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.

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.

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.

Depth-targeted intracortical microstroke by two-photon photothrombosis in rodent brain

Significance: Photothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothromboris model, the photosensitizer Rose Bengal is systemically introduced to the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of Rose Bengal is a one-photon phenomenon and the molecules in the illuminated area (light path) are subject to excitation, targeting of thrombosis is unspecific especially in the depth dimension. We have developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation. Aim: We aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of Rose Bengal so that we confine photothrombosis within a vessel of a target depth. Approach: FITC-dextran is injected into the blood stream to visualize the cerebral blood flow in anesthetized adult mice with a cranial window. After a target vessel is chosen by two-photon imaging (950 nm), Rose Bengal is injected into the blood stream. The scanning wavelength is changed to 720 nm and photothrombosis was induced by scanning the target vessel. Results: Two-photon depth-targeted single vessel photothrombosis was achieved with a success rate of 84.9+/-1.7% within 80 s. Attempts without Rose Bengal (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm. Conclusions: We described a protocol that achieves depth-targeted single vessel photothrombosis by two-photon excitation. Simultaneous imaging of blood flow in the targeted vessel using FITC dextran enabled the confirmation of vessel occlusion and prevention of excess irradiation that possibly induces unintended photodamage.