Abstract
Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1μm fluorescent beads sealed in agarose gel at different depths is improved.
Highlights
Multiphoton excited fluorescence microscopy has been widely utilized for biological imaging since 1990 [1]
Regarding the complexity of spatial and temporal distortions in widefield temporal focusing-based multiphoton microscopy, this study focused on the temporal distortion compensation that restores the excitation pulse width at the focal plane to achieve better widefield optical sectioning
The estimated axial resolutions of the three profiles are 3.6 μm, approximately 5.4 μm, and 3.8 μm, respectively, in full width at the half maximum (FWHM); the axial confinement and two-photon excited fluorescence (TPEF) intensity at the temporal focusing plane are recovered by the wavefront sensorless adaptive optics system (AOS)
Summary
Multiphoton excited fluorescence microscopy has been widely utilized for biological imaging since 1990 [1]. Distortions in the temporal focusing-based multiphoton microscopy or in specimens distort temporal profiles of the excitation pulse at the focal plane of the objective lens [22], leading to reductions in axial resolution, image intensity, and contrast quality. The sensorless AOS is capable of computing the feedback control signals to drive the wavefront corrector via a suitable algorithm to maximize the image quality value, which serves as an indication of the aberration compensation level [31,32]. The AOS uses a 32-element DM as the wavefront corrector, and employs a hill-climbing algorithm to compute an appropriate control signal to drive the DM such that the effects of optical aberrations and specimen-induced temporal distortions are reduced. Contrast enhancements of 1μm fluorescent beads fixed in agarose gel at different sectioning depths can clearly be observed
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