Deep-computer-generated holography with temporal-focusing and a digital propagation matrix for rapid 3D multiphoton stimulation.

Deep learning-based computer-generated holography (DeepCGH) has the ability to generate three-dimensional multiphoton stimulation nearly 1,000 times faster than conventional CGH approaches such as the Gerchberg-Saxton (GS) iterative algorithm. However, existing DeepCGH methods cannot achieve axial confinement at the several-micron scale. Moreover, they suffer from an extended inference time as the number of stimulation locations at different depths (i.e., the number of input layers in the neural network) increases. Accordingly, this study proposes an unsupervised U-Net DeepCGH model enhanced with temporal focusing (TF), which currently achieves an axial resolution of around 5 µm. The proposed model employs a digital propagation matrix (DPM) in the data preprocessing stage, which enables stimulation at arbitrary depth locations and reduces the computation time by more than 35%. Through physical constraint learning using an improved loss function related to the TF excitation efficiency, the axial resolution and excitation intensity of the proposed TF-DeepCGH with DPM rival that of the optimal GS with TF method but with a greatly increased computational efficiency.

Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.

Three-dimensional patterned graphene oxide-quantum dot microstructures via two-photon crosslinking.

The two-photon crosslinking of graphene oxide-quantum dots (GOQDs) adopts rose Bengal as the photoactivator to induce the GOQD assembly process. Based on the Förster resonance energy transfer mechanism with oxygen as the crosslinking medium, three-dimensional patterned GOQD microstructures with near diffraction-limit spatial resolution have been fabricated and analyzed by a multiphoton excited fabrication instrument/microscope.

Running exercise delays neurodegeneration in amygdala and hippocampus of Alzheimer's disease (APP/PS1) transgenic mice.

Alzheimer's disease (AD) is an age-related neurodegenerative disease. Post-mortem examination and brain imaging studies indicate that neurodegeneration is evident in the hippocampus and amygdala of very early stage AD patients. Exercise training is known to enhance hippocampus- and amygdala-associated neuronal function. Here, we investigated the effects of exercise (running) on the neuronal structure and function of the hippocampus and amygdala in APP/PS1 transgenic (Tg) mice. At 4-months-old, an age before amyloid deposition, the amygdala-associated, but not the hippocampus-associated, long-term memory was impaired in the Tg mice. The dendritic complexities of the amygdalar basolateral neurons, but not those in the hippocampal CA1 and CA3 neurons, were reduced. Furthermore, the levels of BDNF/TrkB signaling molecules (i.e. p-TrkB, p-Akt and p-PKC) were reduced in the amygdala, but not in the hippocampus of the 4-month-old Tg mice. The concentrations of Aß40 and Aß42 in the amygdala were higher than those in the hippocampus. Ten weeks of treadmill training (from 1.5- to 4-month-old) increased the hippocampus-associated memory and dendritic arbor of the CA1 and CA3 neurons, and also restored the amygdala-associated memory and the dendritic arbor of amygdalar basolateral neurons in the Tg mice. Similarly, exercise training also increased the levels of p-TrkB, p-AKT and p-PKC in the hippocampus and amygdala. Furthermore, exercise training reduced the levels of soluble Aß in the amygdala and hippocampus. Exercise training did not change the levels of APP or RAGE, but significantly increased the levels of LRP-1 in both brain regions of the Tg mice. In conclusion, our results suggest that tests of amygdala function should be incorporated into subject selection for early prevention trials. Long-term exercise protects neurons in the amygdala and hippocampus against AD-related degeneration, probably via enhancements of BDNF signaling pathways and Aß clearance. Physical exercise may serve as a means to delay the onset of AD.

Differentiation of Col I and Col III isoforms in stromal models of ovarian cancer by analysis of second harmonic generation polarization and emission directionality.

