Auto-ICell: An Accessible and Cost-Effective Integrative Droplet Microfluidic System for Real-Time Single-Cell Morphological and Apoptotic Analysis

The Auto-ICell system, a novel, and cost-effective integrated droplet microfluidic system, is introduced for real-time analysis of single-cell morphology and apoptosis. This system integrates a 3D-printed microfluidic chip with image analysis algorithms, enabling the generation of uniform droplet reactors and immediate image analysis. The system employs a color-based image analysis algorithm in the bright field for droplet content analysis. Meanwhile, in the fluorescence field, cell apoptosis is quantitatively measured through a combination of deep-learning-enabled multiple fluorescent channel analysis and a live/dead cell stain kit. Breast cancer cells are encapsulated within uniform droplets, with diameters ranging from 70 {\mu}m to 240 {\mu}m, generated at a high throughput of 1,500 droplets per minute. Real-time image analysis results are displayed within 2 seconds on a custom graphical user interface (GUI). The system provides an automatic calculation of the distribution and ratio of encapsulated dyes in the bright field, and in the fluorescent field, cell blebbing and cell circularity are observed and quantified respectively. The Auto-ICell system is non-invasive and provides online detection, offering a robust, time-efficient, user-friendly, and cost-effective solution for single-cell analysis. It significantly enhances the detection throughput of droplet single-cell analysis by reducing setup costs and improving operational performance. This study highlights the potential of the Auto-ICell system in advancing biological research and personalized disease treatment, with promising applications in cell culture, biochemical microreactors, drug carriers, cell-based assays, synthetic biology, and point-of-care diagnostics.

Complex-valued universal linear transformations and image encryption using spatially incoherent diffractive networks

As an optical processor, a Diffractive Deep Neural Network (D2NN) utilizes engineered diffractive surfaces designed through machine learning to perform all-optical information processing, completing its tasks at the speed of light propagation through thin optical layers. With sufficient degrees-of-freedom, D2NNs can perform arbitrary complex-valued linear transformations using spatially coherent light. Similarly, D2NNs can also perform arbitrary linear intensity transformations with spatially incoherent illumination; however, under spatially incoherent light, these transformations are non-negative, acting on diffraction-limited optical intensity patterns at the input field-of-view (FOV). Here, we expand the use of spatially incoherent D2NNs to complex-valued information processing for executing arbitrary complex-valued linear transformations using spatially incoherent light. Through simulations, we show that as the number of optimized diffractive features increases beyond a threshold dictated by the multiplication of the input and output space-bandwidth products, a spatially incoherent diffractive visual processor can approximate any complex-valued linear transformation and be used for all-optical image encryption using incoherent illumination. The findings are important for the all-optical processing of information under natural light using various forms of diffractive surface-based optical processors.

Pyramid diffractive optical networks for unidirectional magnification and demagnification

Diffractive deep neural networks (D2NNs) are composed of successive transmissive layers optimized using supervised deep learning to all-optically implement various computational tasks between an input and output field-of-view (FOV). Here, we present a pyramid-structured diffractive optical network design (which we term P-D2NN), optimized specifically for unidirectional image magnification and demagnification. In this P-D2NN design, the diffractive layers are pyramidally scaled in alignment with the direction of the image magnification or demagnification. Our analyses revealed the efficacy of this P-D2NN design in unidirectional image magnification and demagnification tasks, producing high-fidelity magnified or demagnified images in only one direction, while inhibiting the image formation in the opposite direction - confirming the desired unidirectional imaging operation. Compared to the conventional D2NN designs with uniform-sized successive diffractive layers, P-D2NN design achieves similar performance in unidirectional magnification tasks using only half of the diffractive degrees of freedom within the optical processor volume. Furthermore, it maintains its unidirectional image magnification/demagnification functionality across a large band of illumination wavelengths despite being trained with a single illumination wavelength. With this pyramidal architecture, we also designed a wavelength-multiplexed diffractive network, where a unidirectional magnifier and a unidirectional demagnifier operate simultaneously in opposite directions, at two distinct illumination wavelengths. The efficacy of the P-D2NN architecture was also validated experimentally using monochromatic terahertz illumination, successfully matching our numerical simulations. P-D2NN offers a physics-inspired strategy for designing task-specific visual processors.

