Neu259 2006 2 photon

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Video Rate 2-Photon Microscopy James Bouwer UCSD

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Fluorescence Basics

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Definition of Intensity As power increases... intensity increases As the area decreases… intensity increases Watt = energy/time [J/sec] or [photons/sec]

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A Description of 2-Photon Excitation Microscopy The same single photon fluorescent transitions can be excited at twice the wavelength. E2 E1 Single Photon Excitation Two Photon Excitation E1 Linear relationship between excitation and emission intensities Non-linear relationship between excitation and emission intensities Heisenberg

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The Fluorescence Scattering Cross-section Single Photon Excitation: Two Photon Excitation: (sigma) = fluorescence scattering cross-section [cm2] and [cm4 sec/photon] N = number of scattered photons

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Typical Values for Cross-sections Single Photon Excitation: Two Photon Excitation: 2-photon ~ 10-50 cm4 sec/photon single-photon ~ 10-16 cm2

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Reminder of the Principle of Confocal Apertures Single Photon Excitation

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Intensity Profile Near the Focal Plane For a laser focused by a high NA objective only a small illumination volume results for 2-photon. The need for confocal apertures is therefore eliminated!

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Comparison between Continuous Wave and Pulsed IR Laser Lines

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2-photon Lasers Ti - Saphire: 80 MHz pulse rate 100fsec pulse width (10-15 sec) 1 Watt total power output

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Origins of Frequency Broadening in Mode Locked Pulsed Lasers Heisenberg Uncertainty Principle (Quantum Mechanics): h = 6.62 x 10-34 Joule*sec p = momentum (mass*velocity) x = position E = Energy t = time Squeezing pulses in time spreads the pulse energy (ie: spreads the wavelength) Squeezing pulses in time increases instantaneous intensity while keeping average power low

A Small Two-photon Excitation Volume: 

Two-photon microscopy is inherently less damaging to living cells than single photon fluorescence methods Sample A Small Two-photon Excitation Volume Efficiently excite multiple fluorophores with only one laser for cross- correlation applications

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Pulsed IR lasers: provide short pulses of high intensity light ( ≈100fsec at 80MHz) ~ 8 pulses/pixel provide lower average power and high instantaneous intensity broadband excitation (fwhm ≈ 20nm) allows for the simultaneous excitation of multiple fluorophores IR excitation wavelengths: removal of IR excitation from signal is trivial lower energy photons produce less out of plane damage deeper penetration into biological samples I 2 dependence of fluorescence signal and the IR excitation provide some unique opportunities for imaging.

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Original Design of Video Rate 2-Photon Microscope Prototyped at NCMIR

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System Characteristics Image Size 512 x 480 pixels at 8 bits/pixel Frame Rates: 32 frames/sec at full resolution (31msec/frame) for 3 channels 256 frames/sec in 1/8 band scan mode for 3 channels (3ms/frame) Image Processing and Data Storage: high speed image processing boards perform real time ratioing, background subtraction, and channel merging (Gary Fan et al. Biophys. J. 1999) real time hard drive provides 5Gbyte storage in real time

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Bio-Rad’s Improved Design for a Video Rate 2-Photon Microscope RTS2000 Laser in

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NCMIR’s 2-Photon Facility Bio-Rad RTS2000 Prototype 2-Photon

2-Photon Image Collection: 

2-Photon Image Collection Obj. Lens Dichroic Scan Mirrors PMT Millennium Pumped Ti:Sapphire Laser Mai-Tai * 100 fs pulses at 82 MHz 690-1050 nm 0.7-0.9 W Max. frame rate 30 frames/s (480x512 pixels) Sample

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Ratio Imaging of Calcium Wave (Red) in HeLa cells Expressing Yellow-Cameleon exposed to 0.1mM Histamine which triggers Ca2+

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3-D Montage of Filled Astrocyte 4 x 5 (2000 x 2500 x 300 = 1.5x109 pixels)

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FlAsH Microtubule time-lapse (24hrs)

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Triple Labeled Montages using 2-Photon Excitation

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Summary 2-Photon IR excitation is intrinsically less damaging to samples Less out of plane photo-bleaching Best suited for live cell imaging Ability to probe very fast millisecond dynamic time scales Well suited for fluorescence lifetime imaging studies Better light penetration of tissues Lower resolution images

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References: Fan G. Y., Fujisaki H., Miyawaki A., Tsay R. K., Tsien R. Y. and Ellisman M. H. (1999) Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys. J. (USA) 76, 2412-2420. Wiseman P. W., Squier J. A., Ellisman M. H. and Wilson K. R. (2000) Two-photon image correlation spectroscopy and image cross-correlation spectroscopy. J. Microsc. (UK) 200 pt.1, 14-25.