Multiphoton Fluorescence Microscopy Topics Basic Principles of multiphoton

Multi-photon Fluorescence Microscopy

Topics • • • 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. . 700 nm 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 (Mira. X-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 Ti. S ( 100 nm range) computer controlled

General Laser Specifications for MP Microscopy • Pulse Width • Repetition Rate • Average Power <250 fsecs >75 MHz >250 m. W

Comparison of Lasers Available For Multi-Photon Microscopy

Why Femto-second? • High output powers needed in deep imaging higher average power generated by pico-second pulses may generate heating and tweezing effects • 3 P excitation of dyes (DAPI, Indo-1) with pico-second pulses practically impossible • Femto-second pulses may cause 3 P excitation of endogenous cellular compounds - however no evidence that this causes cell toxicity

Relationship between Average Power and Pulse Width

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

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 Nd. Ylf uses p-sec pulses which are then compressed by fibre

Instrument Design

MP Optics Instrument design Detector Confocal Aperture Laser Objective Lens C C Emission Excitation

Choice of Microscope, upright or inverted or both Fentosecond Ti. S laser Beam Control and Monitoring Unit ( Optics Box) Radiance 2000 MP 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 – 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-Photon Microscopy • Deeper sectioning - thick, scattering sections can be imaged to depths not possible in standard confocal • Live cell work - ion measurement (i. e. Ca 2+), 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

Reduction of Emitted Fluorescence due to Scattering Events

Relationship between the Number of Scattering Events and Depth into Aortic Tissue 350 nm 500 nm 700 nm

Scatter light detection improved by External light Detector From Vickie Centonze Frohlich IMR, 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 Imaging

Efficient Simultaneous Detection of Multiple Labels

Following Dynamic Ca 2+ 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 • • 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