Multiphoton Excited Fluorescence Microscopy:Principles, Instrumentation and Some Applications

Outline

1) MPE Photophysics

2) Instrumentation

3) Some Applications of MPE Fluorescence

4) MPE Photodamage Issues

5) 1,2,3 Photon Resolution

Interaction of Light with Matter

...3)3(2)2(1)1( EEEP

P = induced polarization,(n) = nth order non-linear susceptibilityE = electric field 

Linear Processes ·     Simple Absorption/Reflection ·   Rayleigh Scattering

(3) << (2)<< (1) (5-7 orders of magnitude per term)

Second Order Processes

·      Second Harmonic Generation*

·      Sum-Frequency Generation

Third Order Processes

·      Multi-Photon Absorption*

·      Stimulated Raman Scattering

·      Optical Kerr Effect

·      White Light Generation

One and two photon absorption physics

Requires high power:Absorption onlyIn focal plane

Greatly Reduces out of plane bleaching

Simultaneous absorptionVirtual State:Very short lifetime ~10-17 s

Goeppart-Mayer, ~1936

e.g. fluorescein

2-photon excitation of fluorescein: 3D confinement

Absorption, Fluorescence only in middle at focal point

Compare 1 and 2-pAbsorption1-p excites throughout

Advantages of Non-linear Optical Excitation in Imaging ·     Intrinsic 3-Dimensionality (no pinhole)

·    Little Near Infrared and IR Absorption of Biomolecules Greatly Minimizes Out-of-Plane Photo-Bleaching/Damage

 ·    Comparable Lateral (X,Y) and Axial (Z) Resolution to confocal

·     Large Depth of Penetration (scattering decreases in NIR)*5-10 fold

·     Enhanced Contrast and Sensitivity* (non-descanned detection)

 

  * enabling aspects for tissue imaging: brain, connective tissue, muscle

One Photon 2 photon

Absorptionprobability

AbsorptionCoefficient units

(50,000)

(10-16 cm2)

(10-50 cm4s)

10-50 cm4s=1 GM (Goppert-Mayer)

Power (photon)dependence

p P2 (gives rise to sectioning)

Laser Temporaldependence

none 1/

p p2 /

(virtual state)

One and 2-photon absorption characteristics

Cannot use cw lasers (Ar+)

22

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hc

NAPna

2-Photon Absorption Probability

Assume 10 GM cross section (fluorescein)100 femtosecond pulse1.4 NA800 nm80 MHz

Saturation (na≈1) occurs ~50 mW average power

Can propagate same form for three-photon absorption:Need at least 10 fold higher average power

Do most dyes have good enough two-photon cross sections for imaging?

Two-photon cross section measurement

Xu and Webb, 1996

Measure by fluorescence intensity, need quantum yield(same as 1 photon)

One photon absorption simply measured in UV-VIS Spectrometer, Beer’s Law A= εclneed new setup for two-photon absorption: need focused light

Measure wavelength

Measure pulse width

Epi geometry

Measure power

MeasureFluor.

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hc

NAPna

Control power

Two-photon spectrum of rhodamine B:Discrete points, not continuous like UV-VIS for 1-photon

Near-Infrared, rather than visible used for 1-p

Cro

ss

se

ctio

n G

M

Max 820 nmnot 1050 nm

Xu and Webb, 1996

Right slope of 2 atAll wavelengths:2-photon process

Fluorescein and rhodamine

Power Dependence to determine photon number: log-log plots

Same emission spectrumfor 1-p, 2-p excitationShould be, from Quantum MechanicsRelaxation is independent ofMode of excitation

Same emission spectrumFor different 2-p wavelengths:750 and 800 nmJust like 1-photon emission

Xu and Webb, 1996

Verify emission spectrum

Pulse Width Dependence

Correct pulse width dependence

Xu and Webb, 1996

22

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NAPna

Slope of 1

fluorescein Rhodamine (best one)

Indo-1

Good 2-p properties

• Big conjugation

• Donor/Acceptor Pair(push-pull)

• Heteroatom substitution

2-P δ Rhodamine 5x>fluorescein because D/A pairAlso relaxes selection rules over fluorescein

medium

WeakUV absorb Small conj

strong

Conclusion: most dyes used for confocal work forTwo-photon excitation (some better than others)

Some Generalities about multi-photon absorption

1) Emission spectrum is the same as 1-p

2) Emission quantum yield is the same

3) Fluorescence lifetime is the same

4) Spectral positions nominally scale for the same transition: 2-p is twice 1-p wavelength for

5) Selection rules are often different, especially for xanthenes(fluorescein, rhodamine, and derivatives, (calcium green, fluos))

10 SS Nominally forbidden in 2-p

20 SS Nominally forbidden in 1-p: missing in fluoresceinAllowed and stronger in 2-p

10 SS 20 SS

Rhodamine Photophysics

10-12 s

1000 nm TPE

500 nm OPE

800 nm TPE

400 nm OPE

10-9 s

S0

S1

S2

800 nm stronger than 1000 nm band

Reverse of 1-photonFor all xanthenes:Fluorescein,rhodamines

All max ~830 nmNot ~1000 nm

1 and 2-photon bands

mediumstrong

Xu and Webb, 1996

FluoresceinRhodamine B

Fluorescein, RhodamineSimilar band structure

Cro

ss

se

ctio

n G

M

Cro

ss

se

ctio

n G

M

So et al

Simultaneous imaging not possible in one photon absorption :

