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Deep-ultraviolet (UV) microscopy enables label-free, high-resolution, quantitative molecular imaging and enables unique applications in biomedicine, including the potential for fast hematological analysis at the point-of-care. UV microscopy has been shown to quantify hemoglobin content and white blood cells (five-part differential), providing a simple alternative to the current gold standard, the hematological analyzer. Previously, however, the UV system comprised a bulky broadband laser-driven plasma light source along with a large and expensive camera and 3D translation stage. Here, we present a modified deep-UV microscope system with a compact footprint and low-cost components. We detail the novel design with simple, inexpensive optics and hardware to enable fast and accurate automated imaging. We characterize the system, including a modified low-cost web-camera and custom automated 3D translation stage, and demonstrate its ability to scan and capture large area images. We further demonstrate the capability of the system by imaging and analyzing blood smears, using previously trained networks for automatic segmentation, classification (including 5-part white blood cell differential), and colorization. The developed system is approximately 10 times less expensive than previous configurations and can serve as a point-of-care hematology analyzer, as well as be applied broadly in biomedicine as a simple compact, low-cost, quantitative molecular imaging system. 

Neutropenia is a condition comprising an abnormally low number of neutrophils, a type of white blood cell, which puts patients at an increased risk of severe infections. Neutropenia is especially common among cancer patients and can disrupt their treatment or even be life-threatening in severe cases. Therefore, routine monitoring of neutrophil counts is crucial. However, the current standard of care to assess neutropenia, the complete blood count (CBC), is resource-intensive, time-consuming, and expensive, thereby limiting easy or timely access to critical hematological information such as neutrophil counts. Here, we present a simple technique for fast, label-free neutropenia detection and grading via deep-ultraviolet (deep-UV) microscopy of blood cells in polydimethylsiloxane (PDMS)-based passive microfluidic devices. The devices can potentially be manufactured in large quantities at a low cost, requiring only 1 μL of whole blood for operation. We show that the absolute neutrophil counts (ANC) obtained from our proposed microfluidic device-enabled deep-UV microscopy system are highly correlated with those from CBCs using commercial hematology analyzers in patients with moderate and severe neutropenia, as well as healthy donors. This work lays the foundation for the development of a compact, easy-to-use UV microscope system to track neutrophil counts that is suitable for low-resource, at-home, or point-of-care settings. 

Three-dimensional (3D) refractive index (RI) tomography has recently become an exciting new tool for biological studies. However, its limitation to (1) thin samples resulting from a need of transmissive illumination and (2) small fields of view (typically $\sim <\#comment/>50\text{µ<\#comment/>}\mathrm{m}\times <\#comment/>50\text{µ<\#comment/>}\mathrm{m}$) has hindered its utility in broader biomedical applications. In this work, we demonstrate 3D RI tomography with a large field of view in opaque, arbitrarily thick scattering samples (unsuitable for imaging with conventional transmissive tomographic techniques) with a penetration depth of ca. one mean free scattering path length ( $\sim <\#comment/>100\text{µ<\#comment/>}\mathrm{m}$in tissue) using a simple, low-cost microscope system with epi-illumination. This approach leverages a solution to the inverse scattering problem via the general non-paraxial 3D optical transfer function of our quantitative oblique back-illumination microscopy (qOBM) optical system. A theoretical analysis is presented along with simulations and experimental validations using polystyrene beads, and rat and human thick brain tissues. This work has significant implications for the investigation of optically thick, semi-infinite samples in a non-invasive and label-free manner. This unique 3D qOBM approach can extend the utility of 3D RI tomography for translational and clinical medicine. 

Neutropenia is a condition identified by an abnormally low number of neutrophils in the bloodstream and signifies an increased risk of severe infection. Cancer patients are particularly susceptible to this condition, which can be disruptive to their treatment and even life-threatening in severe cases. Thus, it is critical to routinely monitor neutrophil counts in cancer patients. However, the standard of care to assess neutropenia, the complete blood count (CBC), requires expensive and complex equipment, as well as cumbersome procedures, which precludes easy or timely access to critical hematological information, namely neutrophil counts. Here we present a simple, low-cost, fast, and robust technique to detect and grade neutropenia based on label-free multi-spectral deep-UV microscopy. Results show that the developed framework for automated segmentation and classification of live, unstained blood cells in a smear accurately differentiates patients with moderate and severe neutropenia from healthy samples in minutes. This work has significant implications towards the development of a low-cost and easy-to-use point-of-care device for tracking neutrophil counts, which can not only improve the quality of life and treatment-outcomes of many patients but can also be lifesaving. 

Brain tumor surgery involves a delicate balance between maximizing the extent of tumor resection while minimizing damage to healthy brain tissue that is vital for neurological function. However, differentiating between tumor, particularly infiltrative disease, and healthy brain in-vivo remains a significant clinical challenge. Here we demonstrate that quantitative oblique back illumination microscopy (qOBM)—a novel label-free optical imaging technique that achieves tomographic quantitative phase imaging in thick scattering samples—clearly differentiates between healthy brain tissue and tumor, including infiltrative disease. Data from a bulk and infiltrative brain tumor animal model show that qOBM enables quantitative phase imaging of thick fresh brain tissues with remarkable cellular and subcellular detail that closely resembles histopathology using hematoxylin and eosin (H&E) stained fixed tissue sections, the gold standard for cancer detection. Quantitative biophysical features are also extracted from qOBM which yield robust surrogate biomarkers of disease that enable (1) automated tumor and margin detection with high sensitivity and specificity and (2) facile visualization of tumor regions. Finally, we develop a low-cost, flexible, fiber-based handheld qOBM device which brings this technology one step closer to in-vivo clinical use. This work has significant implications for guiding neurosurgery by paving the way for a tool that delivers real-time, label-free, in-vivo brain tumor margin detection. 

Ultraviolet (UV) microscopy has recently re-emerged as an important label-free, molecular imaging technique. This stems from the unique UV absorption properties of many endogenous biomolecules that play a critical role in cell structure and function. However, broadband hyperspectral imaging in this spectral region is challenging due to strong chromatic aberrations inherent in UV systems. Here we apply an intensity-based, two-stage, iterative phase-recovery algorithm that leverages the same chromatic aberrations to overcome this challenge. Importantly, knowledge of samples’ dispersion or absorption properties is not required. We demonstrate that the computationally retrieved phase can be applied to digitally refocus images across a large bandwidth. This enables hyperspectral UV imaging with a simple microscope for quantitative molecular analysis. We validate this method through simulations and through experiments with red blood cells. 

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.