RESUMEN
An aberration correction method is introduced for 3D phase deconvolution microscopy. Our technique capitalizes on multiple illumination patterns to iteratively extract Fourier space aberrations, utilizing the overlapping information inherent in these patterns. By refining the point spread function based on the retrieved aberration data, we significantly improve the precision of refractive index deconvolution. We validate the effectiveness of our method on both synthetic and biological three-dimensional samples, achieving notable enhancements in resolution and measurement accuracy. The method's reliability in aberration retrieval is further confirmed through controlled experiments with intentionally induced spherical aberrations, underscoring its potential for wide-ranging applications in microscopy and biomedicine.
RESUMEN
Lipid droplets (LDs), once considered mere storage depots for lipids, have gained recognition for their intricate roles in cellular processes, including metabolism, membrane trafficking, and disease states like obesity and cancer. This review explores label-free imaging techniques' applications in LD research. We discuss holotomography and vibrational spectroscopic microscopy, emphasizing their potential for studying LDs without molecular labels, and we highlight the growing integration of artificial intelligence. Clinical applications in disease diagnosis and therapy are also considered.
Asunto(s)
Inteligencia Artificial , Gotas Lipídicas , Gotas Lipídicas/metabolismo , Microscopía , Metabolismo de los LípidosRESUMEN
A critical requirement for studying cell mechanics is three-dimensional assessment of cellular shapes and forces with high spatiotemporal resolution. Traction force microscopy with fluorescence imaging enables the measurement of cellular forces, but it is limited by photobleaching and a slow acquisition speed. Here, we present refractive-index traction force microscopy (RI-TFM), which simultaneously quantifies the volumetric morphology and traction force of cells using a high-speed illumination scheme with 0.5-Hz temporal resolution. Without labelling, our method enables quantitative analyses of dry-mass distributions and shear (in-plane) and normal (out-of-plane) tractions of single cells on the extracellular matrix. When combined with a constrained total variation-based deconvolution algorithm, it provides 0.55-Pa shear and 1.59-Pa normal traction sensitivity for a 1-kPa hydrogel substrate. We demonstrate its utility by assessing the effects of compromised intracellular stress and capturing the rapid dynamics of cellular junction formation in the spatiotemporal changes in non-planar traction components.
Asunto(s)
Fenómenos Mecánicos , Tracción , Microscopía de Fuerza Atómica/métodos , AlgoritmosRESUMEN
The precise, quantitative evaluation of intracellular organelles in three-dimensional (3D) imaging data poses a significant challenge due to the inherent constraints of traditional microscopy techniques, the requirements of the use of exogenous labeling agents, and existing computational methods. To counter these challenges, we present a hybrid machine-learning framework exploiting correlative imaging of 3D quantitative phase imaging with 3D fluorescence imaging of labeled cells. The algorithm, which synergistically integrates a random-forest classifier with a deep neural network, is trained using the correlative imaging data set, and the trained network is then applied to 3D quantitative phase imaging of cell data. We applied this method to live budding yeast cells. The results revealed precise segmentation of vacuoles inside individual yeast cells, and also provided quantitative evaluations of biophysical parameters, including volumes, concentration, and dry masses of automatically segmented vacuoles.
RESUMEN
Quantitative phase imaging, integrated with artificial intelligence, allows for the rapid and label-free investigation of the physiology and pathology of biological systems. This review presents the principles of various two-dimensional and three-dimensional label-free phase imaging techniques that exploit refractive index as an intrinsic optical imaging contrast. In particular, we discuss artificial intelligence-based analysis methodologies for biomedical studies including image enhancement, segmentation of cellular or subcellular structures, classification of types of biological samples and image translation to furnish subcellular and histochemical information from label-free phase images. We also discuss the advantages and challenges of artificial intelligence-enabled quantitative phase imaging analyses, summarize recent notable applications in the life sciences, and cover the potential of this field for basic and industrial research in the life sciences.
