Real-time semantic segmentation and anomaly detection of functional images for cell therapy manufacturing.

BACKGROUND AIMS: Cell therapy is a promising treatment method that uses living cells to address a variety of diseases and conditions, including cardiovascular diseases, neurologic disorders and certain cancers. As interest in cell therapy grows, there is a need to shift to a more efficient, scalable and automated manufacturing process that can produce high-quality products at a lower cost. METHODS: One way to achieve this is using non-invasive imaging and real-time image analysis techniques to monitor and control the manufacturing process. This work presents a machine learning-based image analysis pipeline that includes semantic segmentation and anomaly detection capabilities. RESULTS/CONCLUSIONS: This method can be easily implemented even when given a limited dataset of annotated images, is able to segment cells and debris and can identify anomalies such as contamination or hardware failure.

Label-free hematology analysis using deep-ultraviolet microscopy.

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.

Analysis of the Asymmetry between Both Eyes in Early Diagnosis of Glaucoma Combining Features Extracted from Retinal Images and OCTs into Classification Models.

This study aims to analyze the asymmetry between both eyes of the same patient for the early diagnosis of glaucoma. Two imaging modalities, retinal fundus images and optical coherence tomographies (OCTs), have been considered in order to compare their different capabilities for glaucoma detection. From retinal fundus images, the difference between cup/disc ratio and the width of the optic rim has been extracted. Analogously, the thickness of the retinal nerve fiber layer has been measured in spectral-domain optical coherence tomographies. These measurements have been considered as asymmetry characteristics between eyes in the modeling of decision trees and support vector machines for the classification of healthy and glaucoma patients. The main contribution of this work is indeed the use of different classification models with both imaging modalities to jointly exploit the strengths of each of these modalities for the same diagnostic purpose based on the asymmetry characteristics between the eyes of the patient. The results show that the optimized classification models provide better performance with OCT asymmetry features between both eyes (sensitivity 80.9%, specificity 88.2%, precision 66.7%, accuracy 86.5%) than with those extracted from retinographies, although a linear relationship has been found between certain asymmetry features extracted from both imaging modalities. Therefore, the resulting performance of the models based on asymmetry features proves their ability to differentiate healthy from glaucoma patients using those metrics. Models trained from fundus characteristics are a useful option as a glaucoma screening method in the healthy population, although with lower performance than those trained from the thickness of the peripapillary retinal nerve fiber layer. In both imaging modalities, the asymmetry of morphological characteristics can be used as a glaucoma indicator, as detailed in this work.

Optimization of a flexible fiber-optic probe for epi-mode quantitative phase imaging.

Quantitative oblique back-illumination microscopy (qOBM) is an emerging label-free optical imaging technology that enables 3D, tomographic quantitative phase imaging (QPI) with epi-illumination in thick scattering samples. In this work, we present a robust optimization of a flexible, fiber-optic-based qOBM system. Our approach enables in silico optimization of the phase signal-to-noise-ratio over a wide parameter space and obviates the need for tedious experimental optimization which could easily miss optimal conditions. Experimental validations of the simulations are also presented and sensitivity limits for the probe are assessed. The optimized probe is light-weight (∼40g) and compact (8mm in diameter) and achieves a 2µm lateral resolution, 6µm axial resolution, and a 300µm field of view, with near video-rate operation (10Hz, limited by the camera). The phase sensitivity is <20nm for a single qOBM acquisition (at 10Hz) and a lower limit of ∼3 nm via multi-frame averaging. Finally, to demonstrate the utility of the optimized probe, we image (1) thick, fixed rat brain samples from a 9L gliosarcoma tumor model and (2) freshly excised human brain tissues from neurosurgery. Acquired qOBM images using the flexible fiber-optic probe are in excellent agreement with those from a free-space qOBM system (both in-situ), as well as with gold-standard histopathology slices (after tissue processing).

