Optical dispersion would affect ultrashort laser pulse duration and thus decrease energy concentration. The instantaneous peak power will be reduced and affect the nonlinear optical absorption. To detect and compensate the optical dispersion dynamically, we have integrated the real-time ultrashort pulse measurement based on direct optical-dispersion estimation by spectrogram (DOES), the DOES could be used to find the group delay dispersion (GDD) from single shot spectrogram with fast and accurate computation time. the ultrashort pulse compensation system consist of a blazed grating and deformable mirror (DM). The GDD is used to drive the DM compensation by digital PI controller implemented on field programmable gate array (FPGA). The compression system could be operated at 100 Hz in real-time and is implemented to multiphoton excited fluorescence microscopy (MPEFM) system. The experimental result shows the static and dynamic dispersion could be compensated in 50 ms, and the overall nonlinear excited efficiency could increase to 1.4-fold, which is the theoretical limit based on the current setup.
Neural networks present a new approach for solving nonlinear problems and is widely applied in optical science. In this research, we integrate neural network with Shack-Hartmann wavefront sensor (SHWS), not only reconstruct the wavefront but also the intensity of beam profile. This network has the capability to obtain the beam wavefront information without calculating the slop of wavefront, which cost most of the time in traditional algorithm, and also grab the features of beam intensity distribution simultaneously. We also compare the result of reconstruction using single focal spot. The experimental results show that though SHWS pattern training result has slightly better root mean square (RMS), both the reconstructions have high accuracy in wavefront and beam profile.
Significance: Line scanning-based temporal focusing multiphoton microscopy (TFMPM) has superior axial excitation confinement (AEC) compared to conventional widefield TFMPM, but the frame rate is limited due to the limitation of the single line-to-line scanning mechanism. The development of the multiline scanning-based TFMPM requires only eight multiline patterns for full-field uniform multiphoton excitation and it still maintains superior AEC.
Aim: The optimized parallel multiline scanning TFMPM is developed, and the performance is verified with theoretical simulation. The system provides a sharp AEC equivalent to the line scanning-based TFMPM, but fewer scans are required.
Approach: A digital micromirror device is integrated in the TFMPM system and generates the multiline pattern for excitation. Based on the result of single-line pattern with sharp AEC, we can further model the multiline pattern to find the best structure that has the highest duty cycle together with the best AEC performance.
Results: The AEC is experimentally improved to 1.7 μm from the 3.5 μm of conventional TFMPM. The adopted multiline pattern is akin to a pulse-width-modulation pattern with a spatial period of four times the diffraction-limited line width. In other words, ideally only four π / 2 spatial phase-shift scans are required to form a full two-dimensional image with superior AEC instead of image-size-dependent line-to-line scanning.
Conclusions: We have demonstrated the developed parallel multiline scanning-based TFMPM has the multiline pattern for sharp AEC and the least scans required for full-field uniform excitation. In the experimental results, the temporal focusing-based multiphoton images of disordered biotissue of mouse skin with improved axial resolution due to the near-theoretical limit AEC are shown to clearly reduce background scattering.
In this study, we implement temporal-focusing multiphoton selective excitation (TFMPSE) to light field microscopy (LFM), illuminating only the volume of interest, thus significantly reducing the background noise and providing higher contrast and accuracy for the light field image reconstruction; furthermore, offering higher penetration depth in scattering tissue via multiphoton. 3D human-skin in situ immunofluorescence images are used to demonstrate volumetric bioimaging capability. The volume rate of the TFMPSE-LFM can achieve around 100 volumes per second
Mechanical weeding is a necessary means to produce organic soybean in vast area and is the most effective for seedling of weed. However, the growth of weed is inconsistent; we must rely on the quantity of closely weeding to achieve the expected results. Organic cultivation adopts strip planting to reduce the cost of weeding, and uses the cultivator to remove weeds. This operation is slow and with high cost, and the artificial weeding is necessary in the later stage. In contrast, the field weeding robot can provide mechanical or physical prevention of weeds via monitor and image analysis. Therefore, this study aims to effectively increase production and solve the problems of labor shortage and high cost on labors by combining an unmanned ground vehicle with a developing smart laser weeding system for autonomous precise weeding. An intelligent image recognition is adopted to identify the position of the weed, and then through the laser tissue fibrosis to achieve the goal of precise weeding.
