Extending the Flexibility of Multiphoton Microscopy for Multidisciplinary Research: Optimization of Femtosecond Pulse Duration and New Approaches to Precision Beam Control

Karl Garsha

Specialist in Light Microscopy, Imaging Technology Group,  Beckman Institute, UIUC

10/15/2002

3rd Floor Tower, 3269 Beckman

In multiphoton microscopy, the excitation rate depends approximately inversely on the input pulse duration, and lasers for multiphoton work typically operate with pulses in the 60 to 200 femtosecond range. Ultra-short optical pulses have an inherently large frequency bandwidth, and so tend to broaden substantially because of dispersion from propagation through the dispersive elements in a microscope.  The reduction of 2-photon fluorescence emission as a result of pulse width broadening can be as high as 80% and the effects on higher order non-linear events, such as photo-ablation, are even more apparent.  Increasing the average laser power to compensate for pulse lengthening is undesirable in many instances.  Specimen heating due to linear absorption, complications arising from optical trapping of cells or organelles, and the use of average powers which approach the ablation threshold are some of the problems associated with increasing average power to recoup fluorescence intensity.

Ongoing ITG research explores the utility of pulse-dispersion recommendation with regard to diverse imaging situations.  We use a simple setup consisting of a high index prism sequence and a retro reflector to yield sub-100 femtosecond pulse widths at the microscope objective. Pulse-dispersion compensation increases the flexibility of a multiphoton system for use in microfabrication studies, including applications which make use of photo-polymerization. A major potential application of photopolymerization lies in forming three-dimensional optically active structures at the micro-scale. In these applications, it is important to demonstrate high precision beam control in three dimensions as well as to deliver a high peak power in the context of a low average power.  This presentation also introduces recent developments in our efforts to better enable our laser scanning microscope for microfabrication and microsurgery using multiphoton absorption.  Taken together, pulse-dispersion precompensation and beam steering strategies greatly extend the feasibility of developing new and novel applications for multiphoton excitation technology.

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