Label-free deep-UV microscopy detection and grading of neutropenia using a passive microfluidic device.

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.

Ultraviolet hyperspectral microscopy using chromatic-aberration-based iterative phase recovery.

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.

Noninvasive white blood cell quantification in umbilical cord blood collection bags with quantitative oblique back-illumination microscopy.

BACKGROUND: Umbilical cord blood has become an important source of hematopoietic stem and progenitor cells for therapeutic applications. However, cord blood banking (CBB) grapples with issues related to economic viability, partially due to high discard rates of cord blood units (CBUs) that lack sufficient total nucleated cells for storage or therapeutic use. Currently, there are no methods available to assess the likelihood of CBUs meeting storage criteria noninvasively at the collection site, which would improve CBB efficiency and economic viability. MATERIALS AND METHODS: To overcome this limitation, we apply a novel label-free optical imaging method, called quantitative oblique back-illumination microscopy (qOBM), which yields tomographic phase and absorption contrast to image blood inside collection bags. An automated segmentation algorithm was developed to count white blood cells and red blood cells (RBCs) and assess hematocrit. Fifteen CBUs were measured. RESULTS: qOBM clearly differentiates between RBCs and nucleated cells. The cell-counting analysis shows an average error of 13% compared to hematology analysis, with a near-perfect, one-to-one relationship (slope = 0.94) and strong correlation coefficient (r = 0.86). Preliminary results to assess hematocrit also show excellent agreement with expected values. Acquisition times to image a statistically significant number of cells per CBU were approximately 1 minute. CONCLUSION: qOBM exhibits robust performance for quantifying blood inside collection bags. Because the approach is automated and fast, it can potentially quantify CBUs within minutes of collection, without breaching the CBUs' sterile environment. qOBM can reduce costs in CBB by avoiding processing expenses of CBUs that ultimately do not meet storage criteria.

Coherently broadened, high-repetition-rate laser for stimulated Raman scattering-spectroscopic optical coherence tomography.

We present a novel light source specifically tailored for stimulated Raman scattering-spectroscopic optical coherence tomography (SRS-SOCT), which is, to the best of our knowledge, a novel molecular imaging method that combines the molecular sensitivity of SRS with the spatial and spectral multiplexing capabilities of SOCT. The novel laser consists of an 8 W, 450 fs Yb:KGW oscillator, with a repetition rate of 40 MHz, which delivers the Stokes beam for SRS-SOCT and also pumps and amplifies an optical parametric oscillator (OPO). The output of the amplified OPO is then frequency doubled and coherently broadened using a custom-made tapered fiber that generates bandwidth pulses >40 nm, compressible to <50 fs, with the average power over 150 mW, near the shot-noise limit above 250 kHz. The broadened and compressed pulse simultaneously serves as the pump beam and SOCT light source for SRS-SOCT. This light source is assessed for SRS-SOCT, and its implications for other imaging methods are discussed.

A genetically engineered H5 protein expressed in insect cells confers protection against different clades of H5N1 highly pathogenic avian influenza viruses in chickens.

The evolution of highly pathogenic H5N1 avian influenza viruses (HPAI-H5N1) has resulted in the appearance of a number of diverse groups of HPAI-H5N1 based on the presence of genetically similar clusters of their haemagglutinin sequences (clades). An H5 antigen encoded by a recombinant baculovirus and expressed in insect cells was used for oil-emulsion-based vaccine prototypes. In several experiments, vaccination was performed at 10 days of age, followed by challenge infection on day 21 post vaccination (PV) with HPAI-H5N1 clades 2.2, 2.2.1, and 2.3.2. A further challenge infection with HPAI-H5N1 clade 2.2.1 was performed at day 42 PV. High haemagglutination inhibition titres were observed for the recH5 vaccine antigen, and lower haemagglutination inhibition titres for the challenge virus antigens. Nevertheless, the rate of protection from mortality and clinical signs was 100% when challenged at 21 days PV and 42 days PV, indicating protection over the entire broiler chicken rearing period without a second vaccination. The unvaccinated control chickens mostly died between two and five days after challenge infection. A low level of viral RNA was detected by reverse transcription followed by a quantitative polymerase chain reaction in a limited number of birds for a short period after challenge infection, indicating a limited spread of HPAI-H5N1 at flock level. Furthermore, it was observed that the vaccine can be used in a differentiation infected from vaccinated animals (DIVA) approach, based on the detection of nucleoprotein antibodies in vaccinated/challenged chickens. The vaccine fulfilled all expectations of an inactivated vaccine after one vaccination against challenge with different clades of H5N1-HPAI and is suitable for a DIVA approach.