Different transitions, need multiple lasers:390, 490, 540 nm

Multi-color imaging in tissue culture cells:Nucleus (blue), mitochondria (red), actin (green)

Image all 3 simultaneously via 2-photonwith ti:sapphire laser 780 nm: different selection rules for MPE

Confocal detection must be descanned through pinholeto achieve axial discrimination, eliminate out of focus light

Very inefficientOptical budget

Limited depth ofPenetration ~30 microns(based on one scatteringLength in most tissues)

Non-decannedDetection greatlyIncreases theSensitivity

3-5 foldVery significantFewer opticsNo pinhole, Detect scatteredlight

Open pinhole,Not necessaryBut stillConfocal, lossy

2-photon does not Need pinhole at allOr descanning toEliminate out ofFocus light(intrinsic sectioning)

Ti:sapphire100 femtosecond80 MHZ

“The Campagnola”

Very simple beampathRelative to confocal

Lasers for multiphoton excitation

Typical confocals use fixed laser lines,Can be limiting for multi-color imaging

**

*

Lasers for confocal microscopy

All cw: no peak powerNone useful for multiphoton excitationNot tunable or red enoughto match 2-photon bands

Tunable Lasers

Gain Medium with broad emission spectrum gives tunabilityto excite any fluorophoreStill monochromatic when lasing

Organic Dyes (e.g. rhodamines, styryls), titanium sapphire)

Ti:sapphire is almost universally used for 2-photon excitationTi: Al2O3

Titanium Sapphire Ti3+: Al2O3

E 3/2 excited

state

2T2 ground

state

Spin forbidden: long emission lifetime 300 µs

ε=14000, strong for spin-forbidden transition

Huge Stokes shift, Broad emission spectrum

Revolutionized laser industry in ability to make short pulses

Pumped with 532 nm Tunable 700-1000 nm 100 fs, 80 MHz

Modelocked ti:sapphire laser for 2-photon microscope2-photon would not have taken off without this laser

Tuning Range and Power of Ti:Sapphire

100 femtosecond pulses10 nm FWHM bandwidth

Longer wavelengths Less damaging

900 nm is often goodCompromise betweenPower and viability

Gets dodgy after 900Except with big laser,Alignment critical

Excite essentially every dye,Fluorescent proteinWith this wavelength range

Tissue Imaging: MPM enablingDepth and sensitivity

White, Biophys J, 1998

2-photon depth much improved:Reduced scattering 1047 vs 532 nm

1-p 2-p Contrast stretched

2-p descanned here

White, Biophys J, 1998

Non-descanned (direct) detection provides greater sensitivity

Confocal (1-p)<2-p descanned< 2-p direct2-p direct collects ballistic and scattered photons

Still fairlyConfocal

Photons areScattered, misspinhole

Sensitivity (best signals)

X-Zprojection

MFP~20-50 microns

Non-descanned (direct) detection provides greater sensitivity

White, Biophys J, 1998

Important at increasing depth

direct

X-Z

Make or break experiment with Highly scattering tissue

2-photon imaging of retina (salamander)

Fluorescein labeled

X-Z projection

Denk, PNAS, 1999

Too thick for 1-pGood contrast throughout

Svoboda, Neuron, 2006, vol 50, 823(review of 2-P for neuroscience)

Depth capability using 2-photon absorption

Autofluorescence of endogenous species in tissues

Need multi-photon excitation, non-descanned detectionFor enough sensitivity: small cross sections and quantum yields

Autofluorescence in Tumors

Mitochondria:NADH, Flavins

NAD not fluorescentNADH emission toMonitor respiration

NADH good diagnosticOf cell metabolism

Small cross sectionQuantum yield ~10%Small delta ~0.1 GMHigh concentration

Need non-descannedDetection to be viable

Imaging Muscle (NADH)With TPE Fluorescence

Low cross section butHigh concentration

Balaban et al

So et alAnn. Rev. BME2000

2-photon Tissue Imaging (Mouse Ear)

keratinocytesBasal cells

Collagen/elastinfibers

cartilage

Autofluorescence good for many layers

Strata corneum

Keratinocytes

Dermal layer(elastin, collagen)fibers

Human Skin Two-photon imaging

So et alAnn. Rev. BME2000 More versatile than dyes (but weaker)

MPM enabling, very weak in confocal

Endogenous only way for in vivo clinicalApplications.

