Multiphoton Excited Fluorescence Microscopy: Principles, Instrumentation and Some Applications

Outline 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 P = induced polarization, (n) = nth order non-linear susceptibility E = electric field   (3) << (2)<< (1) (5-7 orders of magnitude per term) Linear Processes ·     Simple Absorption/Reflection ·   Rayleigh Scattering Third Order Processes ·      Multi-Photon Absorption* ·      Stimulated Raman Scattering ·      Optical Kerr Effect ·      White Light Generation Second Order Processes ·      Second Harmonic Generation* ·      Sum-Frequency Generation

One and two photon absorption physics Goeppart-Mayer, ~1936 Simultaneous absorption Virtual State: Very short lifetime ~10-17 s Requires high power: Absorption only In focal plane e.g. fluorescein Greatly Reduces out of plane bleaching

2-photon excitation of fluorescein: 3D confinement Absorption, Fluorescence only in middle at focal point Compare 1 and 2-p Absorption 1-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 and 2-photon absorption characteristics One Photon 2 photon d (10-50 cm4s) e (50,000) Absorption Coefficient units 10-50 cm4s= 1 GM (Goppert-Mayer) s (10-16 cm2) Power (photon) dependence p P2 (gives rise to sectioning) Laser Temporal dependence none 1/t (virtual state) Absorption probability p2 s p d /t Cannot use cw lasers (Ar+)

2-Photon Absorption Probability Assume 10 GM cross section (fluorescein) 100 femtosecond pulse 1.4 NA 800 nm 80 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 One photon absorption simply measured in UV-VIS Spectrometer, Beer’s Law A= εcl need new setup for two-photon absorption: need focused light Two-photon cross section measurement Epi geometry Measure Fluor. Measure wavelength Measure pulse width Measure power Control power Xu and Webb, 1996 Measure by fluorescence intensity, need quantum yield (same as 1 photon)

Two-photon spectrum of rhodamine B: Discrete points, not continuous like UV-VIS for 1-photon Max 820 nm not 1050 nm Cross section GM Near-Infrared, rather than visible used for 1-p

Power Dependence to determine photon number: log-log plots Fluorescein and rhodamine Right slope of 2 at All wavelengths: 2-photon process Xu and Webb, 1996

Verify emission spectrum Same emission spectrum for 1-p, 2-p excitation Should be, from Quantum Mechanics Relaxation is independent of Mode of excitation Same emission spectrum For different 2-p wavelengths: 750 and 800 nm Just like 1-photon emission Xu and Webb, 1996

Pulse Width Dependence Slope of 1 Correct pulse width dependence Xu and Webb, 1996

Heteroatom substitution strong medium Rhodamine (best one) fluorescein Good 2-p properties Big conjugation Donor/Acceptor Pair (push-pull) Heteroatom substitution Weak UV absorb Small conj Indo-1 2-P δ Rhodamine 5x>fluorescein because D/A pair Also relaxes selection rules over fluorescein

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

Some Generalities about multi-photon absorption Emission spectrum is the same as 1-p Emission quantum yield is the same Fluorescence lifetime is the same 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)) Nominally forbidden in 2-p Nominally forbidden in 1-p: missing in fluorescein Allowed and stronger in 2-p

1 and 2-photon bands Reverse of 1-photon For all xanthenes: Fluorescein, rhodamines All max ~830 nm Not ~1000 nm

Fluorescein, Rhodamine Similar band structure Rhodamine B Cross section GM Cross section GM strong medium Xu and Webb, 1996

Multi-color imaging in tissue culture cells: Nucleus (blue), mitochondria (red), actin (green) Simultaneous imaging not possible in one photon absorption : Different transitions, need multiple lasers: 390, 490, 540 nm Image all 3 simultaneously via 2-photon with ti:sapphire laser 780 nm: different selection rules for MPE So et al

Confocal detection must be descanned through pinhole to achieve axial discrimination, eliminate out of focus light Very inefficient Optical budget Limited depth of Penetration ~30 microns (based on one scattering Length in most tissues)

Open pinhole, Not necessary But still Confocal, lossy 2-photon does not Need pinhole at all Or descanning to Eliminate out of Focus light (intrinsic sectioning) Ti:sapphire 100 femtosecond 80 MHZ Open pinhole, Not necessary But still Confocal, lossy Non-decanned Detection greatly Increases the Sensitivity 3-5 fold Very significant Fewer optics No pinhole, Detect scattered light

“The Campagnola” Very simple beampath Relative 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 power None useful for multiphoton excitation Not tunable or red enough to match 2-photon bands

Tunable Lasers Gain Medium with broad emission spectrum gives tunability to excite any fluorophore Still monochromatic when lasing Organic Dyes (e.g. rhodamines, styryls), titanium sapphire) Ti:sapphire is almost universally used for 2-photon excitation Ti: 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

Modelocked ti:sapphire laser for 2-photon microscope 2-photon would not have taken off without this laser Pumped with 532 nm Tunable 700-1000 nm 100 fs, 80 MHz

Tuning Range and Power of Ti:Sapphire Longer wavelengths Less damaging 900 nm is often good Compromise between Power and viability Gets dodgy after 900 Except with big laser, Alignment critical Excite essentially every dye, Fluorescent protein With this wavelength range 100 femtosecond pulses 10 nm FWHM bandwidth

