Measurement and correction of in vivo sample aberrations employing a nonlinear guide-star in two-photon excited fluorescence microscopy

Abstract

We demonstrate that sample induced aberrations can be measured in a nonlinear microscope. This uses the fact that two-photon excited fluorescence naturally produces a localized point source inside the sample: the nonlinear guide-star (NL-GS). The wavefront emitted from the NL-GS can then be recorded using a Shack-Hartmann sensor. Compensation of the recorded sample aberrations is performed by the deformable mirror in a single-step. This technique is applied to fixed and in vivo biological samples, showing, in some cases, more than one order of magnitude improvement in the total collected signal intensity.

Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinearin-vivoimaging

“…The level of improvement in signal intensity achieved in this study compares well with previously published data and is generally higher. 13,14,19,22,23,42,43 In this work, using the look-up table approach, the signal intensity was improved, on average up to a depth of 112 μm within organotypic tissue cultures, by a factor of 2.5 AE 0.5 for SHG and 2.8 AE 1.1 for TPEF with peak values of up to 4.1 AE 0.6. The same shapes were used to increase the quality of in vivo images in zebrafish three days after the DMM shapes had been acquired.…”

“…The reference sources employed were either injected fluorescent microspheres or nonlinear guide stars created by the TPEF. [14][15][16] A drawback of those closed loop methods is the loss of power due to the continuous monitoring of the wavefront and/or use of a spatial light modulator as well as the complexity of the setup. Additionally, measuring distorted wavefronts in microscopy is not trivial and generally requires a point source emitter in the sample.…”

Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy

“…Additionally, these measurements are weakly sensitive to odd aberrations [5], due to the double-pass effect [11]. In another solution, instead, the emission from a point source inside the specimen is used to perform ShackHartmann wavefront sensing [12][13][14][15][16]. Here, the difficulty stems from the lack of such reference point sources within the specimen and from the limited number of photons available in the emission signal.…”

Direct wavefront sensing in adaptive optical microscopy using backscattered light

“…Several implementations have used fluorescence emission for aberration measurement. In two-photon excitation fluorescence microscopes, fluorescence is only generated in the focal spot, which can be used as the source for a wavefront sensor [4,5]. Another approach involves the placement of small fluorescent beads in the specimen that can act as point sources for wavefronts that are detected by a Shack-Hartmann wavefront sensor (SHWFS) [6,7].…”