A stage-scanning two-photon microscope equipped with a temporal and a spatial pulse shaper: Enhance fluorescence signal by phase shaping.

Frederik Büchau,Yang Yang,Alexander Patas,Karsten Heyne,Albrecht Lindinger

doi:10.1063/1.5025792

Abstract

Here, we present a stage-scanning two-photon microscope (2PM) equipped with a temporal pulse shaper and a spatial light modulator enabling full control over spectral and spatial phases of the exciting laser pulse. We demonstrate the capability of correcting wavefronts and temporal pulse distortions without cross-dependencies induced by optical elements at the same time enhancing the fluorescence signal. We implemented phase resolved interferometric spectral modulation for temporal pulse shaping and the iterative feedback adaptive compensation technique for spatial pulse modulation as iterative techniques. Sample distortions were simulated by cover glass plates in the optical path and by chirping the exciting laser pulses. Optimization of the spectral and spatial phases results in a signal increase of 30% and nearly complete recovery of the losses. Applying a measured spatial compensation phase within a real leaf sample shows the enhancement in contrast due to wavefront shaping with local fluorescence increase up to 75%. The setup allows full independent control over spatial and spectral phases keeping or improving the spatial resolution of our microscope and provides the optimal tool for sensitive non-linear and coherent control microscopy.

Highlights

Since the presentation of two-photon microscopy by Denk et al.,1 many approaches to increase the imaging quality and signal strength from within samples have been pursued.2–5 Basically there are two possibilities, which are not mutually exclusive: increase the amount of detected photons and/or increase the amount of two-photon excited fluorescence (2PEF) photons
Afterwards we investigated the effect of the spatial light modulator (SLM) and the temporal pulse shaper (TPS): we distorted the wavefront of the incoming light by inserting two 170 μm cover glasses
The presented two-photon microscope with temporal and spatial pulse control starts off with the table-top laser system equipped with a pulse stretcher/compressor module, being capable of providing transform-limited or temporally chirped femtosecond laser pulses

Summary

Introduction

Since the presentation of two-photon microscopy by Denk et al., many approaches to increase the imaging quality and signal strength from within samples have been pursued. Basically there are two possibilities, which are not mutually exclusive: increase the amount of detected photons and/or increase the amount of two-photon excited fluorescence (2PEF) photons. The sample is exposed to additional photons, which might result in out-of-focus 2PEF, spreading the focal volume, or—if they do not take part in the two-photon absorption process—in an increase in photodamage.. The aim is to increase the photon flux through the focal spot and increase the amount of 2PEF at the focus, keeping the total amount of incident photons lower than in the ballistic compensation. One has to compensate for the distortions of the wavefront and of the temporal pulse shape induced by the microscope and the sample. The compensation of the setup-induced distortions can be greatly done by the manufacturer, e.g., by correction collars for wavefront and pulse compressors for temporal distortions. The sample-induced distortions are manifold and might vary strongly with the sample, as well as with the depth within the sample.

Methods

Results

Conclusion