multi-photon fluorescence microscopy. topics basic principles of multi-photon imaging laser systems...
TRANSCRIPT
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Multi-photon Fluorescence Microscopy
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Topics• Basic Principles of multi-photon imaging
• Laser systems
• Multi-photon instrumentation
• Fluorescence probes
• Applications
• Future developments
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Multi-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
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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
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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)
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Multi-Photon Excitation Physical Principles
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Consequence 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
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Classical and confocalfluorescence
Multi-photon fluorescence
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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 seconds)
• Special kind of laser required
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Lasers for MP
Mode-locked femto-second lasers
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CW and Pulsed Lasers
CW
Pulsed
Short Pulse Advantage
Fluorescence proportionalto 1/pulse width x repetition rate
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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
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General Laser Specifications for MP Microscopy
• Pulse Width <250 fsecs• Repetition Rate >75 MHz• Average Power >250 mW
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Comparison of Lasers Available ForMulti-Photon Microscopy
VitesseCoherent
Nd:YLFMicrolase (Coherent)
Ti SapphireCoherent Verdi/MiraSpectra-Physics Millennia/Tsunami
Pulse width <100fsecs 120fsecs <100fsecs
Repetition rate 80MHz 120MHz 82MHz
Wavelength 800nm 1047nm (fixed) 690nm - 1000nm (tunable)
Average output power 200mW 600mW >250mW
Lifetime 5000hrs 5000hrs 5000hrs
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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
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Relationship between Average Power and Pulse Width
0
1
2
3
4
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8
0 1000 2000 3000 4000 5000Pulse Width (fsec)
Pow
er A
vera
ge
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Ratio of 3P excitation to 2P excitation as a Function of Pulse Width
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0 1000 2000 3000 4000 5000
Pulse Width (fsec)
3P e
xcit
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exc
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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
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Instrument Design
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C C C C
Objective Lens Objective Lens
Laser
Confocal Aperture
Detector Detector
Laser
Emission Excitation
MP Optics Instrument design
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Scan head convertible from upright to inverted ( MP ONLY option also available)
Beam Control and Monitoring Unit( Optics Box)
2 or 4 External detector unit
Fentosecond TiS laserChoice of Microscope, upright or inverted or both
Radiance2000MP
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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
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Why 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
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Is not UV confocal the solution?
No - it’s the problem for many of these applications
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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
• 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.
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Strengths 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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Deep Imaging improved by..
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Scattered Light Collection
Iso trop ic em iss io n N o n -sca tterin g
sa m p leS ca tter in gsa m p le
C o llec ted em iss io nem erg es a s p a ra lle l ra y s
C o llec ted em iss io n n o lo n g er p a ra lle l
O b jec tiv ele n s
O b jec tiv ele n s
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Reduction of EmittedFluorescence due to Scattering Events
0102030405060708090
100
0 100 200 300 400
Depth into Tissue (µm)
Flu
ores
cenc
e S
igna
l (%
)
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Relationship between theNumber of Scattering Events and Depth into Aortic
Tissue
0
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2.5
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0 100 200 300 400 500
Depth into Tissue (µm)
Num
ber
of S
catt
erin
g E
ven
ts
350nm
500nm
700nm
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Scatter light detection improved by External light Detector
From Vickie Centonze FrohlichIMR, Madison, WI
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Reduced Photo bleaching...
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MP Fluorochromes and Applications
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Key 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)
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Fluorescent Probes for MP ImagingTiSapphire Laser Nd:YLF Laser
Bodipy AMCACascade Blue BodipyCalcium Crimson Calcium CrimsonCalcium Green Calcium Green (weak)Calcium Orange Congo RedCoumarin 307 DAPI (3-photon)Di-I Di-IDansyl Hydrazine Evans BlueDAPI FITCFura 2 FM4-64FITC GFP (wild type; weak)Flavins (auto-fluorescence) GFP5-65TFluo-3 Hoechst 33258GFP (wild type) Hoechst 33342GFP5-65T Mitotracker RosamineHoechst 33258 Nile JC-1Hoechst 33342 Nile RedLucifer Yellow Oregon GreenNADH (auto-fluorescence) Propidium IodideSerotonin (auto-fluorescence, 3-photon) SafraninTRITC Texas Red
TRITC
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Efficient SimultaneousDetection of Multiple Labels
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Following Dynamic Ca2+ Changes using MP Excitation
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Sources of Tissue Autofluorescence
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Serotonin Distribution in Living Cells
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Imaging of Serotonin Containing Granules Undergoing Secretion
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MP Imaging ofDrug Localisationand Metabolism
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Non Imaging Possibilities
• FRAP (Fluorescence recovery after photobleaching)• Photoactivation • Knock out experiments• FCS (Fluorescence correlation spectroscopy)
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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