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.

Enhanced two-photon excited fluorescence in three-dimensionally crosslinked bovine serum albumin microstructures.

In this study, the intensity of two-photon excited fluorescence (TPEF) of xanthene dye, Rose Bengal (RB), was significantly enhanced via bovine serum albumin (BSA) microstructures fabricated by the two-photon crosslinking (TPC) technique. The RB was utilized as the photoactivator in the TPC processing and the enhanced TPEF intensity correlates with the concentration of fabricated crosslinked BSA microstructures via the power control and pulse selection of the employed femtosecond laser. As a result, fabrication of three-dimensional BSA microstructures can be simultaneously monitored by the use of TPEF intensity. The crosslinked BSA microstructures synthesized may be used as an ordered biomaterial for fluorescence enhancement.

Fabrication of gold nanorods-doped, bovine serum albumin microstructures via multiphoton excited photochemistry.

In this study, three-dimensional (3D) crosslinked bovine serum albumin (BSA) microstructures containing gold nanorods (AuNRs) were fabricated via multiphoton excited photochemistry using Rose Bengal (RB) as the photoactivator. To retain AuNRs in the 3D crosslinked BSA microstructures, the laser wavelength was chosen for two-photon RB absorption for improved two-photon crosslinking efficiency, but not for enhancing the longitudinal plasmon resonance of AuNRs which may result in photothermal damage of AuNRs. Furthermore, with two-photon excitation of RB via AuNRs plasmonics, the laser power can be reduced by about 30%. As a result, 3D BSA microstructures containing AuNRs can be successfully fabricated. The AuNRs-doped BSA microstructures can be applied in biomedical scaffolds with plasmonic properties such as two-photon luminescence imaging and photothermal therapy.

Multiphoton fabrication of freeform polymer microstructures with gold nanorods.

In this study, three-dimensional (3D) polyacrylamide microstructures containing gold nanorods (AuNRs) were fabricated by two-photon polymerization (TPP) using Rose Bengal (RB) as the photoinitiator. To retain AuNRs in the 3D polymer microstructures, the laser wavelength was chosen for two-photon RB absorption for improved TPP efficiency, but not for enhancing the longitudinal plasmon resonance of AuNRs which may result in photothermal damage of AuNRs. After TPP processing, the laser wavelength was tuned for the longitudinal plasmon resonance and the laser power was increased to beyond the damage threshold of the AuNRs for reshaping the AuNRs into gold nanospheres. As a result, AuNRs in designated positions of the fabricated 3D microstructures can be achieved. Two-photon luminescence from the doped AuNRs can also act as contrast agent for the visualization of 3D polymer microstructures.

Surface plasmon-enhanced two-photon fluorescence microscopy for live cell membrane imaging.

A surface plasmon-enhanced two-photon total-internal-reflection fluorescence (TIRF) microscope has been developed to provide fluorescent images of living cell membranes. The proposed microscope with the help of surface plasmons (SPs) not only provides brighter fluorescent images based on the mechanism of local electromagnetic field enhancement, but also reduces photobleaching due to having a shorter fluorophore lifetime. In comparison with a one-photon TIRF, the two-photon TIRF can achieve higher signal-to-noise ratio cell membrane imaging due its smaller excitation volume and lower scattering. By combining the SP enhancement and two-photon excitation TIRF, the microscope has demonstrated it's capability for brighter and more contrasted fluorescence membrane images of living monkey kidney COS-7 fibroblasts transfected with an EYFP-MEM or EGFP-WOX1 construct.

Wavefront sensorless adaptive optics temporal focusing-based multiphoton microscopy.

Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1µm fluorescent beads sealed in agarose gel at different depths is improved.

Gold nanomaterials conjugated with indocyanine green for dual-modality photodynamic and photothermal therapy.

Light-exposure-mediated higher temperatures that markedly accelerate the degradation of indocyanine green (ICG) in aqueous solutions by thermal decomposition have been a serious medical problem. In this work, we present the example of using gold nanorods (Au NRs) and gold nanoparticles (Au NPs) simultaneously serving as photodynamic and photothermal agents to destroy malignant cells. Au NRs and Au NPs were successfully conjugated with hydrophilic photosensitizer, indocyanine green (ICG), to achieve photodynamic therapy (PDT) and photothermal therapy (PTT). We also demonstrated that Au NRs and Au NPs conjugated with ICG displayed high chemical stability and acted as a promising diagnostic probe. Moreover, the photochemical destruction ability would have a gradually increase depending on different sizes of Au NPs. Due to its stability even via higher temperatures mediated by laser irradiation, the combination of PTT and PDT proved to be efficiently killing cancer cells as compared to PTT or PDT treatment alone and enhanced the effectiveness of photodestruction and was demonstrated to enhance its photostability. As a result, the preparation of Au-based nanomaterials conjugated with ICG as well as their use in biomedical applications is valuable developments in multifunctional nanomaterials.

Chronic treadmill exercise in rats delicately alters the Purkinje cell structure to improve motor performance and toxin resistance in the cerebellum.

Although exercise usually improves motor performance, the underlying cellular changes in the cerebellum remain to be elucidated. This study aimed to investigate whether and how chronic treadmill exercise in young rats induced Purkinje cell changes to improve motor performance and rendered the cerebellum less vulnerable to toxin insults. After 1-wk familiarization of treadmill running, 6-wk-old male Wistar rats were divided into exercise and sedentary groups. The exercise group was then subjected to 8 wk of exercise training at moderate intensity. The rotarod test was carried out to evaluate motor performance. Purkinje cells in cerebellar slices were visualized by lucifer yellow labeling in single neurons and by calbindin immunostaining in groups of neurons. Compared with sedentary control rats, exercised rats not only performed better in the rotarod task, but also showed finer Purkinje cell structure (higher dendritic volume and spine density with the same dendritic field). The exercise-improved cerebellar functions were further evaluated by monitoring the long-lasting effects of intraventricular application of OX7-saporin. In the sedentary group, OX7-saporin treatment retarded the rotarod performance and induced ∼60% Purkinje cell loss in 3 wk. As a comparison, the exercise group showed much milder injuries in the cerebellum by the same toxin treatment. In conclusion, exercise training in young rats increased the dendritic density of Purkinje cells, which might play an important role in improving the motor performance. Furthermore, as Purkinje cells in the exercise group were relatively toxin resistant, the exercised rats showed good motor performance, even under toxin-treated conditions.

Adaptive-optics system with liquid-crystal phase-shift interferometer.

We develop an adaptive-optics system based on a Mach-Zehnder radial shearing interferometer with liquid-crystal-device (LCD) phase-shift interferometry (PSI). Using accurate phase calibration and transient nematic driving of the LCD, the developed three-step PSI procedure can be achieved in a time of 5 ms. The proposed Mach-Zehnder radial shearing PSI method reconstructs the phase information using a digital signal processor (DSP). The DSP then computes appropriate control signals to drive a deformable mirror in such a way as to eliminate the wavefront distortion. The current adaptive-optics system is capable of suppressing low-frequency thermal disturbances with a signal-to-noise ratio improvement of more than 20 dB and a steady-state phase error of less than 0.02pi root mean square when the control loop is operated at a frequency of 30 Hz.