Multi-photon Fluorescence Microscopy

Topics• Basic Principles of multi-photon imaging

• Laser systems

• Multi-photon instrumentation

• Fluorescence probes

• Applications

• Future developments

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

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 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

Classical and confocalfluorescence

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 seconds)

• Special kind of laser required

-16

Lasers for MP

Mode-locked femto-second lasers

CW and Pulsed Lasers

CW

Pulsed

Short Pulse Advantage

Fluorescence proportionalto 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 <250 fsecs• Repetition Rate >75 MHz• Average Power >250 mW

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

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

0

1

2

3

4

5

6

7

8

0 1000 2000 3000 4000 5000Pulse Width (fsec)

Pow

er A

vera

ge

Ratio of 3P excitation to 2P excitation as a Function of Pulse Width

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 1000 2000 3000 4000 5000

Pulse Width (fsec)

3P e

xcit

atio

n/2P

exc

itat

ion

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

C C C C

Objective Lens Objective Lens

Laser

Confocal Aperture

Detector Detector

Laser

Emission Excitation

MP Optics Instrument design

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

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– 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

• 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-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

Deep Imaging improved by..

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

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 (%

)

Relationship between theNumber of Scattering Events and Depth into Aortic

Tissue

0

0.5

1

1.5

2

2.5

3

3.5

4

0 100 200 300 400 500

Depth into Tissue (µm)

Num

ber

of S

catt

erin

g E

ven

ts

350nm

500nm

700nm

Scatter light detection improved by External light Detector

From Vickie Centonze FrohlichIMR, Madison, WI

Reduced Photo bleaching...

MP Fluorochromes and Applications

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)

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

Efficient SimultaneousDetection 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 ofDrug Localisationand Metabolism

Non Imaging Possibilities

• 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