Lab-in-a-Tube: A portable imaging spectrophotometer for cost-effective, high-throughput, and label-free analysis of centrifugation processes

Centrifuges serve as essential instruments in modern experimental sciences, facilitating a wide range of routine sample processing tasks that necessitate material sedimentation. However, the study for real time observation of the dynamical process during centrifugation has remained elusive. In this study, we developed an innovative Lab_in_a_Tube imaging spectrophotometer that incorporates capabilities of real time image analysis and programmable interruption. This portable LIAT device costs less than 30 US dollars. Based on our knowledge, it is the first Wi Fi camera built_in in common lab centrifuges with active closed_loop control. We tested our LIAT imaging spectrophotometer with solute solvent interaction investigation obtained from lab centrifuges with quantitative data plotting in a real time manner. Single re circulating flow was real time observed, forming the ring shaped pattern during centrifugation. To the best of our knowledge, this is the very first observation of similar phenomena. We developed theoretical simulations for the single particle in a rotating reference frame, which correlated well with experimental results. We also demonstrated the first demonstration to visualize the blood sedimentation process in clinical lab centrifuges. This remarkable cost effectiveness opens up exciting opportunities for centrifugation microbiology research and paves the way for the creation of a network of computational imaging spectrometers at an affordable price for large scale and continuous monitoring of centrifugal processes in general.

Universal Linear Intensity Transformations Using Spatially-Incoherent Diffractive Processors

Under spatially-coherent light, a diffractive optical network composed of structured surfaces can be designed to perform any arbitrary complex-valued linear transformation between its input and output fields-of-view (FOVs) if the total number (N) of optimizable phase-only diffractive features is greater than or equal to ~2 Ni x No, where Ni and No refer to the number of useful pixels at the input and the output FOVs, respectively. Here we report the design of a spatially-incoherent diffractive optical processor that can approximate any arbitrary linear transformation in time-averaged intensity between its input and output FOVs. Under spatially-incoherent monochromatic light, the spatially-varying intensity point spread functon(H) of a diffractive network, corresponding to a given, arbitrarily-selected linear intensity transformation, can be written as H(m,n;m',n')=|h(m,n;m',n')|^2, where h is the spatially-coherent point-spread function of the same diffractive network, and (m,n) and (m',n') define the coordinates of the output and input FOVs, respectively. Using deep learning, supervised through examples of input-output profiles, we numerically demonstrate that a spatially-incoherent diffractive network can be trained to all-optically perform any arbitrary linear intensity transformation between its input and output if N is greater than or equal to ~2 Ni x No. These results constitute the first demonstration of universal linear intensity transformations performed on an input FOV under spatially-incoherent illumination and will be useful for designing all-optical visual processors that can work with incoherent, natural light.

Roadmap on Deep Learning for Microscopy

Through digital imaging, microscopy has evolved from primarily being a means for visual observation of life at the micro- and nano-scale, to a quantitative tool with ever-increasing resolution and throughput. Artificial intelligence, deep neural networks, and machine learning are all niche terms describing computational methods that have gained a pivotal role in microscopy-based research over the past decade. This Roadmap is written collectively by prominent researchers and encompasses selected aspects of how machine learning is applied to microscopy image data, with the aim of gaining scientific knowledge by improved image quality, automated detection, segmentation, classification and tracking of objects, and efficient merging of information from multiple imaging modalities. We aim to give the reader an overview of the key developments and an understanding of possibilities and limitations of machine learning for microscopy. It will be of interest to a wide cross-disciplinary audience in the physical sciences and life sciences.

Quantitative phase imaging (QPI) through random diffusers using a diffractive optical network

Quantitative phase imaging (QPI) is a label-free computational imaging technique used in various fields, including biology and medical research. Modern QPI systems typically rely on digital processing using iterative algorithms for phase retrieval and image reconstruction. Here, we report a diffractive optical network trained to convert the phase information of input objects positioned behind random diffusers into intensity variations at the output plane, all-optically performing phase recovery and quantitative imaging of phase objects completely hidden by unknown, random phase diffusers. This QPI diffractive network is composed of successive diffractive layers, axially spanning in total ~70 wavelengths; unlike existing digital image reconstruction and phase retrieval methods, it forms an all-optical processor that does not require external power beyond the illumination beam to complete its QPI reconstruction at the speed of light propagation. This all-optical diffractive processor can provide a low-power, high frame rate and compact alternative for quantitative imaging of phase objects through random, unknown diffusers and can operate at different parts of the electromagnetic spectrum for various applications in biomedical imaging and sensing. The presented QPI diffractive designs can be integrated onto the active area of standard CCD/CMOS-based image sensors to convert an existing optical microscope into a diffractive QPI microscope, performing phase recovery and image reconstruction on a chip through light diffraction within passive structured layers.