A developed temporal focusing-based multiphoton microscope (TFMPM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back-focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPM can be condensed from 3.0 μm to 1.5 μm for a 50% improvement. Furthermore, a multiline scanning mechanism based on the DMD can push its AEC nearly equivalent to line scanningbased TFMPM to 1.5 μm from optimal 3.0 μm of the conventional TFMPM. By using the TFMPM with structured illumination and multiline scanning, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression.
Temporal focusing multiphoton excitation microscopy (TFMPEM) is insufficient to perform efficient excitation of fluorophores with the different two-photon absorption spectra in the multi fluorophores imaging via an illuminated configuration of fixed wavelength and polarization direction of the pulse laser because of the a diffraction device. In this study, we overcome and causes it is not useful for in the practical application and its development. In order to implement the temporal focusing microscope system could be able to sever for the different excitation wavelength range of 700-1000 nm, we work on the optical design of the temporal focusing an integration stage and scanning mirror with automatic tuning system for any excitation wavelength. The novel TFMPEM has successfully provided dynamic spectral absorption, 3D two-photon or SHG images with the original ability of the high frame rate.
Temporal focusing multiphoton excitation microscopy has wide field-of-view and optical sectioning. By using digital micromirror device, it provides patterned illumination. However, without filling the back aperture of objective lens, the axial confinement is limited to micron-meters, leading the out-of-focus fluorophores excited and image blurred. In this study, Hilbert-Huang transform is proposed to reduce the background noise. The empirical mode decomposition is first applied to disassemble the image into intrinsic mode functions and then reconstruct by Hilbert transform after diminishing background residues. The axial confinement can be enhanced from 2.79 μm to 0.73 μm with structure frequency in 1.06 μm-1.
Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement of a few microns. To improve the axial confinement of TFMPEM, a binary computer-generated Fourier hologram (CGFH) via a digital-micromirror-device (DMD) was implemented to intrinsically improve the axial confinement by filling the back-focal aperture of the objective lens. Experimental results show that the excitation focal volume can be condensed and the axial confinement improved about 24% according to the DMD holography. In addition, pseudouniform MPE can be achieved using two complementary CGFHs with rapid pulse-width modulation switching via the DMD. Furthermore, bioimaging of CV-1 in origin with SV40 genes-7 cells demonstrates that the TFMPEM with binary DMD holography can improve image quality by enhancing axial excitation confinement and rejecting out-of-focus excitation.
In this study, a field programmable gate array (FPGA)-based Shack-Hartmann wavefront sensor (SHWS) programmed on LabVIEW can be highly integrated into customized applications such as adaptive optics system (AOS) for performing real-time wavefront measurement. Further, a Camera Link frame grabber embedded with FPGA is adopted to enhance the sensor speed reacting to variation considering its advantage of the highest data transmission bandwidth. Instead of waiting for a frame image to be captured by the FPGA, the Shack-Hartmann algorithm are implemented in parallel processing blocks design and let the image data transmission synchronize with the wavefront reconstruction. On the other hand, we design a mechanism to control the deformable mirror in the same FPGA and verify the Shack-Hartmann sensor speed by controlling the frequency of the deformable mirror dynamic surface deformation. Currently, this FPGAbead SHWS design can achieve a 266 Hz cyclic speed limited by the camera frame rate as well as leaves 40% logic slices for additionally flexible design.
Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement (AEC) of a few microns. Herein, a developed TFMPEM with a digital micromirror device (DMD), acting as the blazed grating for light spatial dispersion and simultaneous patterned illumination, has been extended to implement spatially modulated illumination at structured frequency and orientation. By implementing the spatially modulated illumination, the beam coverage at the back-focal aperture of the objective lens can be increased. As a result, the AEC can be condensed from 3.0 μm to 1.5 μm in full width at half maximum for a 2-fold enhancement. Furthermore, by using HiLo microscopy with two structured illuminations at the same spatial frequency but different orientation, biotissue images according to the structured illumination with condensed AEC is obviously superior in contrast and scattering suppression.