A profound remodeling of the extracellular matrix occurs in many epithelial cancers. In ovarian cancer, the minor collagen isoform of Col III becomes upregulated in invasive disease. Here we use second harmonic generation (SHG) imaging microscopy to probe structural differences in fibrillar models of the ovarian stroma comprised of mixtures of Col I and III. The SHG intensity and forward-backward ratios decrease with increasing Col III content, consistent with decreased phasematching due to more randomized structures. We further probe the net collagen α-helix pitch angle within the gel mixtures using what is believed to be a new pixel-based polarization-resolved approach that combines and extends previous analyses. The extracted pitch angles are consistent with those of peptide models and the method has sufficient sensitivity to differentiate Col I from the Col I/Col III mixtures. We further developed the pixel-based approach to extract the SHG signal polarization anisotropy from the same polarization-resolved image matrix. Using this approach, we found that increased Col III results in decreased alignment of the dipole moments within the focal volume. Collectively, the SHG measurements and analysis all indicate that incorporation of Col III results in decreased organization across several levels of collagen organization. Furthermore, the findings suggest that the collagen isoforms comingle within the same fibrils, in good agreement with ultrastructural data. The pixel-based polarization analyses (both excitation and emission) afford determination of structural properties without the previous requirement of having well-aligned fibers, and the approaches should be generally applicable in tissue.

Dynamic particle tracking via temporal focusing multiphoton microscopy with astigmatism imaging.

A three-dimensional (3D) single fluorescent particle tracking strategy based on temporal focusing multiphoton excitation microscopy (TFMPEM) combined with astigmatism imaging is proposed for delivering nanoscale-level axial information that reveals 3D trajectories of single fluorospheres in the axially-resolved multiphoton excitation volume without z-axis scanning. Whereas other scanning spatial focusing multiphoton excitation schemes induce optical trapping interference, temporal focusing multiphoton excitation produces widefield illumination with minimum optical trapping force on the fluorospheres. Currently, the lateral and axial positioning resolutions of the dynamic particle tracking approach are about 14 nm and 21 nm in standard deviation, respectively. Furthermore, the motion behavior and diffusion coefficients of fluorospheres in glycerol solutions with different concentrations are dynamically measured at a frame rate up to 100 Hz. This TFMPEM with astigmatism imaging holds great promise for exploring dynamic molecular behavior deep inside biotissues via its superior penetration, reduced trapping effect, fast frame rate, and nanoscale-level positioning.

Graphene oxide-based micropatterns via high-throughput multiphoton-induced reduction and ablation.

In this study, a developed temporal focusing-based femtosecond laser system provides high-throughput multiphoton-induced reduction and ablation of graphene oxide (GO) films. Integrated with a digital micromirror device to locally control the laser pulse numbers, GO-based micropatterns can be quickly achieved instantly. Furthermore, the degree of reduction and ablation can be precisely adjusted via controlling the laser wavelength, power, and pulse number. Compared to point-by-point scanning laser direct writing, this approach offers a high-throughput and multiple-function approach to accomplish a large area of micro-scale patterns on GO films. The high-throughput micropatterning of GO via the temporal focusing-based femtosecond laser system fulfills the requirement of mass production for GO-based applications in microelectronic devices.

Temporal focusing-based multiphoton excitation microscopy via digital micromirror device.

This Letter presents an enhanced temporal focusing-based multiphoton excitation (MPE) microscope in which the conventional diffraction grating is replaced by a digital micromirror device (DMD). Experimental results from imaging a thin fluorescence film show that the 4.0 µm axial resolution of the microscope is comparable with that of a setup incorporating a 600 lines/mm grating; hence, the optical sectioning ability of the proposed setup is demonstrated. Similar to a grating, the DMD diffracts illuminating light frequencies for temporal focusing; additionally, it generates arbitrary patterns. Since the DMD is placed on the image-conjugate plane of the objective lens' focal plane, the MPE pattern can be projected on the focal plane precisely.

Image-inspired 3D multiphoton excited fabrication of extracellular matrix structures by modulated raster scanning.

Multiphoton excited photochemistry is a powerful 3D fabrication tool that produces sub-micron feature sizes. Here we exploit the freeform nature of the process to create models of the extracellular matrix (ECM) of several tissues, where the design blueprint is derived directly from high resolution optical microscopy images (e.g. fluorescence and Second Harmonic Generation). To achieve this goal, we implemented a new form of instrument control, termed modulated raster scanning, where rapid laser shuttering (10 MHz) is used to directly map the greyscale image data to the resulting protein concentration in the fabricated scaffold. Fidelity in terms of area coverage and relative concentration relative to the image data is ~95%. We compare the results to an STL approach, and find the new scheme provides significantly improved performance. We suggest the method will enable a variety of cell-matrix studies in cancer biology and also provide insight into generating scaffolds for tissue engineering.