Label-Free Imaging of Female Genital Tract Melanocytic Lesions With Pump-Probe Microscopy: A Promising Diagnostic Tool.

OBJECTIVES: Melanomas of the female genital tract present a unique clinical challenge. Not only are these lesions in an anatomically sensitive area, but also they tend to be multifocal and have high recurrence rates. Furthermore, several benign melanocytic proliferations resemble early-stage melanoma clinically and/or histopathologically. Thus, there is a significant need for additional tools that can help correctly diagnose and stage these lesions. Here, we quantitatively and nondestructively analyze the chemical composition of melanin in excised pigmented lesions of the female genital tract using pump-probe microscopy, a high-resolution optical imaging technique that is sensitive to many biochemical properties of melanin. MATERIALS AND METHODS: Thirty-one thin (~5 µm) tissue sections previously excised from female genital tract melanocytic lesions were imaged with pump-probe microscopy and analyzed. RESULTS: We find significant quantitative differences in melanin type and structure between melanoma and nonmalignant melanocytic proliferations. Our analysis also suggests a link between the molecular signatures of melanins and lesion-specific genetic mutations. Finally, significant differences are found between metastatic and nonmetastatic melanomas. The limitations of this work include the fact that molecular information is restricted to melanin pigment and the sample size is relatively small. CONCLUSIONS: Pump-probe microscopy provides unique information regarding the biochemical composition of genital tract melanocytic lesions, which can be used to improve the diagnosis and staging of vulvar melanomas.

Dispersion-based stimulated Raman scattering spectroscopy, holography, and optical coherence tomography.

Stimulated Raman scattering (SRS) enables fast, high resolution imaging of chemical constituents important to biological structures and functional processes, both in a label-free manner and using exogenous biomarkers. While this technology has shown remarkable potential, it is currently limited to point scanning and can only probe a few Raman bands at a time (most often, only one). In this work we take a fundamentally different approach to detecting the small nonlinear signals based on dispersion effects that accompany the loss/gain processes in SRS. In this proof of concept, we demonstrate that the dispersive measurements are more robust to noise compared to amplitude-based measurements, which then permit spectral or spatial multiplexing (potentially both, simultaneously). Finally, we illustrate how this method may enable different strategies for biochemical imaging using phase microscopy and optical coherence tomography.

Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR.

Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial strains. Twenty-two of the strains represented all of the presently available 13 phenotypic variants of G. anatis originating from different geographical locations. Nine strains represented each of the additional six Gallibacterium species and 21 strains represented other poultry associated bacterial species of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae. Regarding specificity none of non-G. anatis strains tested positive with the proposed assay. To test and compare the qPCR method's ability to detect G. anatis from field samples, the sensitivity was compared to a previously published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10(8) colony forming units (CFU)/ml to as low as 10(0) CFU/ml copies. The proposed qPCR method is thus highly specific, sensitive and reproducible. In conclusion, we have developed a qPCR method that allows species-specific identification of G. anatis.

Femtosecond pulse shaping enables detection of optical Kerr-effect (OKE) dynamics for molecular imaging.

