Two-photon patterned photostimulation with low-power, high-efficiency and reliable single-cell optogenetic control

Two-photon optogenetics enables selectively stimulating individual cells for manipulating neuronal ensembles. As the general photostimulation strategy, the patterned two-photon excitation has enabled millisecond-timescale activation for single or multiple neurons, but its activation efficiency is suffered from high laser power due to low beam-modulation efficiency. Here, we develop a high-efficiency beam-shaping method based on the Gerchberg-Saxton (GS) algorithm with spherical-distribution initial phase (GSSIP) to reduce the patterned two-photon excitation speckles and intensity. It can well control the phase of shaped beams to attain speckle-free accurate patterned illumination with an improvement of 44.21% in the modulation efficiency compared with that of the traditional GS algorithm. A combination of temporal focusing and the GSSIP algorithm (TF-GSSIP) achieves patterned focusing through 500-μm-thickness mouse brain slices, which is 2.5 times deeper than the penetration depth of TF-GS with the same signal-to-noise ratio (SNR). With our method, the laser power can be reduced to only 55.56% of that with traditional method (the temporal focusing with GS, TF-GS) to reliably evoke GCaMP6s response in C1V1-expressing cultured neurons with single-cell resolution. Besides, the photostimulation efficiency is remarkably increased by 80.19% at the same excitation density of 0.27 mW/μm2. This two-photon stimulation method with low-power, reliable and patterned illumination may pave the way for analyzing neural circuits and neural coding and decoding mechanism.

Enhancement of image quality in planar Airy light-sheet microscopy via subtraction method

The use of propagation-invariant Airy beams enables a light-sheet microscopy with a large field-of-view. Without relying upon two-photon excitation or deconvolution-based processing to eliminate out-of focus blur caused by the side lobes, here, we present how the subtraction method is applied to enhance the image quality in digital scanned light-sheet microscopy with Airy beam. In the proposed method, planar Airy beam with the symmetric transversal structure is used to excite the sample. A hollow Airy beam with zero intensity at the focal plane is created, which is mainly used to excite the out-of-focus signal. By scanning the sample twice with the normal planar Airy beam and the hollow Airy beam, digital post-processing of the obtained images by subtraction allows for the rejection of out-of-focus blur and improves the optical sectioning, the axial resolution and the intensity distribution uniformity of the light-sheet microscopy.

Efficiency of Plasmon-Induced Dual-Mode Fluorescence Enhancement upon Two-Photon Excitation

Anisotropic noble metal nanoparticles supporting more than one localized surface plasmon resonance can be tailored for efficient dual-mode fluorescence enhancement by ensuring an adequate coupling to both absorption and emission bands of fluorophores. This approach is naturally extended to two-photon excitation fluorescence, where a molecule is excited by simultaneous nonlinear absorption of two photons. However, the relative impact of plasmon coupling to excitation and emission on the overall fluorescence enhancement can be very different in this case. Here, by using the finite-difference time-domain method, we study the two-photon excitation fluorescence of near-infrared fluorescent protein (NirFP) eqFP670, which is the most red-shifted NirFP to date, in proximity to a silver nanobar. By optimizing the length and aspect ratio of the particle, we reach a fluorescence enhancement factor of 103. We show that the single mode coupling regime with highly tuned near-field significantly outperforms the dual-mode coupling enhancement. The plasmon-induced amplification of the fluorophore’s excitation rate becomes of utmost importance due to its quadratic dependence on light intensity, defining the fluorescence enhancement upon two-photon excitation. Our results can be used for the rational design of hybrid nanosystems based on NirFP and plasmonic nanoparticles with greatly improved brightness important for developing whole-body imaging techniques.

Direct Two-Photon Excitation of Isomeric Transition in Thorium-229 Nucleus

A possibility of the two-photon excitation of an isomeric state in a nucleus of thorium-229 has been discussed. The fluorescence intensity of the excitation is demonstrated to be identical for the irradiation of nuclei with either monochromatic light or polychromatic radiation consisting of a sequence of short lightpulses of the same intensity. The two-photon excitation of Th3+ ion in an electromagnetic trap with a focused laser beam with a wavelength of about 320 nm and power of 100 mW can lead to the absorption saturation, at which the fluorescence emission with the frequency of the transition in a nucleus is maximal. In crystals doped with Th4+ to a concentration of about 1018 cm-3 and irradiated with a laser radiation 10 W in power, the emission of several photons persecond with a wavelength of about 160 nm becomes possible.

Multi-Photon Microscopy for the Evaluation of Interstitial Fibrosis in Extended Criteria Donor Kidneys: A Proof-of-Concept Study

Introduction:This study evaluated the initial use of label-free second harmonic generation (SHG) imaging with two-photon excitation (2PE) auto-fluorescence for the quantification of collagen/fibrosis on pre-implantation biopsies of extended criteria donors (ECD).Methods:A total of 20 core ECD kidney transplant biopsy specimen tissues from 7 ECD donors, taken at the time of pre-implantation were retrieved, cut into 5-micron sections, mounted on slides and deparaffinized. The core needle biopsies were imaged with 2X and 20X objective using the commercially available laser-based Genesis® 200 (Histoindex Pte Ltd and Clinnovate Pte Ltd, Singapore). The entire core was selected as the Region of Interest (ROI). Corresponding clinical information from transplant donors were retrieved. Histopathological review was performed, and all biopsies had Interstitial Fibrosis (IF)/Tubular Atrophy (TA) scores > 0. Collagen parameters measured included quantification by the Collagen Area Ratio in Tissue (CART) and qualitative measurements by Collagen Reticulation Index (CRI).Results: 20 explant core biopsies were extracted from 10 donor kidney samples, of which originated from 7 donors. Table 1 depicts the baseline ECD characteristics of the donors. For the kidneys with multiple biopsies done, we obtained an average score of the collagen parameters across the different samples. Biopsies classified with > 85% KDPI score had significantly higher CAR and CART than biopsies with ≤ 85% KDPI score.Conclusion:MPM is an evolving technology that enables the quantification of the amount (CART) and quality (CRI) of collagen deposition in unstained explant biopsies of ECD kidneys. This initial evaluation found significant differences in both parameters between ECD kidneys with more or less than 85% KDPI scores.

Photoisomerization transition state manipulation by entangled two-photon absorption

We demonstrate how two-photon excitation with quantum light can influence elementary photochemical events. The azobenzene trans → cis isomerization following entangled two-photon excitation is simulated using quantum nuclear wave packet dynamics. Photon entanglement modulates the nuclear wave packets by coherently controlling the transition pathways. The photochemical transition state during passage of the reactive conical intersection in azobenzene photoisomerization is strongly affected with a noticeable alteration of the product yield. Quantum entanglement thus provides a novel control knob for photochemical reactions. The distribution of the vibronic coherences during the conical intersection passage strongly depends on the shape of the initial wave packet created upon quantum light excitation. X-ray signals that can experimentally monitor this coherence are simulated.