescence lifetime less than control donor fluorescence lifetime (tD)
• But isn’t it easier to image decreases in donor fluorescence intensity rather than measure fluorescence lifetime?
KT = (1/τD) • [R0/r]6
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FRET and FLIM
• Remember all those nonlinearities from last lecture?
• Brightness (or intensity) of fluorophore, as measured on your image, more than just Q
1. Local concentration of fluorophore2. Optical path of microscope3. Local excitation light intensity4. Local fluorescence detection efficiency
• FLIM provides independent measure of local donor lifetime
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Going back to those problems with FRET:These drawbacks can all be used to make sensors
Change in FRET for changes in:• Orientation
• cameleon dye for Ca++
• Local environment• Phosphate near fluorophore• Membrane voltage (flash)
• Change in lifetime of donor• Binding of molecule displacing water
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Cameleon: FRET-based and genetically-encoded calcium probe
Miyawaki et al, Nature, 1997
Calmodulin bonds Ca2+
and changes its conformation
[Ca2+]
Cameleon family: calmodulin-based indicators of [Ca2+] using FRET isosbestic point
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Paper to read
• Pearson, H., 2007. The good, the bad and the ugly. Nature 447, 138-140.
• http://www.nature.com/nature/journal/v447/n7141/full/447138a.html
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Spatial Resolution of Biological Imaging Techniques
• Resolution is diffraction limited.
• Abbe (1873) reported that smallest resolvable distance between two points (d) using a conventional microscope may never be smaller than half the wavelength of the imaging light (~200 nm)
Ernst Abbe (1840-1905)
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Super-resolution microscopy• Most recent Nobel prize
in Chemistry• Many ways to achieve• Some more super than
others.
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Spatial Resolution of Biological Imaging Techniques
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Super-resolution microscopy1. “True” super-resolution techniques
• Subwavelength imaging• Capture information in evanescent waves• Quantum mechanical phenomenon
2. “Functional” super-resolution techniques1. Deterministic
• Exploit nonlinear responses of fluorophores2. Stochastic
• Exploit the complex temporal behaviors of fluorophores
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Spatial Resolution of Biological Imaging Techniques
“True” super-resolution
“Functional”
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Near-Field Scanning Optical Microscopy (NSOM)
• Scanning Near-Field Optical Microscopy (SNOM)• Likely the super-resolution technique with the
highest resolution• But only for superficial structures• A form of Scanning Probe Microscopy (SPM)
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Scanning Tunneling Microscopy• Images surface at
atomic level• Developed in 1981• Binning and Rohrer won
Nobel for its development
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Scanning Tunneling Microscopy• Images surface at
atomic level• Developed in 1981• Binning and Rohrer won
Nobel for its development
• Works via quantum tunneling
• Schrödinger equation
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Near-Field Scanning Optical Microscopy (NSOM)
Break the diffraction limit by working in the near-field
Launch light through small aperture
Illuminated “spot” is smaller than diffraction limit
(about the size of the tip for a distance equivalent to tip
diameter) Near-field = distance of a couple of tip diameters
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NSOM working in the near-field
• Aperture diameter less than the wavelength of light
• In 1993 Eric Betzig and Robert Chichester used NSOM for repetitive single molecule imaging
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NSOM working in the near-field
• Near-field near surface of object, < λ of light
• Near-field consists of light as evanescent wave
• Evanescent waves higher frequency, more information
• Evanescent waves quantum tunneling phenomenon
• Product of Schrödinger wave equations
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Near-Field Scanning Optical Microscopy (NSOM)
How to make an NSOM tip
Tip of pulled quartz fiber
Very small fraction of light makes it through small
(50nm) aperture
Aluminize tip to minimize loss of light
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Near-Field Scanning Optical Microscopy (NSOM)
SEM of tip
Tip shining on sample(can detect with wide-field)
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How to move the tip? Steal from AFM
Atomic Force Microscopy (AFM)
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Atomic Force Microscopy (AFM)• Child of STM• Invented by Gerd
Binnig, first experiments 1986
• 1000 times better resolution than optical microscopes
• Scan specimen surface with very sharp tip
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AFM tips
• Most made of silicon but borosilicate glass and silicon nitride also used
Silicon Nitride Sharp tip Super tip
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Atomic Force Microscopy (AFM)• Big advantage over SEM is
that can image in liquid• Requires liquid cell for AFM
Two patches with different micelle orientation
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AFM has two types of imaging modes
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Modification to do tapping or non-contact mode
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AFM (tapping mode) of IgG
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AFM does have some disadvantages
1. Imaging area is small2. Scan speed slow3. Can be affected by
nonlinearities4. Image artifacts, e.g.
steep walls or overhangs
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Near-Field Scanning Optical Microscopy (NSOM)
Break the diffraction limit by working in the near-field
• Like AFM can do NSOM with tapping mode
• Requires bent tip• Move tip up and down
like AFM• Not best way of doing
NSOM• Hard to make probe• Bend causes loss of light
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If not tapping like AFM how else to scan tip in NSOM?
Shear force mode. Advantage: don’t need laser to keep track of probe.
To keep tip in near-field, need to be ~50nm from surface
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Sense presence of surface from dithering tip (lateral)(Increased shear force when surface is near)
Keep dithering amplitude low <10 nm
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Shear force mode with non optical feedback
• Use real-time feedback to keep probe in near-field range but not touching
• Tip can be oscillated at resonance frequency
• Tip can be straight• Easier to make• Cheaper• But surface needs to be
relatively flat
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NSOM instrument
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NSOM tips
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NSOM images
Single molecules of DiI on glass surface
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NSOM images
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NSOM disadvantages
• Practically zero working distance and small depth of field.
• Extremely long scan times for high resolution images or large specimen areas.
• Very low little light through such a tiny aperture.
• Only features at surface of specimens can be studied.
• Fiber optic probes are somewhat problematic for imaging soft materials due to their high spring constants, especially in shear-force mode.
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CLSM
Depth(um)
Resolution(um)
LM
OCT
NSOM
MRI
SPIM
SIM/STP
Performance range of optical microscopy
TIRF
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Homework 5
There are so many different ways to do super-resolution microscopy. Interestingly, an entirely novel method was just published this year in Science called expansion microscopy.
Question: What makes this super-resolution technique so novel compared to all the others?
Hint: see this figure from Ke, M.-T., Fujimoto, S., Imai, T., 2013. Nat Neurosci 16, 1154-1161.