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Multi-photon Fluorescence
Microscopy
Topics
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Basic Principles of multi-photon imaging
Laser systems
Multi-photon instrumentation
Fluorescence probes
Applications
Future developments
Multi-photon Excitation
A non-linear process
• Excitation caused by 2 or more photons
interacting simultaneously
• Fluorescence intensity proportional to
(laser intensity)n , n = number of photons
• fluorescence localised to focus region
History - Multi-photon
• Originally proposed by Maria
Goeppert-Mayer in 1931
• First applications in molecular
spectroscopy (1970’s)
• Multi-photon microscopy first
demonstrated by Denk, Strickler and
Webb in 1989 (Cornell University,
USA)
• With Cornell, Bio-Rad is the first to
commercial develop the technology in
1996
Multi-photon microscopy
• The only contrast mode is fluorescence ( IR
transmission/DIC is possible)
• Lateral and axial resolution are determined by
the excitation process
• Red or far red laser illumination is used to
excite UV and visible wavelength probes
(e.g.. 700nm for DAPI)
Multi-Photon Excitation
Physical Principles
Consequence of multi photon excitation
1-Photon
* Excitation occurs everywhere
that the laser beam interacts
with samples
* Excitation efficiency
proportional to the intensity
2-Photon
* Excitation localised
* Excitation efficiency proportional
the square of laser intensity
* Emission highest in focal region
where intensity is highest
Classical and
confocal
fluorescence
Multi-photon
fluorescence
Key points for multi photon excitation
• Wavelength of light used is approximately 2 x that used in a
conventional system. (i.e. red light can excite UV probes)
• Excitation process depends on 2-Photons arriving in a very short
space of time (i.e. 10 -16 seconds)
• Special kind of laser required
Lasers for MP
Mode-locked femto-second lasers
CW and Pulsed Lasers
CW
Pulsed
Short Pulse Advantage
Fluorescence proportional
to 1/pulse width x repetition rate
Laser Options
• Coherent, Verdi-Mira (MiraX-BIO) X-Wave Optics, good
beam pointing, beam reducer needed
• Spectra Physics, Millennia/Tsunami Established system,
extended tuning optics, good beam diameter
• Coherent Vitesse & Nd:Ylf Turn-key, fixed wavelength
lasers, small footprint
• Coherent Vitesse XT and Spectra physics Mai Tai - small
footprint, limited tuning TiS ( 100 nm range) computer
controlled
General Laser Specifications for
MP Microscopy
• Pulse Width
• Repetition Rate
• Average Power
<250 fsecs
>75 MHz
>250 mW
Comparison of Lasers Available For
Multi-Photon Microscopy
Vitesse
Coherent
Pulse width
<100fsecs
Nd:YLF
Ti Sapphire
Microlase (Coherent) Coherent Verdi/Mira
Spectra-Physics Millennia/Tsunami
120fsecs
<100fsecs
Repetition rate
80MHz
120MHz
82MHz
Wavelength
800nm
1047nm (fixed)
690nm - 1000nm (tunable)
Average output power 200mW
600mW
>250mW
Lifetime
5000hrs
5000hrs
5000hrs
Why Femto-second?
• High output powers needed in deep imaging higher average power generated by pico-second
pulses may generate heating and tweezing effects
• 3P excitation of dyes (DAPI, Indo-1) with pico-second
pulses practically impossible
• Femto-second pulses may cause 3P excitation of
endogenous cellular compounds - however
no evidence that this causes cell toxicity
Relationship between Average Power
and Pulse Width
8
Power Average
7
6
5
4
3
2
1
0
0
1000
2000
3000
4000
Pulse Width (fsec)
5000
3P excitation/2P excitation
Ratio of 3P excitation to 2P excitation as a
Function of Pulse Width
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
1000
2000
3000
Pulse Width (fsec)
4000
5000
What about Fibre-delivery of Pulsed Lasers
• Advantage - alignment and system footprint
• Problem - average power output combined with
short pulses for a tuneable laser suffer considerable
power loss, and realignemnt of laser with each
wavelength change ( repointing)
• problem less with fixed wavelength. ie NdYlf uses p-sec pulses
which are then compressed by fibre
Instrument Design
MP Optics Instrument design
Detector
Detector
Confocal Aperture
Laser
Laser
Objective Lens
Objective Lens
C
C
Emission
Excitation
Choice of Microscope, upright or inverted or both
Fentosecond TiS laser
Beam Control and Monitoring Unit
( Optics Box)
Radiance2000MP
Scan head convertible from upright to inverted ( MP ONLY option also available)
2 or 4 External detector unit
Key specifications
• Adaptable to a wide range of microscopes - Nikon, Olympus and
Zeiss
• Compatible with six femtosecond pulsed lasers
• Beam conditioning units range from basic functionality to flexible
fully featured units
• Beam delivery systems for single ‘scopes and to switch between
‘scopes
• Non-descanned and descanned detector options
• Reduced system footprints
• Multi-Photon ONLY scan head version available
Why all this trouble?