Dual-resonant scanning multiphoton microscope with ultrasound lens and resonant mirror for rapid volumetric imaging.

A dual-resonant scanning multiphoton (DRSM) microscope incorporating a tunable acoustic gradient index of refraction lens with a resonant mirror is developed for high-speed volumetric imaging. In the proposed microscope, the pulse train signal of a femtosecond laser is used to trigger an embedded field programmable gate array to sample the multiphoton excited fluorescence signal at the rate of one pixel per laser pulse. It is shown that a frame rate of around 8000 Hz can be obtained in the x-z plane for an image region with a size of 256 × 80 pixels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 343 × 343 × 120 µm3 with an image size of 256 × 256 × 80 voxels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 256 × 256 × 80 voxels, which represents 343 × 343 × 120 µm3 in field-of-view. The rapid volumetric imaging rate eliminates the aliasing effect for observed temporal frequencies lower than 15 Hz. The practical feasibility of the DRSM microscope is demonstrated by observing the mushroom bodies of a drosophila brain and performing 3D dynamic observations of moving 10-µm fluorescent beads.

Biocompatible zeolite-dye composites with anti-amyloidogenic properties.

One of the most attractive approaches in biomedicine and pharmacy is the application of multifunctional materials. The mesoporous structure of clinoptilolite (CZ) absorbs various types of substances and can be used as a model for studying the carriers for targeted drug delivery with controlled release. CZ-dye composites are fabricated by incorporation into clinoptilolite pores commonly used dyes, aluminum phthalocyanine, zinc porphine, and hypericin. We examined and compared the effect of pure dyes and CZ-dye composites on insulin amyloidogenesis. The formation of insulin amyloid fibrils and the disassembly of preformed fibrils is significantly affected by any of the three compounds, however, the strongest effect is observed for aluminum phthalocyanine indicating a structurally-dependent anti-amyloidogenic activity of the dyes. The incorporation of dyes into CZ particles resulted in enhanced anti-amyloidogenic activity in comparison to pure CZ particles. The cell metabolic activity, biocompatibility and fluorescence biodistribution of the dyes entrapped in the composites were tested in vitro (U87 MG cells) and in vivo in the quail chorioallantoic membrane model. Considering the photoactive properties of the dyes used, we assume their applicability in photodiagnostics and photodynamic therapy. It can also be expected that their anti-amyloidogenic potential can be enhanced by photodynamic effect.

Temporal focusing multiphoton microscopy with cross-modality multi-stage 3D U-Net for fast and clear bioimaging.

Temporal focusing multiphoton excitation microscopy (TFMPEM) enables fast widefield biotissue imaging with optical sectioning. However, under widefield illumination, the imaging performance is severely degraded by scattering effects, which induce signal crosstalk and a low signal-to-noise ratio in the detection process, particularly when imaging deep layers. Accordingly, the present study proposes a cross-modality learning-based neural network method for performing image registration and restoration. In the proposed method, the point-scanning multiphoton excitation microscopy images are registered to the TFMPEM images by an unsupervised U-Net model based on a global linear affine transformation process and local VoxelMorph registration network. A multi-stage 3D U-Net model with a cross-stage feature fusion mechanism and self-supervised attention module is then used to infer in-vitro fixed TFMPEM volumetric images. The experimental results obtained for in-vitro drosophila mushroom body (MB) images show that the proposed method improves the structure similarity index measures (SSIMs) of the TFMPEM images acquired with a 10-ms exposure time from 0.38 to 0.93 and 0.80 for shallow- and deep-layer images, respectively. A 3D U-Net model, pretrained on in-vitro images, is further trained using a small in-vivo MB image dataset. The transfer learning network improves the SSIMs of in-vivo drosophila MB images captured with a 1-ms exposure time to 0.97 and 0.94 for shallow and deep layers, respectively.

Intravascular laser irradiation of blood as novel migraine treatment: an observational study.