We apply femtosecond pulse shaping to generate optical pulse trains that directly access a material's nonlinear refractive index (n2) and can thus determine time-resolved optical Kerr-effect (OKE) dynamics. Two types of static pulse trains are discussed: The first uses two identical fields delayed in time, plus a pump field at a different wavelength. Time-resolved OKE dynamics are retrieved by monitoring the phase of the interference pattern produced by the two identical fields in the Fourier-domain (FD) as a function of pump-probe-time-delay (where the probe is one of the two identical fields). The second pulse train uses three fields with equal time delays, but with the center field phase shifted by π/2. In this pulse scheme, changes on a sample's nonlinear refractive index produce a new frequency in the FD signal, which in turn yields background-free intensity changes in the conjugate (time) domain and provides superior signal-to-noise ratios. The demonstrated sensitivity improvements enable, for the first time to our knowledge, molecular imaging based on OKE dynamics.

Near-infrared excited state dynamics of melanins: the effects of iron content, photo-damage, chemical oxidation, and aggregate size.

Ultrafast pump-probe measurements can discriminate the two forms of melanin found in biological tissue (eumelanin and pheomelanin), which may be useful for diagnosing and grading melanoma. However, recent work has shown that bound iron content changes eumelanin's pump-probe response, making it more similar to that of pheomelanin. Here we record the pump-probe response of these melanins at a wider range of wavelengths than previous work and show that with shorter pump wavelengths the response crosses over from being dominated by ground-state bleaching to being dominated by excited-state absorption. The crossover wavelength is different for each type of melanin. In our analysis, we found that the mechanism by which iron modifies eumelanin's pump-probe response cannot be attributed to Raman resonances or differences in melanin aggregation and is more likely caused by iron acting to broaden the unit spectra of individual chromophores in the heterogeneous melanin aggregate. We analyze the dependence on optical intensity, finding that iron-loaded eumelanin undergoes irreversible changes to the pump-probe response after intense laser exposure. Simultaneously acquired fluorescence data suggest that the previously reported "activation" of eumelanin fluorescence may be caused in part by the dissociation of metal ions or the selective degradation of iron-containing melanin.

Non-Invasive Label-free Analysis Pipeline for In Situ Characterization of Differentiation in 3D Brain Organoid Models.

Brain organoids provide a unique opportunity to model organ development in a system similar to human organogenesis in vivo. Brain organoids thus hold great promise for drug screening and disease modeling. Conventional approaches to organoid characterization predominantly rely on molecular analysis methods, which are expensive, time-consuming, labor-intensive, and involve the destruction of the valuable 3D architecture of the organoids. This reliance on end-point assays makes it challenging to assess cellular and subcellular events occurring during organoid development in their 3D context. As a result, the long developmental processes are not monitored nor assessed. The ability to perform non-invasive assays is critical for longitudinally assessing features of organoid development during culture. In this paper, we demonstrate a label-free high-content imaging approach for observing changes in organoid morphology and structural changes occurring at the cellular and subcellular level. Enabled by microfluidic-based culture of 3D cell systems and a novel 3D quantitative phase imaging method, we demonstrate the ability to perform non-destructive high-resolution imaging of the organoid. The highlighted results demonstrated in this paper provide a new approach to performing live, non-destructive monitoring of organoid systems during culture.

Label-free functional analysis of root-associated microbes with dynamic quantitative oblique back-illumination microscopy.

The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable agricultural practices. Nitrogen-fixing bacteria, known as diazotrophs, offer a potential solution by converting atmospheric nitrogen into bioavailable forms, reducing the reliance on synthetic fertilizers. However, a deeper understanding of their interactions with plants and other microbes is needed. In this study, we introduce a recently developed label-free 3D quantitative phase imaging technology called dynamic quantitative oblique back-illumination microscopy (DqOBM) to assess the functional dynamic activity of diazotrophs in vitro and in situ. Our experiments involved three different diazotrophs (Sinorhizobium meliloti, Azotobacter vinelandii, and Rahnella aquatilis) cultured on media with amendments of carbon and nitrogen sources. Over 5 days, we observed increased dynamics in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize microbial dynamics within the root cap and elongation zone of Arabidopsis thaliana primary roots. This allowed us to identify distinct areas of microbial infiltration in plant roots without the need for fluorescent markers. Our findings demonstrate that DqOBM can effectively characterize microbial dynamics and provide insights into plant-microbe interactions in situ, offering a valuable tool for advancing our understanding of sustainable agriculture.

