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.

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.

Easily implementable field programmable gate array-based adaptive optics system with state-space multichannel control.

In this paper, an easily implementable adaptive optics system (AOS) based on a real-time field programmable gate array (FPGA) platform with state-space multichannel control programmed by LabVIEW has been developed, and also integrated into a laser focusing system successfully. To meet the requirements of simple programming configuration and easy integration with other devices, the FPGA-based AOS introduces a standard operation procedure including AOS identification, computation, and operation. The overall system with a 32-channel driving signal for a deformable mirror (DM) as input and a Zernike polynomial via a lab-made Shack-Hartmann wavefront sensor (SHWS) as output is optimally identified to construct a multichannel state-space model off-line. In real-time operation, the FPGA platform first calculates the Zernike polynomial of the optical wavefront measured from the SHWS as the feedback signal. Then, a state-space multichannel controller according to the feedback signal and the identified model is designed and implemented in the FPGA to drive the DM for phase distortion compensation. The current FPGA-based AOS is capable of suppressing low-frequency thermal disturbances with a steady-state phase error of less than 0.1 π within less than 10 time steps when the control loop is operated at a frequency of 30 Hz.

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.