Neutropenia is a blood disorder characterized by an abnormally low number of neutrophils in the bloodstream. This condition signifies an increased risk of infections, and thus can lead to life-threatening medical emergencies in severe cases. Therefore, it is critical to routinely monitor neutrophil counts in cancer patients. However, the current clinical standard-of-care for blood cell enumeration to assess neutropenia relies on complete blood count (CBC) which requires expensive and complex equipment, multiple reagents, and cumbersome procedures, impeding easy and timely access to critical hematological information. Here, we demonstrate the application of a microfluidic device which, along with deep-ultraviolet microscopy, enables stain-free and fixative-free hematological assessment of neutropenia. We demonstrate the capabilities of our approach in detection and staging of neutropenia by imaging samples obtained from healthy donors as well as moderate and severe neutropenia patients while verifying the results against CBC findings. This work has significant implications towards the development of a low-cost, and easy-to-use point-of-care device for tracking neutrophil counts.
Significance: The morphological properties and hemoglobin (Hb) content of red blood cells (RBCs) are essential biomarkers to diagnose or monitor various types of hematological disorders. Label-free mass mapping approaches enable accurate Hb quantification from individual cells, serving as promising alternatives to conventional hematology analyzers. Deep ultraviolet (UV) microscopy is one such technique that allows high-resolution, molecular imaging, and absorption-based mass mapping.
Aim: To compare UV absorption-based mass mapping at four UV wavelengths and understand variations across wavelengths and any assumptions necessary for accurate Hb quantification.
Approach: Whole blood smears are imaged with a multispectral UV microscopy system, and the RBCs’ dry masses are computed. This approach is compared to quantitative phase imaging-based mass mapping using data from an interferometric UV imaging system.
Results: Consistent Hb mass and mean corpuscular Hb values are obtained at all wavelengths, with the precision of the single-cell mass measurements being nearly identical at 220, 260, and 280 nm but slightly lower at 300 nm.
Conclusions: A full hematological analysis (including white blood cell identification and characterization, and Hb quantification) may be achieved using a single UV illumination wavelength, thereby improving the speed and cost-effectiveness.
Prostate cancer is the most common non-cutaneous cancer in men. Moreover, identifying the most effective treatment strategy for an individual with prostate cancer strongly depends on the Gleason score (Grade group) of the disease. However, this task continues to be a significant clinical challenge in a subset of patients. Currently, the gold standard for grading prostate cancer is the Pathologist’s visual assessment of hematoxylin-and-eosin-stained histological sections, and designation of a Gleason score (Grade group) based on the top two most common Gleason grades. However, this process is subjective and thus prone to error and high variability, especially amongst non-Urologic Pathologists or those who are not aware of recent modifications to the grading system. In addition, variations in protocols for staining, could make quantitative analysis of stained tissues challenging in some cases. Therefore, there is a significant clinical need to develop additional complementary quantitative methods that can provide robust, objective, reproducible, and accurate information of the aggressiveness and grade of prostate cancer. Here we address this issue by imaging unstained tissue sections using multi-spectral deep-UV microscopy. This method enables us to obtain valuable quantitative insight into the aggressiveness and grade of the disease due to its subcellular spatial resolution and high sensitivity to many endogenous biomolecules, including nucleic acid and proteins. The approach uses a simple, fast and cost effective wide-field imaging configuration that is well-suited for pathology applications. Spectral signatures form wavelengths ranging from 220 nm to 450 nm are analyzed using a geometrical representation of principal component analysis. Our results reveal distinct morphological and molecular alterations in tissue as they progress from benign to cancerous, and as they become more aggressive (higher grade). In this research project, we delineated the design of the multispectral, deep UV microscope and described our quantitative and qualitative image analysis. Our results show that multispectral deep UV microscopy provides a quantitative measure to differentiate between prostate cancer patients with varying grades of cancer.