Cannot use dyes (toxicity) cannot penetrate tissues or GFP expressions

Trend is multimodal: fluorescence + scattering fluorescence + CT fluorescence + PET

Needs multiphoton for depth of penetration and Sensitivity due to weak signals

Multiphoton for FRAP, uncaging, photoactivation

Multiphoton bleaching

Need 3D treatment, both radial, axial PSF

Calcein in RBL cells

Calcein In solution

2-photon FRAP in cells, solution

Webb et al

2-P photobleaching and fluorescence recoveryIn starfish oocyte

10 kD dye- DextranCross nuclear envelope

70 kD does not cross

Better Cell viability than 1-p due to confinement

Line scan bleach, page scan recovery

2-photon uncaging glutamate

Svoboda, Neuron, 2006

Fluo-5 calcium sensitiveAlexa Ca insensitive

Need 2-p localizationFor this

2-photon photoactivation of GFP

Svoboda, Neuron, 2006

Uncaging cross sections very smallFraction of 1 GMRequires high power, short wavelengthsPA FP can be more efficient

Measure of diffusion

Choice of Excitation Wavelength ·     Redder is always better for cell viability, imaging depth in tissue ·     Selection Rules different for One and Two-photon Excitation for

many dyes: check published literature (some not right)•  Fluorescein 2-p maximum is 820 nm, but that band is invisible in 1-p excitation

•2-P absorption coefficients do not always scale with 1-p absorption Fluorescein 5x weaker 2-p absorber than Rhodamine All flourescein like dyes are somewhat weak in 2-P:

Calcium Green, Calcein, Fluos

·     Ti:Sapphire is tunable 700-1000 nm, but only one wavelength at a time:

Optimize if multiple fluorophores, can usually do thisNot usually possible by 1-photon   

Optical Considerations  

Objective Lenses ·     Throughput: most lenses were designed for Visible Excitation, not near- Infrared (but changing) ·     Highly corrected lenses have losses of 2-4 fold, depending on wavelength (worse to the red) and square dependence on NLO= big losses ·     Neofluars worse transmission than Fluars ·     Some New lenses are available optimized for near- infrared   Registration Issues

 ·     Focus of White light vs Laser often different by 10-20 microns (dispersion)  ·     Overlapping visible and near-infrared lasers difficult for uncaging  

Practical Considerations of Multi-photon Excited Fluorescence Microscopy 

 MPE good for long term imaging because of sectioning but there are drawbacks ·     Still bleaches in plane just like one photon ·     New problems can arise from high peak power giving rise to unwanted non-linear effects Plasma formation leading to cell destruction (makes holes) Accidental 3 photon absorption of proteins and nucleic acids (700-800 nm) (abnormal cell division) damage thresholds highly wavelength dependentdetermined by cell division or live cell/dead cell membrane assay 700-800 nm ~10 mW at 1.4 NA is good limit at sample(Scales for lower NA) >850 nm little damage (still bleaches in-plane)

  

Damage can arise from Higher Order Absorption

Plasma formation:Very damaging

Bleaching, Free radical damageIn plane, same for 1,2 photonsFor first triplet

Piston, Biophys J. 2000

488 nm 1-photon

Slope=1.2

Bleaching of fluorescein dextran in droplets

710 nm 2-photon

Slope=1.9 (low power)

Like absorption probability

Two-photon Bleaching as function of average power, exposure

Piston,Biophys J.2000

Bleaching highlyNonlinear:16.5 mW>>8.3Much higher rateThan quadraticScalingGoes to higher states

Nonlinearity would be decreased at longer wavelengths

Piston,2000

NADH=3.65

Coumarin=5.1

Indo-1=3.5

Highly nonlinear:Higher order processesExcitation to higher states

Non-linear bleaching (ctd)

For same transition 2-pDoes not bleach moreThan 1-p!

Photodamage and Average Power in live cells (Neher, 2002)

Two criteria yieldSlope=2.4 (log-log)

Some 3-p contribution

visual

Fura 2-AM

Neher and Photodamage continued

Photodamage~-1.5

Also highly nonlinear

Linear with pixel dwell time:No change in peak powerConsistent with highly nonlineardamage

Pulse width dependence Pixel Dwell time dependence

RAYLEIGH CRITERION for Resolution for 2 Objects

Not resolved Barely resolved Completely Resolved

Determination of Point Spread Function of Microscope

NAd

2

22.1min

Abbe` Limit

175 nm fluorescent BeadSub-resolution

Volume is EllipsoidAxial ~NA2

PSF is measured size of infinitely smallPoint source of light

One and Two-photon axial discrimination

widefield

1-p confocal

2-photonLonger wavelength

2-photon confocal

1-p and 2-p Confocal geometries about the same resolution1-p confocal better than 2-P nonconfocal

Radial PSF

Axial PSF

Comparison of 1 and 2 photon PSFs2P wavelength twice that of one photon

One photon is better becauseShorter wavelength, but 2-p Better than Abbe limit(not twice 1-photon)NLO not diffraction limitedCooperative effect

Smaller PSF=Better resolution

Optical Resolution of the Campagnola Microscope

Imaging sub-resolution 100 nm fluorescent beads

Both agree well with theory

 Is 2-photon excitation really necessary or advantageous for the experiment?

Weakly Fluorescent (autofluorescence) samples:

yes, sensitivity non-descanned detection can make or break experiment

Thick and turbid samples: yes, sensitivity, detection Reduced scattering  

FRAP, FRET, Uncaging: yes, 3-D

 

routine Cell Imaging: NO!!