We present a novel approach to achieve high-speed depth-resolved two-photon imaging through the development of a deep-learning-based temporal-focusing two-photon microscope utilizing the De-scattering with Excitation Patterning (DEEP) method, referred to as DEEP-Line. DEEP-Line incorporates a line-scanning scheme, widefield detection utilizing a high-speed Silicon Photomultiplier array, and employs deep-learning-based image reconstruction. The performance of our system is validated using diverse biological samples. Our imaging method achieves orders of magnitude improvement in speed by reducing excitation patterns to several tens and employing MHz parallel detections. Furthermore, our approach can enable fluorescence lifetime imaging and enhances axial resolution.
We present in situ monitoring of two-photon lithography with optical diffraction tomography (ODT) in an integrated home-built system. We show how two-photon lithographic system and intensity-based ODT can be organically integrated to provide reconstruction of 3D refractive index distribution of the printed structure at a diffraction-limited resolution. Due to ODT’s label-free nature, our method realizes in situ quality inspection of printed structure without specific sample preparation. In this way, our in situ observing solution can provide a timely feedback to the fabrication quality of two-photon lithography and potentially enables closed-loop optimizing and planning of printing parameters on-the-fly.
Multiphoton microscopy is the gold standard for deep tissue fluorescence imaging. Long wavelengths enable hundreds of microns deep penetration of excitation light, but the emission fluorescence at shorter wavelengths encounters scattering before detection. While not being an issue for point scanning geometries, for wide-field geometries emission light scattering degrades the image quality. In this work, we use temporally focused pattered excitations to spatially encode image information before emission light scattering. Upon detection, images are reconstructed computationally by solving a linear inverse problem. We further improve our results by learning inverse solvers and optimal patterns through physics-based deep learning.
A new optical microscopy technique, termed high spatial and temporal resolution synthetic aperture phase microscopy (HISTR-SAPM), is proposed to improve the lateral resolution of wide-field coherent imaging. Under plane wave illumination, the resolution is increased by twofold to around 260 nm, while achieving millisecond-level temporal resolution. In HISTR-SAPM, digital micromirror devices are used to actively change the sample illumination beam angle at high speed with high stability. An off-axis interferometer is used to measure the sample scattered complex fields, which are then processed to reconstruct high-resolution phase images. Using HISTR-SAPM, we are able to map the height profiles of subwavelength photonic structures and resolve the period structures that have 198 nm linewidth and 132 nm gap (i.e., a full pitch of 330 nm). As the reconstruction averages out laser speckle noise while maintaining high temporal resolution, HISTR-SAPM further enables imaging and quantification of nanoscale dynamics of live cells, such as red blood cell membrane fluctuations and subcellular structure dynamics within nucleated cells. We envision that HISTR-SAPM will broadly benefit research in material science and biology.
We propose a high-throughput 3D imaging cytometer for fast quantification of DNA double strand break (DSB) frequency in cells for DNA damage study. With structured illumination enabled depth contrast and a fast focus tunable lens enabled scanning, this system generates a three-dimensional stack of clustered nuclei γH2AX foci with submicron resolution at a speed of 800 cells/second. Moreover, we unify the stack construction with the deep neural network, which largely improve quantification accuracy as well as the processing speed. Compared to previous 2D imaging approach, the addition of z-resolution in our 3D method provides an extra dimension of contrast and thus allows for more accurate DNA DSB quantification.
We demonstrate a high-speed and high-resolution quantitative phase imaging method by implementing a synthetic aperture technique through using digital micromirror devices (DMDs). DMDs provide high speed steering of the illumination beam angle upon the sample that induces sample frequency shift, which is the basis of achieving high resolution in the quantitative phase imaging (QPI) system. With a high-speed camera for image acquisition, our QPI system achieves synthetic aperture imaging at >25 frame per second (fps). The high-speed imaging capability of the system allows for better observation of samples dynamics, especially in live biological structures, where motions could result in degraded imaging quality. In experiments, our synthetic aperture-based QPI system has resolved sub-diffraction limited structures of 220 nm periods and quantified red blood cell membrane fluctuations, which opens new avenues in material metrology and biological imaging applications.
Diffraction Phase Microscopy (DPM) is a widely used quantitative phase imaging method, whose common-path nature endows it with low noise and high sensitivity. Current applications of DPM include biological topography as well as biological dynamics for its nondestructive feature. Many different forms of DPM based on the original idea have appeared according to the different demands. In this paper, both the principle and the DPM classification will be given a comprehensive description. Furthermore, the future trend of DPM development is also discussed.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.