Tissue Imaging: MPM enabling Depth and sensitivity

2-photon depth much improved: Reduced scattering 1047 vs 532 nm Contrast stretched 2-photon depth much improved: Reduced scattering 1047 vs 532 nm 2-p descanned here White, Biophys J, 1998

Non-descanned (direct) detection provides greater sensitivity Photons are Scattered, miss pinhole Still fairly Confocal MFP~20-50 microns X-Z projection Sensitivity (best signals) Confocal (1-p)<2-p descanned< 2-p direct 2-p direct collects ballistic and scattered photons White, Biophys J, 1998

Non-descanned (direct) detection provides greater sensitivity Important at increasing depth X-Z direct Make or break experiment with Highly scattering tissue White, Biophys J, 1998

2-photon imaging of retina (salamander) Fluorescein labeled X-Z projection Too thick for 1-p Good contrast throughout Denk, PNAS, 1999

Depth capability using 2-photon absorption Svoboda, Neuron, 2006, vol 50, 823 (review of 2-P for neuroscience)

Autofluorescence of endogenous species in tissues Need multi-photon excitation, non-descanned detection For enough sensitivity: small cross sections and quantum yields

Autofluorescence in Tumors Mitochondria: NADH, Flavins NAD not fluorescent NADH emission to Monitor respiration NADH good diagnostic Of cell metabolism Small cross section Quantum yield ~10% Small delta ~0.1 GM High concentration Need non-descanned Detection to be viable

Imaging Muscle (NADH) With TPE Fluorescence Low cross section but High concentration Balaban et al

2-photon Tissue Imaging (Mouse Ear) keratinocytes Basal cells Collagen/elastin fibers cartilage So et al Ann. Rev. BME 2000 Autofluorescence good for many layers

Human Skin Two-photon imaging Strata corneum Keratinocytes Dermal layer (elastin, collagen) fibers So et al Ann. Rev. BME 2000 More versatile than dyes (but weaker) MPM enabling, very weak in confocal

Endogenous only way for in vivo clinical Applications. 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

2-photon FRAP in cells, solution Calcein in RBL cells Calcein In solution Webb et al

2-P photobleaching and fluorescence recovery In starfish oocyte 10 kD dye- Dextran Cross nuclear envelope 70 kD does not cross Line scan bleach, page scan recovery Better Cell viability than 1-p due to confinement

2-photon uncaging glutamate Fluo-5 calcium sensitive Alexa Ca insensitive Need 2-p localization For this Svoboda, Neuron, 2006

2-photon photoactivation of GFP Uncaging cross sections very small Fraction of 1 GM Requires high power, short wavelengths PA FP can be more efficient Svoboda, Neuron, 2006 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 this Not usually possible by 1-photon

Objective Lenses Registration Issues 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

  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 dependent determined 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) Practical Considerations of Multi-photon Excited Fluorescence Microscopy   Registration Issues   ·     Focus of White light vs Laser often different by 10-20 microns ·     Overlapping visible and near-infrared lasers difficult for uncaging ·     Second Harmonic Generation Alignment is different than Laser

Damage can arise from Higher Order Absorption Plasma formation: Very damaging Bleaching, Free radical damage In plane, same for 1,2 photons For first triplet

Bleaching of fluorescein dextran in droplets 488 nm 1-photon 710 nm 2-photon Slope=1.2 Slope=1.9 (low power) Like absorption probability Piston, Biophys J. 2000

decreased at longer wavelengths Two-photon Bleaching as function of average power, exposure Bleaching highly Nonlinear: 16.5 mW>>8.3 Much higher rate Than quadratic Scaling Goes to higher states Nonlinearity would be decreased at longer wavelengths Piston,Biophys J.2000

Non-linear bleaching (ctd) NADH=3.65 Coumarin=5.1 Indo-1=3.5 Highly nonlinear: Higher order processes Excitation to higher states For same transition 2-p Does not bleach more Than 1-p! Piston,2000

Photodamage and Average Power in live cells (Neher, 2002) Fura 2-AM visual Two criteria yield Slope=2.4 (log-log) Some 3-p contribution

Neher and Photodamage continued Pulse width dependence Pixel Dwell time dependence Linear with pixel dwell time: No change in peak power Consistent with highly nonlinear damage Photodamage~-1.5 Also highly nonlinear

RAYLEIGH CRITERION for Resolution for 2 Objects Barely resolved Not resolved Completely Resolved

Determination of Point Spread Function of Microscope Abbe` Limit PSF is measured size of infinitely small Point source of light 175 nm fluorescent Bead Sub-resolution Volume is Ellipsoid Axial ~NA2

One and Two-photon axial discrimination widefield 1-p confocal 2-photon Longer wavelength 2-photon confocal 1-p and 2-p Confocal geometries about the same resolution 1-p confocal better than 2-P nonconfocal

Comparison of 1 and 2 photon PSFs 2P wavelength twice that of one photon Radial PSF Smaller PSF= Better resolution Axial PSF One photon is better because Shorter wavelength, but 2-p Better than Abbe limit (not twice 1-photon) NLO not diffraction limited Cooperative effect

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!!