Neutropenia is a condition where the hematopoietic system has a suppressed production of neutrophils, a type of white blood cell that is critical for fighting infections. This condition affects half to nearly eighty percent of cancer patients receiving chemotherapy, depending on the type of malignancy. Neutropenia can also be congenital or acquired from autoimmune disorders or nutritional deficits, in addition to cancer. Neutropenia, formally defined as <1500 neutrophils/µL in peripheral blood, puts patients at an increased risk of life-threatening infections. Thus, it is critical to constantly monitor neutrophil counts for many patients. Hematological analysis of neutropenia is performed by highly trained personnel at certified laboratories via complete blood count (CBC) and visual inspection which require complex, time-consuming, and expensive sample preparation and instrumentation. Thus, an easy-to-use, label- and reagent-free, and inexpensive hematology analysis device is highly desirable to circumvent these limitations and allow point-of-care disease monitoring and diagnosis. In this work, we demonstrate the application of deep-ultraviolet (UV) microscopy as label-free method for rapid and facile neutropenia detection. Our approach provides key hematological information and enables quantitative assessment of live blood cells based on their molecular and structural signatures in minutes. Here we show the ability of deep-UV microscopy to clearly identify patients with moderate and severe neutropenia based on an automated blood smear analysis. We also demonstrate a pseudo-colorization scheme which recapitulates the gold-standard Giemsa stains and allows visual inspection and enumeration of various blood cells types. This work has significant implications for developing a simple and low-cost point-of-care device that can ultimately improve the care and quality of life of many neutropenia and cancer patients.
Deep ultraviolet microscopy (UV) enables high-resolution, label-free imaging of biological samples and yields diagnostically relevant quantitative molecular and structural information. We recently demonstrated that deep UV microscopy can serve as a simple, fast, and low-cost alternative to modern hematology analyzers that assess variations in the morphological, molecular, and cytogenetic properties of blood cells to monitor and diagnose blood disorders. We also introduced a pseudocolorization scheme that uses multi-spectral UV images (acquired at three different wavelengths) to generate images whose colors accurately recapitulate those produced by conventional Giemsa staining, and can thus be used for visual hematological analysis. Here, we present a deeplearning framework to virtually stain single-channel UV images acquired at 260 nm, providing a factor of three improvement in imaging speed without sacrificing accuracy. We train a generative adversarial network (GAN) using image pairs consisting of single-channel UV images of blood smears and their corresponding pseudocolorized images to generate realistic, virtually stained images. The virtual stained images are post-processed to improve contrast and yield consistent background colors. We quantify the performance of our framework in terms of the structural similarity index (SSIM) for each color channel. Our virtual staining scheme is the first step towards a completely automated hematological analysis pipeline that includes segmentation and classification of different blood cell types to compute metrics of diagnostic value. Our method eliminates the need to acquire images at different wavelengths and could potentially lead to the development of a faster and more compact label-free, point-of-care hematology analyzer.
Correctly diagnosing and staging prostate cancer continues to be a significant clinical challenge. Currently, the standard of care consists of a pathologist’s visual assessment of hematoxylin-and-eosin-stained (HE) histological sections, and designation of a Gleason score based on the top two most common patterns. However, this process is subjective and thus prone to error. Further, lack of standard protocols for staining, makes quantitative analysis of stained tissues difficult. Therefore, there is a significant need to develop new quantitative methods that can provide robust, objective, and accurate information of the aggressiveness and stage of prostate cancer. In this work, we seek to address this challenge using multi-spectral deep-UV microscopy of unstained tissue sections. This method yields valuable insight into the aggressiveness and stage of the disease due to its subcellular spatial resolution and high sensitivity to many endogenous biomolecules, including nucleic acid and proteins. In our approach we use a simple and cost effective wide-field imaging configuration with sequential illumination at multiple wavelengths ranging from 220 nm to 450 nm. Spectral signatures are analyzed in conjunction with the morphology using a geometrical representation of principal component analysis and principles of mathematical morphology. Our results reveal distinct morphological and molecular alterations in the tissue as cancer becomes more aggressive. In this presentation we will detail the design of the multispectral, deep UV microscope; describe our quantitative image analysis; and show preliminary results.
Clinical hematological practice often relies on analysis of the peripheral blood based on microscopic evaluation of blood smears and complete blood count (CBC). Accurate examination of blood cell abnormalities using such methods necessitates complex, time-consuming, and expensive sample preparation as well as instruments which require a many reagents and intensive maintenance. Further, hematology analysis is performed at healthcare centers by trained personnel which significantly limits monitoring frequency for patients with severe conditions and can compromise the treatment outcome. Therefore, a portable, easy-to-use, and inexpensive hematology analysis device can potentially improve quality of life for patients with blood diseases and allow point-of-care monitoring and diagnosis. In this work, we demonstrate label-free blood cell assessment based on deep-ultraviolet (UV) microscopy. Our approach provides quantitative endogenous molecular information from live cells and enables assessment and differentiation of blood cell types based on their molecular and structural signatures. We show the ability of our method by performing classification of polymorphonuclear leukocyte (PMNL) subtypes based on features extracted from deep-UV images. In addition, we demonstrate a pseudo-colorization scheme which accurately mimics the colors produced by standard Giemsa staining and enable visual examination of blood smears. The results of our work paves the way for development of a low-cost and easy-to-use hematological analysis device that can be used for point-of-care applications.
