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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.

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slide1

Multiphoton Excited Fluorescence Microscopy:Principles, Instrumentation and Some Applications

slide2

Outline

  • MPE Photophysics
  • 2) Instrumentation
  • 3) Some Applications of MPE Fluorescence
  • 4) MPE Photodamage Issues
  • 5) 1,2,3 Photon Resolution
slide3

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

slide4

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

slide5

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

slide6

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

slide7

One and 2-photon absorption characteristics

One Photon2 photon

d (10-50 cm4s)

e (50,000)

Absorption

Coefficient units

10-50 cm4s=

1 GM (Goppert-Mayer)

s (10-16 cm2)

p

P2 (gives rise to sectioning)

Power (photon)

dependence

Laser Temporal

dependence

(virtual state)

1/t

none

Absorption

probability

p2

s

p

d

/t

Cannot use cw lasers (Ar+)

slide8

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?

slide9

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)

slide10

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

slide11

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

slide12

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

slide13

Pulse Width Dependence

Slope of 1

Correct pulse width dependence

Xu and Webb, 1996

slide14

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

slide15

Conclusion: most dyes used for confocal work for

Two-photon excitation (some better than others)

slide16

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

slide17

1 and 2-photon bands

Reverse of 1-photon

For all xanthenes:

Fluorescein,

rhodamines

All max ~830 nm

Not ~1000 nm

slide18

Fluorescein, Rhodamine

Similar band structure

Fluorescein

Rhodamine B

Cross section GM

Cross section GM

strong

medium

Xu and Webb, 1996

slide19

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

slide21

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)

slide22

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

slide23

“The Campagnola”

Very simple beampath

Relative to confocal

slide25

Lasers for multiphoton excitation

Typical confocals use fixed laser lines,

Can be limiting for multi-color imaging

slide26

Lasers for confocal microscopy

*

*

*

All cw: no peak power

None useful for multiphoton excitation

Not tunable or red enough

to match 2-photon bands

slide27

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

slide28

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

slide29

Modelocked ti:sapphire laser for 2-photon microscope

2-photon would not have taken off without this laser

100 fs, 80 MHz

Tunable 700-1000 nm

Pumped with 532 nm

slide30

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

slide31

Tissue Imaging: MPM enabling

Depth and sensitivity

slide32

1-p

2-p

Contrast stretched

2-photon depth much improved:

Reduced scattering

1047 vs 532 nm

2-p descanned here

White, Biophys J, 1998

slide33

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

slide34

Non-descanned (direct) detection provides greater sensitivity

Important at increasing depth

X-Z

direct

White, Biophys J, 1998

Make or break experiment with

Highly scattering tissue

slide35

2-photon imaging of retina (salamander)

Fluorescein labeled

X-Z projection

Too thick for 1-p

Good contrast throughout

Denk, PNAS, 1999

slide36

Depth capability using 2-photon absorption

Svoboda, Neuron, 2006, vol 50, 823

(review of 2-P for neuroscience)

slide38

Autofluorescence of endogenous species in tissues

Need multi-photon excitation, non-descanned detection

For enough sensitivity: small cross sections and quantum yields

slide39

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

slide40

Imaging Muscle (NADH)

With TPE Fluorescence

Low cross section but

High concentration

Balaban et al

slide41

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

slide42

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

slide43

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 bleaching
Multiphoton bleaching

Need 3D treatment, both radial, axial PSF

slide46

2-photon FRAP in cells, solution

Calcein in RBL cells

Calcein In solution

Webb et al

slide47

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

slide48

2-photon uncaging glutamate

Fluo-5 calcium sensitive

Alexa Ca insensitive

Need 2-p localization

For this

Svoboda, Neuron, 2006

slide49

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

slide51

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
slide52

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

slide53

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

slide55

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

slide56

Bleaching of fluorescein dextran in droplets

488 nm 1-photon

710 nm 2-photon

Slope=1.9 (low power)

Slope=1.2

Like absorption probability

Piston, Biophys J. 2000

slide57

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

slide58

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

slide59

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

slide60

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

slide62

RAYLEIGH CRITERION for Resolution for 2 Objects

Barely resolved

Completely Resolved

Not resolved

slide63

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

slide64

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

slide65

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

slide66

Optical Resolution of the Campagnola Microscope

Imaging sub-resolution 100 nm fluorescent beads

Both agree well with theory

slide67

 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!!