Adaptive optics light-sheet microscopy based on direct wavefront sensing without any guide star

Abstract

We propose an Adaptive Optics Light-Sheet Fluorescence Microscope (AO-LSFM) for closed-loop aberrations correction at the emission path, providing intrinsic instrumental simplicity and high accuracy when compared to previously reported schemes. The approach is based on direct wavefront sensing i.e. not on time-consuming iterative algorithms, and does not require the use of any guide star thus reducing instrumental complexity and/or sample preparation constraints. The design is based on a modified Shack-Hartmann wavefront sensor providing compatibility with extended sources such as images from optical sectioning microscopes. We report an AO-LSFM setup based on such sensor including characterization of the sensor performance, and demonstrate for the first time significant contrast improvement on neuronal structures of the ex-vivo adult drosophila brain in depth.

Single-shot quantitative aberration and scattering length measurements in mouse brain tissues using an extended-source Shack-Hartmann wavefront sensor

“…The fluorescence emitted was collected through the objective, its back focal plane being conjugated with the ESSH microlens array by a pair of achromatic lenses (focal lengths: 300 mm and 80 mm). The ESSH was similar to the device reported in our previous publication [23], and was composed of an array of 17 x 23 microlenses, with a focal length of 5.1 mm, the CMOS sensor being in the focal plane of the microlenses. A squared field diaphragm was placed in front of the ESSH, leading to a field of view of 120 x 120 µm in the object plane, for each microlens, thus selecting the correlation area and avoiding crosstalk between adjacent thumbnails.…”

“…The present experiment is nevertheless a clear demonstration of the capability of an ESSH-driven AO loop to provide a correction in low SBR situations, whatever the origin of such SBR is, and on real biological objects of interest such as neurons. Also, there was no consideration here of the impact of the isoplanetic patch with the use of SA3, since this topic was already discussed in detail in our previous work [23]. Finally, our ESSH sensor doesn't require descanning of the fluorescence, as in centroid measurement [14], nor requires the use of a specific scanning arrangement and of an ultrafast laser for the wavefront measurement when the technique is applied outside non-linear microscopy [22].…”

“…The approach was successfully used to drive AO in Lattice Light-Sheet [15], as well as in two-photon [14] and in Structured Illumination Microscopy (SIM) [26]. Recently, we demonstrated the use of an Extended-Source SH wavefront sensor (ESSH) to enable AO in light-sheet microscopy [23], by adapting pioneer work from astronomy to the constraints of fluorescence microscopy. The method relies on the cross-correlation of images of an extended source obtained through a microlens array.…”

“…In fluorescence microscopy, when coupled to an optical sectioning method such as two-photon microscopy or light-sheet fluorescence microscopy, the ESSH uses the fluorescence signal as a guide plane, providing lower instrumental complexity and cost than the previous approach based on the scan and descan of a non-linear signal. Its efficiency has been proven when coupled to light-sheet for AO-enhanced neuroimaging in the adult drosophila brain in weekly scattering conditions [23].…”

Smart imaging to empower brain-wide neuroscience at single-cell levels

“…Aside from these hardware adaptions, AI techniques are vastly adopted to automate and speed up the imaging procedure as well as to improve the imaging quality [ 63 – 68 ]. For instance, to automatically adjust the illumination at real-time and minimize the need of imaging parameters tuning [ 64 ], to correct the aberrations using wave-front sensing method [ 65 ], to tackle the defocusing of the light sheet microscope with adaptive refocusing method [ 68 ]. In addition, content-aware imaging [ 63 , 69 , 70 ] and stitching [ 71 , 72 ] are developed to suppress sample degradation, speed up the imaging, and enlarge the sample coverage.…”