• Conventional confocal has many limitations
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limited depth penetration
short life times for cell observation
problems with light scatter especially in dense cells
limitations with live cell work
Is not UV confocal the solution?
No - it’s the problem for many of these
applications
Why has UV confocal seen such little popularity worldwide
Despite being available for nearly 10 years, only a
small number of systems have been installed
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Chromatic errors
High Toxicity to cells and tissues
Poor penetration
Enhances autofluorescence
Almost unusable in plant sciences
High scattering
User safety
Limited options with lenses
In two years the installed base of MP systems have
doubled over all UV systems world wide.
Strengths of Multi-Photon
Microscopy
• Deeper sectioning - thick, scattering sections
can be imaged to depths not possible in
standard confocal
• Live cell work - ion measurement (i.e. Ca2+),
GFP, developmental biology - reduced toxicity
from reduced full volume bleaching allows
longer observation
• Autofluorescence - NADH, seratonin,
connective tissue, skin and deep UV excitation
Deep Imaging improved by..
Scattered Light Collection
Collected emission
emerges as
parallel rays
Collected emission
no longer parallel
Objective
lens
Isotropic
emission
Non-scattering
sample
Objective
lens
Scattering
sample
Fluorescence Signal (%)
Reduction of Emitted
Fluorescence due to Scattering Events
100
90
80
70
60
50
40
30
20
10
0
0
100
200
300
Depth into Tissue (µm)
400
Relationship between the
Number of Scattering Events and Depth into Aortic
Tissue
Number of Scattering Events
4
350nm
3.5
500nm
3
2.5
2
700nm
1.5
1
0.5
0
0
100
200
300
400
Depth into Tissue (µm)
500
Scatter light detection improved by
External light Detector
From Vickie Centonze Frohlich
IMR, Madison, WI
Reduced Photo bleaching...
MP Fluorochromes and
Applications
Key issues
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Most commonly used probes can be imaged
MP is effectively exciting at UV/blue wavelengths
Excitation spectra are broader than for 1-photon
Emission spectra are the same as in 1-photon excitation
All probes are excited simultaneously at the same wavelength
Probe combinations must be chosen so that they are separated
by emission spectra
• Co-localization is exact even between UV and visible probes
• Can use objective lenses which are not full achromats (e.g. z
focus shift)
Fluorescent Probes for MP Imaging
TiSapphire Laser
Bodipy
Cascade Blue
Calcium Crimson
Calcium Green
Calcium Orange
Coumarin 307
Di-I
Dansyl Hydrazine
DAPI
Fura 2
FITC
Flavins (auto-fluorescence)
Fluo-3
GFP (wild type)
GFP5-65T
Hoechst 33258
Hoechst 33342
Lucifer Yellow
NADH (auto-fluorescence)
Serotonin (auto-fluorescence, 3-photon)
TRITC
Nd:YLF Laser
AMCA
Bodipy
Calcium Crimson
Calcium Green (weak)
Congo Red
DAPI (3-photon)
Di-I
Evans Blue
FITC
FM4-64
GFP (wild type; weak)
GFP5-65T
Hoechst 33258
Hoechst 33342
Mitotracker Rosamine
Nile JC-1
Nile Red
Oregon Green
Propidium Iodide
Safranin
Texas Red
TRITC
Efficient Simultaneous
Detection of Multiple Labels
Following Dynamic Ca2+ Changes using
MP Excitation
Sources
of Tissue
Autofluorescence
Serotonin Distribution in Living Cells
Imaging of Serotonin Containing
Granules Undergoing Secretion
MP Imaging of
Drug Localisation
and Metabolism
Non Imaging Possibilities
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FRAP (Fluorescence recovery after photobleaching)
Photoactivation
Knock out experiments
FCS (Fluorescence correlation spectroscopy)
MP in a “nutshell”
• Multi-Photon microscopy allows optical
section imaging deeper into samples than
other methods, even in the presence of strong
light scattering
• Multi-Photon microscopy allows the study of
live samples for longer periods of time than
other methods, reducing cytotoxic damage