Design of a multi-modality DMD-based two-photon microscope system.

We present the modular design and characterization of a multi-modality video-rate two-photon excitation (TPE) microscope based on integrating a digital micromirror device (DMD), which functions as an ultrafast beam shaper and random-access scanner, with a pair of galvanometric scanners. The TPE microscope system realizes a suite of new imaging functionalities, including (1) multi-layer imaging with 3D programmable imaging planes, (2) DMD-based wavefront correction, and (3) multi-focus optical stimulation (up to 22.7 kHz) with simultaneous TPE imaging, all in real-time. We also report the detailed optomechanical design and software development that achieves high level system automation. To verify the performance of different microscope functions, we have devised and performed imaging experiments on Drosophila brain, mouse kidney and human stem cells. The results not only show improved imaging resolution and depths via the DMD-based adaptive optics, but also demonstrate fast multi-focus stimulation for the first time. With the new imaging capabilities, e.g., tools for optogenetics, the multi-modality TPE microscope may play a critical role in the applications pertinent to neuroscience and biophotonics.

Aberration-free 3D imaging via DMD-based two-photon microscopy and sensorless adaptive optics.

In this Letter, we present a new, to our knowledge, aberration-free 3D imaging technique based on digital micromirror device (DMD)-based two-photon microscopy and sensorless adaptive optics (AO), where 3D random-access scanning and modal wavefront correction are realized using a single DMD chip at 22.7 kHz. Specifically, the DMD is simultaneously used as a deformable mirror to modulate a distorted wavefront and a fast scanner to maneuver the laser focus in a 3D space by designed binary holograms. As such, aberration-free 3D imaging is realized by superposing the wavefront correction and 3D scanning holograms. Compared with conventional AO devices and methods, the DMD system can apply optimal wavefront correction information to different imaging regions or even individual pixels without compromising the scanning speed and device resolution. In the experiments, we first focus the laser through a diffuser and apply sensorless AO to retrieve a corrected focus. After that, the DMD performs 3D scanning on a Drosophila brain labeled with green fluorescent protein. The two-photon imaging results, where optimal wavefront correction information is applied to 3×3 separate regions, demonstrate significantly improved resolution and image quality. The new DMD-based imaging solution presents a compact, low-cost, and effective solution for aberration-free two-photon deep tissue imaging, which may find important applications in the field of biophotonics.

Compressive sensing for fast 3-D and random-access two-photon microscopy.

3-D two-photon excitation (TPE) microscopy has been a critical tool for biological study since its introduction. Yet, the speed is largely limited by its point detector, e.g., photomultiplier tube (PMT), which requires a point-scanning imaging sequence. In this Letter, we present a multi-focus compressive sensing (CS) method for 3-D and random-access TPE microscopy based on a digital micromirror device (DMD). This new platform combines CS with a unique holography-based DMD random-access scanner to enhance the imaging speed by three to five times for imaging arbitrarily selected regions in 3-D specimens without sacrificing the resolution. In the experiments, 1-20 randomly selected foci are generated by modulating the wavefront of a femtosecond laser via binary holography, where the combined intensity is recorded by a PMT. By exploiting CS algorithms, 3-D images at arbitrarily selected sites can be reconstructed. Simulations and imaging experiments on different samples have been performed to verify the principle and identify the optimal processing parameters, including the number of laser foci and sampling ratios. The results show that high-resolution images can be obtained by using a 25% sampling ratio and five foci. The new CS-based TPE imaging method may find important applications in biological studies, e.g., neuronal imaging and optogenetics.

Spatially resolved random-access pump-probe microscopy based on binary holography.

In this Letter, we present a spatially resolved pump-probe microscope based on a digital micromirror device (DMD). The microscope system enables the measurements of ultrafast transient processes at arbitrarily selected regions in a 3-D specimen. To achieve random-access scanning, the wavefront of the probe beam is modulated by the DMD via binary holography. By switching the holograms stored in the DMD memory, the laser focus can be rapidly moved in space in a discrete fashion. The microscope system has a field of view of 65×130×155 µm3 in the x, y, and z axes, respectively; and a scanning speed of 8 kHz which is limited by the response time of the lock-in amplifier. To demonstrate the pump-probe system, we measured the ultrafast transient reflectivity of 2-D gold patterns on a silicon substrate and on silicon nitride cantilever beams. The results show an excellent signal-to-noise ratio and spatial-temporal resolution, as well as the 3-D random scanning capability. The new pump-probe microscope is a versatile instrument to characterize ultrafast 3-D phenomena with high spatial and temporal resolution, e.g., the propagation of localized surface plasmon resonance on curved surfaces.