Asunto(s)
Inteligencia Artificial , Disciplinas de las Ciencias Biológicas , Aumento de la Imagen , Imagenología Tridimensional/métodosRESUMEN
Many important microscopy samples, such as liquid crystals, biological tissue, or starches, are birefringent in nature. They scatter light differently depending on the polarization of the light and the orientation of the molecules. The complete characterization of a birefringent sample is a challenging task because its 3 × 3 dielectric tensor must be reconstructed at every three-dimensional position. Moreover, obtaining a birefringent tomogram is more arduous for thick samples, where multiple light scattering should also be considered. In this study, we developed a new dielectric tensor tomography algorithm that enables full characterization of highly scattering birefringent samples by solving the vectoral inverse scattering problem while accounting for multiple light scattering. We proposed a discrete image-processing theory to compute the error backpropagation of vectorially diffracting light. Finally, our theory was experimentally demonstrated using both synthetic and biologically birefringent samples.
RESUMEN
An ideal holographic camera measures the amplitude and phase of the light field so that the focus can be numerically adjusted after the acquisition, and depth information about an imaged object can be deduced. The performance of holographic cameras based on reference-assisted holography is significantly limited owing to their vulnerability to vibration and complex optical configurations. Non-interferometric holographic cameras can resolve these issues. However, existing methods require constraints on an object or measurement of multiple-intensity images. In this paper, we present a holographic image sensor that reconstructs the complex amplitude of scattered light from a single-intensity image using reciprocal diffractive imaging. We experimentally demonstrate holographic imaging of three-dimensional diffusive objects and suggest its potential applications by imaging a variety of samples under both static and dynamic conditions.
RESUMEN
One major challenge associated with lung cancer organoids (LCOs) is their predominant derivation from surgical specimens of patients with early-stage lung cancer. However, patients with advanced lung cancer, who are in need of chemotherapy, often cannot undergo surgery. Therefore, there is an urgent need to successfully generate LCOs from biopsy specimens. Conventional lung biopsy techniques, such as transthoracic needle biopsy and forceps biopsy, only yield small amounts of lung tissue, resulting in a low success rate for culturing LCOs from biopsy samples. Furthermore, potential complications, like bleeding and pneumothorax, make it difficult to obtain sufficient tissue. Another critical issue is the overgrowth of normal lung cells in later passages of LCO culture, and the optimal culture conditions for LCOs are yet to be determined. To address these limitations, we attempted to create LCOs from cryobiopsy specimens obtained from patients with lung cancer (n = 113). Overall, the initial success rate of establishing LCOs from cryobiopsy samples was 40.7% (n = 46). Transbronchial cryobiopsy enables the retrieval of significantly larger amounts of lung tissue than bronchoscopic forceps biopsy. Additionally, cryobiopsy can be employed for peripheral lesions, and it is aided via radial endobronchial ultrasonography. This study significantly improved the success rate of LCO culture and demonstrated that the LCOs retained characteristics that resembled the primary tumors. Single-cell RNA sequencing confirmed high cancer cell purity in early passages of LCOs derived from patients with advanced lung cancer. Furthermore, the three-dimensional structure and intracellular components of LCOs were characterized using three-dimensional holotomography. Finally, drug screening was performed using a specialized micropillar culture system with cryobiopsy-derived LCOs. LCOs derived from cryobiopsy specimens offer a promising solution to the critical limitations of conventional LCOs. Cryobiopsy can be applied to patients with lung cancer at all stages, including those with peripheral lesions, and can provide sufficient cells for LCO generation. Therefore, we anticipate that cryobiopsy will serve as a breakthrough strategy for the clinical application of LCOs in all stages of lung cancer.
Asunto(s)
Criocirugía , Neoplasias Pulmonares , Humanos , Broncoscopía/métodos , Criocirugía/métodos , Neoplasias Pulmonares/patología , Pulmón/patología , Organoides/patologíaRESUMEN
We developed a machine learning algorithm (MLA) that can classify human thyroid cell clusters by exploiting both Papanicolaou staining and intrinsic refractive index (RI) as correlative imaging contrasts and evaluated the effects of this combination on diagnostic performance. Thyroid fine-needle aspiration biopsy (FNAB) specimens were analyzed using correlative optical diffraction tomography, which can simultaneously measure both, the color brightfield of Papanicolaou staining and three-dimensional RI distribution. The MLA was designed to classify benign and malignant cell clusters using color images, RI images, or both. We included 1535 thyroid cell clusters (benign: malignancy = 1128:407) from 124 patients. Accuracies of MLA classifiers using color images, RI images, and both were 98.0%, 98.0%, and 100%, respectively. As information for classification, the nucleus size was mainly used in the color image; however, detailed morphological information of the nucleus was also used in the RI image. We demonstrate that the present MLA and correlative FNAB imaging approach has the potential for diagnosing thyroid cancer, and complementary information from color and RI images can improve the performance of the MLA.
