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Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique in biomedical research that uses the fluorophore decay rate to provide additional contrast in fluorescence microscopy. However, at present, the calculation, analysis, and interpretation of FLIM is a complex, slow, and computationally expensive process. Machine learning (ML) techniques are well suited to extract and interpret measurements from multi-dimensional FLIM data sets with substantial improvement in speed over conventional methods. In this topical review, we first discuss the basics of FILM and ML. Second, we provide a summary of lifetime extraction strategies using ML and its applications in classifying and segmenting FILM images with higher accuracy compared to conventional methods. Finally, we discuss two potential directions to improve FLIM with ML with proof of concept demonstrations.
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Fluorescence lifetime imaging microscopy (FLIM) systems are limited by their slow processing speed, low signal-to-noise ratio (SNR), and expensive and challenging hardware setups. In this work, we demonstrate applying a denoising convolutional network to improve FLIM SNR. The network will be integrated with an instant FLIM system with fast data acquisition based on analog signal processing, high SNR using high-efficiency pulse-modulation, and cost-effective implementation utilizing off-the-shelf radio-frequency components. Our instant FLIM system simultaneously provides the intensity, lifetime, and phasor plots textit{in vivo} and textit{ex vivo}. By integrating image denoising using the trained deep learning model on the FLIM data, provide accurate FLIM phasor measurements are obtained. The enhanced phasor is then passed through the K-means clustering segmentation method, an unbiased and unsupervised machine learning technique to separate different fluorophores accurately. Our experimental textit{in vivo} mouse kidney results indicate that introducing the deep learning image denoising model before the segmentation effectively removes the noise in the phasor compared to existing methods and provides clearer segments. Hence, the proposed deep learning-based workflow provides fast and accurate automatic segmentation of fluorescence images using instant FLIM. The denoising operation is effective for the segmentation if the FLIM measurements are noisy. The clustering can effectively enhance the detection of biological structures of interest in biomedical imaging applications.
An image dataset of 10 different size molecules, where each molecule has 2,000 structural variants, is generated from the 2D cross-sectional projection of Molecular Dynamics trajectories. The purpose of this dataset is to provide a benchmark dataset for the increasing need of machine learning, deep learning and image processing on the study of scattering, imaging and microscopy.
We report the cell biological applications of a recently developed multiphoton fluorescence lifetime imaging microscopy system using a streak camera (StreakFLIM). The system was calibrated with standard fluorophore specimens and was shown to have high accuracy and reproducibility. We demonstrate the applicability of this instrument in living cells for measuring the effects of protein targeting and point mutations in the protein sequence which are not obtainable in conventional intensity based fluorescence microscopy methods. We discuss the relevance of such time resolved information in quantitative energy transfer microscopy and in measurement of the parameters characterizing intracellular physiology.
Advances in computing power, deep learning architectures, and expert labelled datasets have spurred the development of medical imaging artificial intelligence systems that rival clinical experts in a variety of scenarios. The National Institutes of Health in 2018 identified key focus areas for the future of artificial intelligence in medical imaging, creating a foundational roadmap for research in image acquisition, algorithms, data standardization, and translatable clinical decision support systems. Among the key issues raised in the report: data availability, need for novel computing architectures and explainable AI algorithms, are still relevant despite the tremendous progress made over the past few years alone. Furthermore, translational goals of data sharing, validation of performance for regulatory approval, generalizability and mitigation of unintended bias must be accounted for early in the development process. In this perspective paper we explore challenges unique to high dimensional clinical imaging data, in addition to highlighting some of the technical and ethical considerations in developing high-dimensional, multi-modality, machine learning systems for clinical decision support.
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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