An open source three-mirror laser scanning holographic two-photon lithography system.
Summary Two-photon polymerization (TPP) is a leading technique for fabricating 2D and 3D nanostructures with sub-diffraction-limit features. This study introduces an open-hardware, open-software holographic multibeam TPP system featuring a phase-only spatial light modulator (SLM) and a three-mirror scan head. The novel three-reflective surface design—with two mirrors scanning along the same axis—resolves virtual conjugation loss in […]
Revealing architectural order with quantitative label-free imaging and deep learning
Summary We report quantitative label-free imaging with phase and polarization (QLIPP) for simultaneous measurement of density, anisotropy, and orientation of structures in unlabeled live cells and tissue slices, using advanced image processing techniques. We combine QLIPP with deep neural networks and machine learning models to predict fluorescence images of diverse cell and tissue structures, enhancing […]
Rapid learning of neural circuitry from holographic ensemble stimulation enabled by model-based compressed sensing
Summary Discovering how neural computations are implemented in the cortex at the level of monosynaptic connectivity requires probing for the existence of synapses from possibly thousands of presynaptic candidate neurons. Two-photon optogenetics has been shown to be a promising technology for mapping such monosynaptic connections via serial stimulation of neurons with single-cell resolution. However, this […]
The logic of recurrent circuits in the primary visual cortex
Summary Incoming visual signals. Despite the importance of recurrent circuits for cortical processing, the basic rules that govern how nearby cortical neurons influence each other remain enigmatic. We used two-photon holographic optogenetics to activate ensembles of neurons in Layer 2/3 of the primary visual cortex (V1) in the absence of external stimuli to isolate the […]
High-performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks
Summary The biophysical limitations of existing optogenetic tools restrict the scale, speed, and precision of neural control. Using structure-guided mutagenesis, we have engineered high-potency opsins that retain fast kinetics, enabling large-scale, spatiotemporally precise control of neuronal populations. These novel opsins have been extensively benchmarked against existing optogenetic tools and characterized under two-photon illumination, providing a […]
Deconvolution for multimode fiber imaging: modeling of spatially variant PSF
Summary Wavefront-controlled light focusing through step-index multimode optical fibers (MMFs) enables minimally invasive endoscopic imaging of biological tissue. However, the spatially variant point spread function (PSF) of MMF imaging systems poses challenges for traditional deconvolution algorithms, which assume a uniform PSF. To address this, we developed svmPSF, an open-source Java-based framework for ImageJ that models […]
Three-dimensional structured illumination microscopy with enhanced axial resolution
Summary We present two distinct, complementary methods for improving axial resolution in three-dimensional structured illumination microscopy (3D SIM) with minimal or no modification to the optical system. First, we show that placing a mirror directly opposite the sample enables 4-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with ~120 […]