Asunto(s)
Neoplasias de la Tiroides , Nódulo Tiroideo , Humanos , Nódulo Tiroideo/patología , Biopsia con Aguja Fina/métodos , Refractometría , Neoplasias de la Tiroides/patología , Aprendizaje Automático , Sensibilidad y EspecificidadRESUMEN
The refractive index (RI) of cells and tissues is crucial in pathophysiology as a noninvasive and quantitative imaging contrast. Although its measurements have been demonstrated using three-dimensional quantitative phase imaging methods, these methods often require bulky interferometric setups or multiple measurements, which limits the measurement sensitivity and speed. Here, we present a single-shot RI imaging method that visualizes the RI of the in-focus region of a sample. By exploiting spectral multiplexing and optical transfer function engineering, three color-coded intensity images of a sample with three optimized illuminations were simultaneously obtained in a single-shot measurement. The measured intensity images were then deconvoluted to obtain the RI image of the in-focus slice of the sample. As a proof of concept, a setup was built using Fresnel lenses and a liquid-crystal display. For validation purposes, we measured microspheres of known RI and cross-validated the results with simulated results. Various static and highly dynamic biological cells were imaged to demonstrate that the proposed method can conduct single-shot RI slice imaging of biological samples with subcellular resolution.
RESUMEN
For patients with acute ischemic stroke, histological quantification of thrombus composition provides evidence for determining appropriate treatment. However, the traditional manual segmentation of stained thrombi is laborious and inconsistent. In this study, we propose a label-free method that combines optical diffraction tomography (ODT) and deep learning (DL) to automate the histological quantification process. The DL model classifies ODT image patches with 95% accuracy, and the collective prediction generates a whole-slide map of red blood cells and fibrin. The resulting whole-slide composition displays an average error of 1.1% and does not experience staining variability, facilitating faster analysis with reduced labor. The present approach will enable rapid and quantitative evaluation of blood clot composition, expediting the preclinical research and diagnosis of cardiovascular diseases.
Asunto(s)
Isquemia Encefálica , Aprendizaje Profundo , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Trombosis , Humanos , Accidente Cerebrovascular/diagnóstico por imagen , Isquemia Encefálica/patología , Trombosis/diagnóstico por imagen , Trombosis/patología , TomografíaRESUMEN
Biomolecular condensates play a key role in organizing cellular reactions by concentrating a specific set of biomolecules. However, whether condensate formation is accompanied by an increase in the total mass concentration within condensates or by the demixing of already highly crowded intracellular components remains elusive. Here, using refractive index imaging, we quantify the mass density of several condensates, including nucleoli, heterochromatin, nuclear speckles, and stress granules. Surprisingly, the latter two condensates exhibit low densities with a total mass concentration similar to the surrounding cyto- or nucleoplasm. Low-density condensates display higher permeability to cellular protein probes. We find that RNA tunes the biomolecular density of condensates. Moreover, intracellular structures such as mitochondria heavily influence the way phase separation proceeds, impacting the localization, morphology, and growth of condensates. These findings favor a model where segregative phase separation driven by non-associative or repulsive molecular interactions together with RNA-mediated selective association of specific components can give rise to low-density condensates in the crowded cellular environment.
Asunto(s)
Núcleo Celular , ARN , ARN/metabolismo , Núcleo Celular/metabolismo , Nucléolo Celular/metabolismo , Heterocromatina/metabolismoRESUMEN
Owing to its unique penetrating power and high-resolution capability, X-ray imaging has been an irreplaceable tool since its discovery. Despite the significance, the resolution of X-ray imaging has largely been limited by the technical difficulties on X-ray lens making. Various lensless imaging methods have been proposed, but are yet relying on multiple measurements or additional constraints on measurements or samples. Here we present coherent speckle-correlation imaging (CSI) using a designed X-ray diffuser. CSI has no prerequisites for samples or measurements. Instead, from a single shot measurement, the complex sample field is retrieved based on the pseudorandomness of the speckle intensity pattern, ensured through a diffuser. We achieve a spatial resolution of 13.9 nm at 5.46 keV, beating the feature size of the diffuser used (300 nm). The high-resolution imaging capability is theoretically explained based on fundamental and practical limits. We expect the CSI to be a versatile tool for navigating the unexplored world of nanometer.