AI algorithm combined with RNA editing-based blood biomarkers to discriminate bipolar from major depressive disorders in an external validation multicentric cohort.

Bipolar disorder (BD) is a leading cause of disability worldwide, as it can lead to cognitive and functional impairment and premature mortality. The first episode of BD is usually a depressive episode and is often misdiagnosed as major depressive disorder (MDD). Growing evidence indicates that peripheral immune activation and inflammation are involved in the pathophysiology of BD and MDD. Recently, by developing a panel of RNA editing-based blood biomarkers able to discriminate MDD from depressive BD, we have provided clinicians a new tool to reduce the misdiagnosis delay observed in patients suffering from BD. The present study aimed at validating the diagnostic value of this panel in an external independent multicentric Switzerland-based cohort of 143 patients suffering from moderate to major depression. The RNA-editing based blood biomarker (BMK) algorithm developped allowed to accurately discriminate MDD from depressive BD in an external cohort, with high accuracy, sensitivity and specificity values (82.5 %, 86.4 % and 80.8 %, respectively). These findings further confirm the important role of RNA editing in the physiopathology of mental disorders and emphasize the possible clinical usefulness of the biomarker panel for optimization treatment delay in patients suffering from BD.

Pump-probe nonlinear phase dispersion spectroscopy.

Pump-probe microscopy is an imaging technique that delivers molecular contrast of pigmented samples. Here, we introduce pump-probe nonlinear phase dispersion spectroscopy (PP-NLDS), a method that leverages pump-probe microscopy and spectral-domain interferometry to ascertain information from dispersive and resonant nonlinear effects. PP-NLDS extends the information content to four dimensions (phase, amplitude, wavelength, and pump-probe time-delay) that yield unique insight into a wider range of nonlinear interactions compared to conventional methods. This results in the ability to provide highly specific molecular contrast of pigmented and non-pigmented samples. A theoretical framework is described, and experimental results and simulations illustrate the potential of this method. Implications for biomedical imaging are discussed.

Partially coherent broadband 3D optical transfer functions with arbitrary temporal and angular power spectra.

Optical diffraction tomography is a powerful technique to produce 3D volumetric images of biological samples using contrast produced by variations in the index of refraction in an unlabeled specimen. While this is typically performed with coherent illumination from a variety of angles, interest has grown in partially coherent methods due to the simplicity of the illumination and the computation-free axial sectioning provided by the coherence window of the source. However, such methods rely on the symmetry or discretization of a source to facilitate quantitative analysis and are unable to efficiently handle arbitrary illumination that may vary asymmetrically in angle and continuously in the spectrum, such as diffusely scattered or thermal sources. A general broadband theory may expand the scope of illumination methods available for quantitative analysis, as partially coherent sources are commonly available and may benefit from the effects of spatial and temporal incoherence. In this work, we investigate partially coherent tomographic phase microscopy from arbitrary sources regardless of angular distribution and spectrum by unifying the effects of spatial and temporal coherence into a single formulation. This approach further yields a method for efficient computation of the overall systems' optical transfer function, which scales with O(n 3), down from O(mn 4) for existing convolutional methods, where n 3 is the number of spatial voxels in 3D space and m is the number of discrete wavelengths in the illumination spectrum. This work has important implications for enabling partially coherent 3D quantitative phase microscopy and refractive index tomography in virtually any transmission or epi-illumination microscope.