BACKGROUND: Migraine is one of four major chronic diseases that cause disability. Decreases in regional cerebral blood flow (rCBF) occur during migraine attacks. Laser therapy is extensively employed in treating other vascular diseases; nevertheless, its effectiveness in migraine management remains largely unknown. Therefore, we evaluated the effect of low-level intravascular laser irradiation of blood (ILIB) therapy in patients with migraine. METHODS: We performed an observational case-control study in 24 patients suffering from migraine. Patients were divided into an ILIB treatment group and a traditional rehabilitation group. This study performed clinical assessments and single-photon emission computed tomography (SPECT) prior to and after the treatment and 1 month later. Changes in rCBF-SPECT between groups and between timepoints were compared to clinical outcomes. RESULTS: Nine patients undergoing rehabilitation and fifteen patients undergoing ILIB were studied from baseline to 1 month follow-up. The ILIB group, visual analog scale for pain (P = 0.001), Montreal Cognitive Assessment (P = 0.003), and Athens Insomnia Scale (P < 0.001) symptom scores significantly improved after treatment. SPECT imaging showed a 1.27 ± 0.27 fold increase in rCBF after ILIB treatment, and no significant differences in the rehabilitation group. CONCLUSIONS: Low-level ILIB therapy is associated with better clinical and vascular outcomes, and may be a feasible treatment option for migraine. Although our sample size was small, our data provide a starting point for migraine laser therapy research.

Categorization of collagen type I and II blend hydrogel using multipolarization SHG imaging with ResNet regression.

Previously, the discrimination of collagen types I and II was successfully achieved using peptide pitch angle and anisotropic parameter methods. However, these methods require fitting polarization second harmonic generation (SHG) pixel-wise information into generic mathematical models, revealing inconsistencies in categorizing collagen type I and II blend hydrogels. In this study, a ResNet approach based on multipolarization SHG imaging is proposed for the categorization and regression of collagen type I and II blend hydrogels at 0%, 25%, 50%, 75%, and 100% type II, without the need for prior time-consuming model fitting. A ResNet model, pretrained on 18 progressive polarization SHG images at 10° intervals for each percentage, categorizes the five blended collagen hydrogels with a mean absolute error (MAE) of 0.021, while the model pretrained on nonpolarization images exhibited 0.083 MAE. Moreover, the pretrained models can also generally regress the blend hydrogels at 20%, 40%, 60%, and 80% type II. In conclusion, the multipolarization SHG image-based ResNet analysis demonstrates the potential for an automated approach using deep learning to extract valuable information from the collagen matrix.

Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned excitation.

One of the limits of conventional scanning multiphoton microfabrication is its low throughput due to point-by-point processing. In order to surpass this limit, a multiphoton microfabrication system based on spatiotemporal focusing and patterned excitation has been developed to quickly provide three-dimensional (3D) freeform polymer microstructures. 3D freeform polymer microstructures using Rose Bengal as the photoinitiator are created by sequentially stacking two-dimensional fabricating patterns. The size of each fabrication area can be larger than 300 × 170 µm2 (full width at half maximum). Compared to conventional scanning multiphoton excitation and fixed mask pattern generation, this approach offers freeform microstructures and a greater than three-order increase in fabrication speed. Furthermore, the system is capable of optically sectioning the fabricated microstructures for providing 3D inspection.

Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning.

In this study, a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. Key features of this microscope are the integrations of a 10 kHz repetition rate ultrafast amplifier featuring high instantaneous peak power (maximum 400 µJ/pulse at a 90 fs pulse width) and a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled camera into a spatiotemporal focusing microscope. This configuration can produce multiphoton images with an excitation area larger than 200 × 100 µm² at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 µm were observed in real-time with a lateral spatial resolution of less than 0.5 µm and an axial resolution of approximately 3.5 µm. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.

Investigation of two-photon excited fluorescence increment via crosslinked bovine serum albumin.