The ultraviolet region of the spectrum offers unique capabilities for label-free molecular imaging of biological samples by providing highly-specific, quantitative information of many important endogenous biomolecules. However, the application of UV spectral imaging to biomedicine has been limited. To this end, we have recently introduced ultraviolet hyperspectral interferometric (UHI) microscopy, which applies interferometry to overcome significant challenges associated with UV spectroscopy when applied to molecular imaging. Here we present an alternative approach for UV multi-spectral microscopy which enables faster wide-field imaging at the expense of fewer spectral data points. Instead of line-scanning to recover high-resolution spectral information with an imaging spectrometer, we detect a wide field-of-view using a UV-sensitive camera and recover the spectral information using several (>5) UV-filters. Moreover, rather than using interferometry to recover the phase to correct for chromatic aberrations, we leverage the chromatic aberrations themselves to obtain a stack of through-focus intensity images (at various wavelengths) and then apply an iterative solution of the Transport of Intensity (TIE) equation to recover the phase and produce in-focus images at all wavelengths without moving the sample or objective. This configuration greatly simplifies the instrumentation, reducing its footprint and making it less expensive, while enabling fast, wide area imaging with better photon efficiency. We assess the capabilities of this technique through a series of simulations and experiments on red blood cells, which show good quantitative agreement with UHI and tabulated hemoglobin absorption properties. Potential biomedical applications are also discussed.
Polymorphonuclear leukocyte (PMNL) count is employed as an immune status indicator for diagnosis of numerous medical conditions. Currently, assessment of PMNLs (i.e., neutrophils, eosinophils, basophils) is a part of complete blood count (CBC) that is performed by trained technicians at healthcare centers and involves sample preparation which is costly and time consuming, both of which limits monitoring frequency. A prominent application of PMNL counting is in identification of neutropenia—a condition describing an abnormally low number of neutrophils in the bloodstream (<1500/μL)—common among cancer patients receiving chemotherapy. Susceptibility to infections in neutropenia patients puts them at an increased risk for medical emergencies, and thus requires constant monitoring of their neutrophil count. Therefore, a portable and easy-to-use, in-home device can potentially circumvent these requirements and enable neutropenia diagnosis. In this work, we demonstrate the feasibility of accurately identifying PMNL subtypes using deep-ultraviolet (UV) microscopy as label-free molecular imaging technique. Our approach benefits from quantitative endogenous molecular information provided by deep-UV imaging, to enable assessment of different cell types based on their molecular and structural signatures. We show the ability of our system to measure neutrophil count in samples containing a mixture of PMNL subtypes as well as whole blood samples by extracting various features from deep-UV images and performing classification to obtain cell count for each subtype. Finally, we will discuss the potential of this technology to empower cancer patients and improve their quality of life via a simple and relatively inexpensive device for point-of-care neutropenia assessment.
Early detection of the most prevalent oral disease worldwide, i.e., dental caries, still remains as one of the major challenges in dentistry. The current dental standard of care relies on caries detection methods, such as visual inspection and x-ray radiography, which lack the sufficient specificity and sensitivity to detect caries at early stages of formation when they can be healed. We report on the feasibility of early caries detection in a clinically and commercially viable thermophotonic imaging system. The system incorporates intensity-modulated laser light along with a low-cost long-wavelength infrared (LWIR; 8 to 14 μm) camera, providing diagnostic contrast based on the enhanced light absorption of early caries. The LWIR camera is highly suitable for integration into clinical platforms because of its low weight and cost. In addition, through theoretical modeling, we show that LWIR detection enhances the diagnostic contrast due to the minimal LWIR transmittance of enamel and suppression of the masking effect of the direct thermal Planck emission. Diagnostic performance of the system and its detection threshold are experimentally evaluated by monitoring the inception and progression of artificially induced occlusal and smooth surface caries. The results are suggestive of the suitability of the developed LWIR system for detecting early dental caries.
Dental caries is one of the most prevailing oral diseases which can be healed if detected in early stages of formation. In this paper, we present a clinically and commercially viable thermophotonic imaging technology for detection of early enamel caries using an inexpensive long-wavelength infrared (LWIR) camera. The efficacy of the system is verified through theoretical simulations as well as experiments carried out on extracted teeth with natural and artificially-induced caries.
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