1. Multiphoton Excited Fluorescence Microscopy: Principles, Instrumentation and Some Applications

2. Outline
MPE Photophysics
2) Instrumentation
3) Some Applications of MPE Fluorescence
4) MPE Photodamage Issues
5) 1,2,3 Photon Resolution

3. 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

4. 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

5. 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

6. 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

7. 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+)

8. 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?

9. 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)

10. 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

11. 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

12. 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

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

14. 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

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

16. 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

17. 1 and 2-photon bands
Reverse of 1-photon
For all xanthenes:
Fluorescein,
rhodamines
All max ~830 nm
Not ~1000 nm

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

19. 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

21. 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)

22. 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

23. “The Campagnola”
Very simple beampath
Relative to confocal

25. Lasers for multiphoton excitation
Typical confocals use fixed laser lines,
Can be limiting for multi-color imaging

26. Lasers for confocal microscopy* * *
All cw: no peak power
None useful for multiphoton excitation
Not tunable or red enough
to match 2-photon bands

27. 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

28. 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

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

30. 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

31. Tissue Imaging: MPM enabling
Depth and sensitivity

32. 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

33. 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

34. 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

35. 2-photon imaging of retina (salamander)
Fluorescein labeled
X-Z projection
Too thick for 1-p
Good contrast throughout
Denk, PNAS, 1999

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

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

39. 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

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

41. 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

42. 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

43. 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

44. Multiphoton for FRAP, uncaging, photoactivation

45. Multiphoton bleaching
Need 3D treatment, both radial, axial PSF

46. 2-photon FRAP in cells, solution
Calcein in RBL cells
Calcein In solution
Webb et al

47. 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

48. 2-photon uncaging glutamate
Fluo-5 calcium sensitive
Alexa Ca insensitive
Need 2-p localization
For this
Svoboda, Neuron, 2006

49. 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

51. 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 nm, but only one wavelength at a time:
Optimize if multiple fluorophores, can usually do this
Not usually possible by 1-photon

52. 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 microns (dispersion)
·     Overlapping visible and near-infrared lasers difficult for uncaging

53. 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 ( nm) (abnormal cell division)
damage thresholds highly wavelength dependent
determined by cell division or live cell/dead cell membrane assay
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 microns
·     Overlapping visible and near-infrared lasers difficult for uncaging
·     Second Harmonic Generation Alignment is different than Laser

55. 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

56. 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

57. 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

58. 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

59. 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

60. 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

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

63. 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

64. 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

65. 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

66. Optical Resolution of the Campagnola Microscope
Imaging sub-resolution 100 nm fluorescent beads
Both agree well with theory

67.  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!!