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Video microscopy has a long history of providing insights and breakthroughs for a broad range of disciplines, from physics to biology. Image analysis to extract quantitative information from video microscopy data has traditionally relied on algorithmic approaches, which are often difficult to implement, time consuming, and computationally expensive. Recently, alternative data-driven approaches using deep learning have greatly improved quantitative digital microscopy, potentially offering automatized, accurate, and fast image analysis. However, the combination of deep learning and video microscopy remains underutilized primarily due to the steep learning curve involved in developing custom deep-learning solutions. To overcome this issue, we introduce a software, DeepTrack 2.0, to design, train and validate deep-learning solutions for digital microscopy. We use it to exemplify how deep learning can be employed for a broad range of applications, from particle localization, tracking and characterization to cell counting and classification. Thanks to its user-friendly graphical interface, DeepTrack 2.0 can be easily customized for user-specific applications, and, thanks to its open-source object-oriented programming, it can be easily expanded to add features and functionalities, potentially introducing deep-learning-enhanced video microscopy to a far wider audience.
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Quantitative phase imaging (QPI) has been widely applied in characterizing cells and tissues. Spatial light interference microscopy (SLIM) is a highly sensitive QPI method, due to its partially coherent illumination and common path interferometry geometry. However, its acquisition rate is limited because of the four-frame phase-shifting scheme. On the other hand, off-axis methods like diffraction phase microscopy (DPM), allows for single-shot QPI. However, the laser-based DPM system is plagued by spatial noise due to speckles and multiple reflections. In a parallel development, deep learning was proven valuable in the field of bioimaging, especially due to its ability to translate one form of contrast into another. Here, we propose using deep learning to produce synthetic, SLIM-quality, high-sensitivity phase maps from DPM, single-shot images as input. We used an inverted microscope with its two ports connected to the DPM and SLIM modules, such that we have access to the two types of images on the same field of view. We constructed a deep learning model based on U-net and trained on over 1,000 pairs of DPM and SLIM images. The model learned to remove the speckles in laser DPM and overcame the background phase noise in both the test set and new data. Furthermore, we implemented the neural network inference into the live acquisition software, which now allows a DPM user to observe in real-time an extremely low-noise phase image. We demonstrated this principle of computational interference microscopy (CIM) imaging using blood smears, as they contain both erythrocytes and leukocytes, in static and dynamic conditions.
Fluorescence microscopy has enabled a dramatic development in modern biology by visualizing biological organisms with micrometer scale resolution. However, due to the diffraction limit, sub-micron/nanometer features are difficult to resolve. While various super-resolution techniques are developed to achieve nanometer-scale resolution, they often either require expensive optical setup or specialized fluorophores. In recent years, deep learning has shown the potentials to reduce the technical barrier and obtain super-resolution from diffraction-limited images. For accurate results, conventional deep learning techniques require thousands of images as a training dataset. Obtaining large datasets from biological samples is not often feasible due to the photobleaching of fluorophores, phototoxicity, and dynamic processes occurring within the organism. Therefore, achieving deep learning-based super-resolution using small datasets is challenging. We address this limitation with a new convolutional neural network-based approach that is successfully trained with small datasets and achieves super-resolution images. We captured 750 images in total from 15 different field-of-views as the training dataset to demonstrate the technique. In each FOV, a single target image is generated using the super-resolution radial fluctuation method. As expected, this small dataset failed to produce a usable model using traditional super-resolution architecture. However, using the new approach, a network can be trained to achieve super-resolution images from this small dataset. This deep learning model can be applied to other biomedical imaging modalities such as MRI and X-ray imaging, where obtaining large training datasets is challenging.
Microscopy is a powerful visualization tool in biology, enabling the study of cells, tissues, and the fundamental biological processes; yet, the observed images typically suffer from blur and background noise. In this work, we propose a unifying framework of algorithms for Gaussian image deblurring and denoising. These algorithms are based on deep learning techniques for the design of learnable regularizers integrated into the Wiener-Kolmogorov filter. Our extensive experimentation line showcases that the proposed approach achieves a superior quality of image reconstruction and surpasses the solutions that rely either on deep learning or on optimization schemes alone. Augmented with the variance stabilizing transformation, the proposed reconstruction pipeline can also be successfully applied to the problem of Poisson image deblurring, surpassing the state-of-the-art methods. Moreover, several variants of the proposed framework demonstrate competitive performance at low computational complexity, which is of high importance for real-time imaging applications.
A well-trained deep neural network is shown to gain capability of simultaneously restoring two kinds of images, which are completely destroyed by two distinct scattering medias respectively. The network, based on the U-net architecture, can be trained by blended dataset of speckles-reference images pairs. We experimentally demonstrate the power of the network in reconstructing images which are strongly diffused by glass diffuser or multi-mode fiber. The learning model further shows good generalization ability to reconstruct images that are distinguished from the training dataset. Our work facilitates the study of optical transmission and expands machine learnings application in optics.
In transmission X-ray microscopy (TXM) systems, the rotation of a scanned sample might be restricted to a limited angular range to avoid collision to other system parts or high attenuation at certain tilting angles. Image reconstruction from such limited angle data suffers from artifacts due to missing data. In this work, deep learning is applied to limited angle reconstruction in TXMs for the first time. With the challenge to obtain sufficient real data for training, training a deep neural network from synthetic data is investigated. Particularly, the U-Net, the state-of-the-art neural network in biomedical imaging, is trained from synthetic ellipsoid data and multi-category data to reduce artifacts in filtered back-projection (FBP) reconstruction images. The proposed method is evaluated on synthetic data and real scanned chlorella data in $100^circ$ limited angle tomography. For synthetic test data, the U-Net significantly reduces root-mean-square error (RMSE) from $2.55 times 10^{-3}$ {mu}m$^{-1}$ in the FBP reconstruction to $1.21 times 10^{-3}$ {mu}m$^{-1}$ in the U-Net reconstruction, and also improves structural similarity (SSIM) index from 0.625 to 0.920. With penalized weighted least square denoising of measured projections, the RMSE and SSIM are further improved to $1.16 times 10^{-3}$ {mu}m$^{-1}$ and 0.932, respectively. For real test data, the proposed method remarkably improves the 3-D visualization of the subcellular structures in the chlorella cell, which indicates its important value for nano-scale imaging in biology, nanoscience and materials science.
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