Topics Acoustic Optical Modulator Faster scanning methods Laser trapping Fluorescence lifetime imaging.

Topics

• Acoustic Optical Modulator

• Faster scanning methods

• Laser trapping

• Fluorescence lifetime imaging

Acousto-optic modulator

Bragg Diffraction: sound waves diffract lightCan achieve ~90% diffraction efficiency into 1st order spot

RF (100 MHz) onTransducer sets upAcoustic wave inSecond crystal,Forms grating0th order

Condition for Constructive interference:

Double-slit Experiment

a sinθ = nλ

n = 0, 1, 2, 3 …

After focusing:

d = f λ / a

Applications of Acousto-optic Modulators in microscopes

1) Select Wavelength (tunable filter AOTF): vary drive frequency:Achieve same angle of deflection (wavelength dependent, spacing of grating)

2) Control Laser Power: vary RF power to change diffraction fraction

3) Control Beam angle for fast scanning: vary frequency for same , fixed power (achieves different deflected angle)

AOTF to select laser line and power(drive frequency and RF power, respectively)

Laser line selection

Acousto-optic beam deflector

Scanning in a confocal microscope: very fastCompared to galvo mirrors ~100 fold (paper next week)

Sweep beam byChanging deflection(linearized)

Faster Imaging than with two galvos: line scanning + one galvo

Linear CCDSlit pinholes

Detection on line-scanning microscope

Scanning via spinning disk

Spinning disk microscopy

Uses White light: convenient but very poor light budget

Microlens focuses onPinholes, conjugateTo specimen plane

CCD detection,Much higherquantum efficiencyThan PMT

Modern Design

Light contamination between adjacent pinholes

Spinning disk microscopy

Advantages:1. Can image very rapidly ( image collection not limited

by scanning mirrors

2. Use of cooled CCD camera yields lower noise than PMT (un-cooled) higher quantum yield

Disadvantages:

1. Light path not efficient (need powerful light source)2. Fixed pixel size3. Disk needs to match objective4. Lose spatial control of excitation field5. Problem with very thick samples

Laser Trapping

Light Can Be Bent by Air

• Dielectric material

• n > n(surroundings)

• Force range is in pN

How to measure the force?

xxxF )(Langevin equationStochastic force

xxxF )(Langevin equation

power spectrum )(

)(222c

B

ff

TkfS

Position sensing with Quadrant photodiodes

x = [(B+D) - (A+C)] / [A+B+C+D]y = [(A+B) - (C+D] / [A+B+C+D]

Direct observation of base-pair stepping by RNA polymerase

Nature. 2005 Nov 24;438(7067):460-5

Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM

www.bact.wisc.edu/landick/research.htm

Simple

But low resolution

Stepping size per base pair = 3.4 Å

The Dumbbell Setup

The Concept of Force Clamp

Summary:

1. Decouple from stage

2. Helium environment

3. Passive force clamp

HOT

HolographicOpticalTweezers

Sensitive to environment: pH, ions, potentialSNARF, Calcium Green, CameleonsPerform in vitro calibrations

• Results Not sensitive to bleaching artifacts

• Not sensitive to uneven staining (unless self-quenched)

• Not sensitive to alignment (intensity artifacts)

Fluorescence Lifetime Imaging

iscf

f

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k

nonradiscf

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iscf kk 1

Fluorescence Quantum Yield φ: important for dyesRatio of emitted to absorbed photons

Measured lifetime is sum ofRates of natural lifetime and non radiative decay paths

(k is rate,(k is rate,Inverse of time)Inverse of time)

Quantum Yield:Quantum Yield:

fk 1

0 Natural lifetime

Unquenched emission:Normal QY, lifetime

Quenched emissionDecreased QY, lifetimee.g. metals, aggregation

Unquenched and Quenched Emission

iscf

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k

nonradiscf

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2 general approaches: time domain and frequency domain

Short pulse laser modulate CW laser

Frequency Domain Methods for Lifetime Measurements:Modulate laser and measure phase change of fluorescence

Use cw laser (e.g. argon ion)Modulate at rate nearInverse of emission lifetime10-100 MHz

Measure phase changewith Lockin amplifier

Time-domain Widefield Lifetime imaging with ICCD

Variable delayed gate is scannedTo sample exponential decay:Many frames

ICCD has no time intrinsic response: slow readoutGated gain

Two-photon has short pulse laser for time-gating

Time-correlated single photon counting:•most flexibility, most accurate,•samples whole decay•Best time response

Measures time of flight of photonsAfter excitation pulse

Bins data at each time intervalRather than gating

Collect enough photons to approximate exponential:

Slower than gating butBetter measurement, Can separate biexponentials: Multiple components

Principles of time-correlated single photon counting

TAC or TDC measures time of flight, bins photons

B&H addon to Zeiss Laser scanning confocal

Electronics all in one PCI board, ~50K addon

Time gating measurements of fluorescence decayTemporal Resolution defined by IRF (laser, detector, electronics)

IRF=instrument response function,Must be (much) shorter than fluorescence lifetime (delta function)to avoid convolution

Measure IRF with reflectionor known short lifetimee.g. Rose Bengal (90 ps)

Ideal IRF Real IRF

Gate away from IRF (laser pulse, PMT response)Lose photons

PMT Detectors for Lifetime measurements

~300 picosecond resolutionBetter with deconvolutionCost ~$500

~30 picosecond resolutionNo dispersionCost ~$15000fragile

PMTS have low quantum yield(10-20%), MCP worse ~5%

Microchannel plate photomultiplier: full of holes, kick off electrons

Dispersion in time of flightacross 14 dynodesLimits time response

Intensity vs fluorescence lifetime image

Same dye, different lifetime because of environment

FRET Outcomes

Donor decreases

Acceptorincreases

LifetimeIntensity

CFP and YFP FRET by Lifetime Imaging

Channel changes conformation, distance changes, Donor quenching occurs due to FRET

Short lifetime is FRET from DonorFor given pixel Ratio of fast to slow decay coefficientsis estimate of FRET efficiency

FLIM as Diagnostic of Joint Disorder

H&E staining

Widefieldfluorescence

WidefieldFLIM

Little info

Detail revealed by FLIM

Fixed, thin sections

Effects of Pinhole Size

Intensity and lifetime measurements

CFP-YFP linked by short peptide chainEnergy is transferred from CFP to YFPLifetime reveals info intensity does not