Title: Multi-photon Fluorescence Microscopy
1Multi-photon Fluorescence Microscopy
2Topics
- Basic Principles of multi-photon imaging
- Laser systems
- Multi-photon instrumentation
- Fluorescence probes
- Applications
- Future developments
3Multi-photon ExcitationA 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
4History - Multi-photon
- Originally proposed by Maria Goeppert-Mayer in
1931 - First applications in molecular spectroscopy
(1970s) - 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
5 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)
6Multi-Photon Excitation Physical Principles
7Consequence of multi photon excitation
- 1-Photon 2-Photon
- Excitation occurs everywhere Excitation
localised - that the laser beam interacts
- with samples Excitation efficiency
proportional
the square of laser intensity - Excitation efficiency
- proportional to the intensity
Emission highest in focal region
where intensity is highest -
8Classical and confocal fluorescence
Multi-photon fluorescence
9Key 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
seconds) - Special kind of laser required
-16
10Lasers for MP
- Mode-locked femto-second lasers
11CW and Pulsed Lasers
CW
Pulsed
Short Pulse Advantage Fluorescence
proportional to 1/pulse width x repetition rate
12- 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 NdYlf 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
13General Laser Specifications for MP Microscopy
- Pulse Width lt250 fsecs
- Repetition Rate gt75 MHz
- Average Power gt250 mW
14Comparison of Lasers Available ForMulti-Photon
Microscopy
15Why 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
16Relationship between Average Power and Pulse Width
17Ratio of 3P excitation to 2P excitation as a
Function of Pulse Width
18What 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
19Instrument Design
20MP Optics Instrument design
Detector
Detector
Confocal Aperture
Laser
Laser
Objective Lens
Objective Lens
C
C
C
C
Excitation
Emission
21Choice of Microscope, upright or inverted or both
Fentosecond TiS laser
Beam Control and Monitoring Unit ( Optics Box)
Radiance2000MP
2 or 4 External detector unit
Scan head convertible from upright to inverted (
MP ONLY option also available)
22Key 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
23Why all this trouble?
- Conventional confocal has many limitations
- limited depth penetration
- short life times for cell observation
- problems with light scatter especially in dense
cells - limitations with live cell work
24Is not UV confocal the solution?
- No - its the problem for many of these
applications
25Why has UV confocal seen such little popularity
worldwide
- Despite being available for nearly 10 years, only
a small number of systems have been installed - 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.
26Strengths of Multi-PhotonMicroscopy
- 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
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28Deep Imaging improved by..
29Scattered Light Collection
30Reduction of EmittedFluorescence due to
Scattering Events
31Relationship between theNumber of Scattering
Events and Depth into Aortic Tissue
350nm
500nm
700nm
32Scatter light detection improved by External
light Detector
From Vickie Centonze Frohlich IMR, Madison, WI
33Reduced Photo bleaching...
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35MP Fluorochromes and Applications
36Key issues
- 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)
37Fluorescent Probes for MP Imaging
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39Efficient SimultaneousDetection of Multiple
Labels
40Following Dynamic Ca2 Changes using MP Excitation
41Sources of Tissue Autofluorescence
42Serotonin Distribution in Living Cells
43Imaging of Serotonin Containing Granules
Undergoing Secretion
44MP Imaging ofDrug Localisationand Metabolism
45Non Imaging Possibilities
- FRAP (Fluorescence recovery after photobleaching)
- Photoactivation
- Knock out experiments
- FCS (Fluorescence correlation spectroscopy)
46MP 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