KEYWORDS: 3D microstructuring, Pulsed laser operation, Digital micromirror devices, Optical amplifiers, Microfabrication, Beam controllers, Control systems, 3D image processing, Ultrafast phenomena, Imaging systems
A developed temporal focusing-based multiphoton excitation system with additional patterned excitation and local laser control can provide high-throughput fabrication of three-dimensional gray-level biomicrostructures via two-photon cross-linking with rose bengal (RB) as the photoactivator. Multiple bovine serum albumin (BSA) structures of different concentrations were simultaneously achieved by selecting different pulse numbers in the designated regions with an appropriate femtosecond laser power within a few seconds. Intensity of the RB two-photon excited fluorescence (TPEF) is correlated to the concentration of the fabricated cross-linked BSA microstructure. Hence, the fabricated BSA microstructure can be monitored online by utilizing the RB TPEF as contrast agent. It suggests that this approach with its unique capability of high-speed, gray-level, and online-inspection fabrication meets the requirements of the biomedical researches involved in extracellular matrix.
One of the limits of a conventional multiphoton microfabrication is its low throughput due to the sequential nature of
scanning process. In this study, a multiphoton microfabrication system based on spatiotemporal focusing and patterned
excitation has been developed to provide freeform polymer microstructures fast. The system integrates a 10 kHz
repetition rate ultrafast amplifier featuring strong instantanrror device generating designed patterns at the focal plane. As the result, three-dimensional freeform polymer microstructures using Rose Bengal as the photoinitiator are created by sequentially stacking up two-dimensional (2D) structures layer-by-layer. The size of each 2D fabrication area can be larger than 100 × 100 μm2. Compared with scanning process or fixed mask pattern generation, this approach provides two- or three-fold fabrication speed and freeform microstructures. Furthermore, the system is capable of optical sectioning the fabricated microstructures for providing 3D inspection.
Unlike conventional multiphoton microscopy according to pixel by pixel point scanning, a widefield multiphoton
microscope based on spatiotemporal focusing has been developed to provide fast optical sectioning images at a frame
rate over 100 Hz. In order to overcome the aberrations of the widefield multiphoton microscope and the wavefront
distortion from turbid biospecimens, an image-based adaptive optics system (AOS) was integrated. The feedback
control signal of the AOS was acquired according to locally maximize image intensity, which were provided by the
widefield multiphoton excited microscope, by using a hill climbing algorithm. Then, the control signal was utilized to
drive a deformable mirror in such a way as to eliminate the aberration and distortion. A R6G-doped PMMA thin film is
also increased by 3.7-fold. Furthermore, the TPEF image quality of 1 μm fluorescent beads sealed in agarose gel at
different depths is improved.
Conventional multiphoton microscopy employs beam scanning; however, in this study a microscope based on
spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. The microscope integrates a 10 kHz repetition rate ultrafast amplifier featuring strong instantaneous peak power (maximum 400 μJ/pulse at 90 fs pulse width) with a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled device camera. This configuration can produce multiphoton excited images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz. Brownian motions of fluorescent microbeads as small as 0.5 μm have been instantaneously observed with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Moreover, we combine the widefield multiphoton microscopy with structure illuminated technique named HiLo to reject the background scattering noise to get better quality for bioimaging.
This study investigated theoretically and experimentally that two-photon excited fluorescence is enhanced and
quenched via surface plasmons (SPs) excited by total internal reflection with a silver film. The fluorescence intensity is
fundamentally affected by the local electromagnetic field enhancement and the quantum yield change according to the
surrounding structure and materials. By utilizing the Fresnel equation and classical dipole radiation modeling, local
electric field enhancement, fluorescence quantum yield, and fluorescence emission coupling yield via SPs were
theoretically analyzed at different dielectric spacer thicknesses between the fluorescence dye and the metal film. The
fluorescence lifetime was also decreased substantially via the quenching effect. A two-photon excited total internal
reflection fluorescence (TIRF) microscopy with a time-correlated single photon counting device has been developed to
measure the fluorescence lifetimes, photostabilities, and enhancements. The experimental results demonstrate that the
fluorescence lifetimes and the trend of the enhancements are consistent with the theoretical analysis. The maximum
fluorescence enhancement factor in the surface plasmon-total internal reflection fluorescence (SP-TIRF) configuration
can be increased up to 30 fold with a suitable thickness SiO2 spacer. Also, to compromise for the fluorescence
enhancement and the fluorophore photostability, we find that the SP-TIRF configuration with a 10 nm SiO2 spacer can
provide an enhanced and less photobleached fluorescent signal via the assistance of enhanced local electromagnetic
field and quenched fluorescence lifetime, respectively.
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