The two-photon excited fluorescence (TPEF) increments of two dyes via bovine serum albumin (BSA) microstructures fabricated by the two-photon crosslinking technique were investigated. One is Rose Bengal (RB) with a high non-radiative decay rate, while the other is Eosin Y with a low non-radiative decay rate. Experimental results demonstrate that the quantum yield and lifetime of RB are both augmented via crosslinked BSA microstructures. Compared with theoretical analysis, this result indicates that the non-radiative decay rate of RB is decreased; hence, the quenched effect induced by BSA solution is suppressed. However, the fluorescence lifetime of Eosin Y is acutely abated despite the augmented quantum yield for the two-photon crosslinking processing from BSA solution. This result deduces that the radiative decay rate increased. Furthermore, the increased TPEF intensity and lifetime of RB correlated with the concentration of fabricated crosslinked BSA microstructures through pulse selection of the employed femtosecond laser is demonstrated and capable of developing a zone-plate-like BSA microstructure.

Different types of exercise induce differential effects on neuronal adaptations and memory performance.

Different exercise paradigms show differential effects on various forms of memory. We hypothesize that the differential effects of exercises on memory performance are caused by different neuroplasticity changes in relevant brain regions in response to different exercise trainings. We examined the effects of treadmill running (TR) and wheel running (WR) on the Pavlovian fear conditioning task that assesses learning and memory performance associated with the amygdala (cued conditioning) and both the amygdala and hippocampus (contextual conditioning). The skeletal muscle citrate synthase activity, an indicator of aerobic capacity, was elevated in rats received 4 w of TR, but not WR. While both TR and WR elevated the contextual conditional response, only TR facilitated the cued conditional response. Using a single-neuron labeling technique, we found that while both TR and MR enlarged the dendritic field and increased the spine density in hippocampal CA3 neurons, only TR showed these effects in basolateral amygdalar neurons. Moreover, both types of exercise upregulated synaptic proteins (i.e., TrkB and SNAP-25) in the hippocampus; however only TR showed similar effects in the amygdala. Injection of K252a, a TrkB kinase inhibitor, in the dorsal hippocampus or basolateral amygdala abolished the exercise-facilitated contextual or cued fear learning and memory performance, respectively, regardless of the types of exercise. In summary, our results supported that different types of exercise affect the performance of learning and memory via BDNF-TrkB signaling and neuroplasticity in specific brain regions. The brain region-specific neuronal adaptations are possibly induced by various levels of intensity/stress elicited by different types of exercise.

Image improvement of temporal focusing multiphoton microscopy via superior spatial modulation excitation and Hilbert-Huang transform decomposition.

Temporal focusing-based multiphoton excitation microscopy (TFMPEM) just provides the advantage of widefield optical sectioning ability with axial resolution of several micrometers. However, under the plane excitation, the photons emitted from the molecules in turbid tissues undergo scattering, resulting in complicated background noise and an impaired widefield image quality. Accordingly, this study constructs a general and comprehensive numerical model of TFMPEM utilizing Fourier optics and performs simulations to determine the superior spatial frequency and orientation of the structured pattern which maximize the axial excitation confinement. It is shown experimentally that the optimized pattern minimizes the intensity of the out-of-focus signal, and hence improves the quality of the image reconstructed using the Hilbert transform (HT). However, the square-like reflection components on digital micromirror device leads to pattern residuals in the demodulated image when applying high spatial frequency of structured pattern. Accordingly, the HT is replaced with Hilbert-Huang transform (HHT) in order to sift out the low-frequency background noise and pattern residuals in the demodulation process. The experimental results obtained using a kidney tissue sample show that the HHT yields a significant improvement in the TFMPEM image quality.

Using deep learning to identify maturity and 3D distance in pineapple fields.

Pineapples are an important agricultural economic crop in Taiwan. Considerable human resources are required to protect pineapples from excessive solar radiation, which could otherwise lead to overheating and subsequent deterioration. Note that simple covering all of the fruit with a paper bag is not a viable solution, due to the fact that it makes it impossible to determine whether the fruit is ripe. This paper proposes a system by which to automate the detection of ripe pineapples. The proposed deep learning architecture enables detection regardless of lighting conditions, achieving accuracy of more than 99.27% with error of less than 2% at distances of 300 ~ 800 mm. This proposed system using an Nvidia TX2 is capable of 15 frames per second, thereby making it possible to mount the device on machines that move at walking speed.