Data class-specific all-optical transformations and encryption

Diffractive optical networks provide rich opportunities for visual computing tasks since the spatial information of a scene can be directly accessed by a diffractive processor without requiring any digital pre-processing steps. Here we present data class-specific transformations all-optically performed between the input and output fields-of-view (FOVs) of a diffractive network. The visual information of the objects is encoded into the amplitude (A), phase (P), or intensity (I) of the optical field at the input, which is all-optically processed by a data class-specific diffractive network. At the output, an image sensor-array directly measures the transformed patterns, all-optically encrypted using the transformation matrices pre-assigned to different data classes, i.e., a separate matrix for each data class. The original input images can be recovered by applying the correct decryption key (the inverse transformation) corresponding to the matching data class, while applying any other key will lead to loss of information. The class-specificity of these all-optical diffractive transformations creates opportunities where different keys can be distributed to different users; each user can only decode the acquired images of only one data class, serving multiple users in an all-optically encrypted manner. We numerically demonstrated all-optical class-specific transformations covering A-->A, I-->I, and P-->I transformations using various image datasets. We also experimentally validated the feasibility of this framework by fabricating a class-specific I-->I transformation diffractive network using two-photon polymerization and successfully tested it at 1550 nm wavelength. Data class-specific all-optical transformations provide a fast and energy-efficient method for image and data encryption, enhancing data security and privacy.

Unidirectional Imaging using Deep Learning-Designed Materials

A unidirectional imager would only permit image formation along one direction, from an input field-of-view (FOV) A to an output FOV B, and in the reverse path, the image formation would be blocked. Here, we report the first demonstration of unidirectional imagers, presenting polarization-insensitive and broadband unidirectional imaging based on successive diffractive layers that are linear and isotropic. These diffractive layers are optimized using deep learning and consist of hundreds of thousands of diffractive phase features, which collectively modulate the incoming fields and project an intensity image of the input onto an output FOV, while blocking the image formation in the reverse direction. After their deep learning-based training, the resulting diffractive layers are fabricated to form a unidirectional imager. As a reciprocal device, the diffractive unidirectional imager has asymmetric mode processing capabilities in the forward and backward directions, where the optical modes from B to A are selectively guided/scattered to miss the output FOV, whereas for the forward direction such modal losses are minimized, yielding an ideal imaging system between the input and output FOVs. Although trained using monochromatic illumination, the diffractive unidirectional imager maintains its functionality over a large spectral band and works under broadband illumination. We experimentally validated this unidirectional imager using terahertz radiation, very well matching our numerical results. Using the same deep learning-based design strategy, we also created a wavelength-selective unidirectional imager, where two unidirectional imaging operations, in reverse directions, are multiplexed through different illumination wavelengths. Diffractive unidirectional imaging using structured materials will have numerous applications in e.g., security, defense, telecommunications and privacy protection.

Deep Learning-enabled Virtual Histological Staining of Biological Samples

Histological staining is the gold standard for tissue examination in clinical pathology and life-science research, which visualizes the tissue and cellular structures using chromatic dyes or fluorescence labels to aid the microscopic assessment of tissue. However, the current histological staining workflow requires tedious sample preparation steps, specialized laboratory infrastructure, and trained histotechnologists, making it expensive, time-consuming, and not accessible in resource-limited settings. Deep learning techniques created new opportunities to revolutionize staining methods by digitally generating histological stains using trained neural networks, providing rapid, cost-effective, and accurate alternatives to standard chemical staining methods. These techniques, broadly referred to as virtual staining, were extensively explored by multiple research groups and demonstrated to be successful in generating various types of histological stains from label-free microscopic images of unstained samples; similar approaches were also used for transforming images of an already stained tissue sample into another type of stain, performing virtual stain-to-stain transformations. In this Review, we provide a comprehensive overview of the recent research advances in deep learning-enabled virtual histological staining techniques. The basic concepts and the typical workflow of virtual staining are introduced, followed by a discussion of representative works and their technical innovations. We also share our perspectives on the future of this emerging field, aiming to inspire readers from diverse scientific fields to further expand the scope of deep learning-enabled virtual histological staining techniques and their applications.