RESUMEN
Clear cell renal cell carcinoma (ccRCC) is a common histopathological subtype of renal cancer and is notorious for its poor prognosis. Its accurate diagnosis by histopathology, which relies on manual microscopic inspection of stained slides, is challenging. Here, we present a correlative approach to utilize stained images and refractive index (RI) tomography and demonstrate quantitative assessments of the structural heterogeneities of ccRCC slides obtained from human patients. Machine-learning-assisted segmentation of nuclei and cytoplasm enabled the quantification at the subcellular level. Compared to benign regions, malignant regions exhibited a considerable increase in structural heterogeneities. The results demonstrate that RI tomography provides quantitative information in synergy with stained images on the structural heterogeneities in ccRCC.
RESUMEN
Dielectric tensor tomography reconstructs the three-dimensional dielectric tensors of microscopic objects and provides information about the crystalline structure orientations and principal refractive indices. Because dielectric tensor tomography is based on transmission measurement, it suffers from the missing cone problem, which causes poor axial resolution, underestimation of the refractive index, and halo artifacts. In this study, we study the application of total variation and positive semi-definiteness regularization to three-dimensional tensor distributions. In particular, we demonstrate the reduction of artifacts when applied to dielectric tensor tomography.
RESUMEN
Three-dimensional (3D) quantitative phase imaging (QPI) enables long-term label-free tomographic imaging and quantitative analysis of live individual bacteria. However, the Brownian motion or motility of bacteria in a liquid medium produces motion artifacts during 3D measurements and hinders precise cell imaging and analysis. Meanwhile, existing cell immobilization methods produce noisy backgrounds and even alter cellular physiology. Here, we introduce a protocol that utilizes hydrogels for high-quality 3D QPI of live bacteria maintaining bacterial physiology. We demonstrate long-term high-resolution quantitative imaging and analysis of individual bacteria, including measuring the biophysical parameters of bacteria and responses to antibiotic treatments.
Asunto(s)
Hidrogeles , Imagenología Tridimensional , Imagenología Tridimensional/métodos , BacteriasRESUMEN
Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium. Understanding the biological features of various parasite forms is important for the optical diagnosis and defining pathological states, which are often constrained by the lack of ambient visualization approaches. Here, we employ a label-free tomographic technique to visualize the host red blood cell (RBC) remodeling process and quantify changes in biochemical properties arising from parasitization. Through this, we provide a quantitative body of information pertaining to the influence of host cell environment on growth, survival, and replication of P. falciparum and P. vivax in their respective host cells: mature erythrocytes and young reticulocytes. These exquisite three-dimensional measurements of infected red cells demonstrats the potential of evolving 3D imaging to advance our understanding of Plasmodium biology and host-parasite interactions.
Asunto(s)
Malaria , Plasmodium , Humanos , Malaria/parasitología , Eritrocitos/parasitología , Procesamiento de Imagen Asistido por Computador , TomografíaRESUMEN
A groundbreaking work in 1970 by Arthur Ashkin paved the way for developing various optical trapping techniques. Optical tweezers have become an established method for the manipulation of biological objects, due to their noninvasiveness and precise controllability. Recent innovations are accelerating and now enable single-cell manipulation through holographic light structuring. In this review, we provide an overview of recent advances in optical tweezer techniques for studies at the individual cell level. Our review focuses on holographic optical tweezers that utilize active spatial light modulators to noninvasively manipulate live cells. The versatility of the technology has led to valuable integrations with microscopy, microfluidics, and biotechnological techniques for various single-cell studies. We aim to recapitulate the basic principles of holographic optical tweezers, highlight trends in their biophysical applications, and discuss challenges and future prospects.
RESUMEN
The isolation of white blood cells (WBCs) from whole blood constitutes a pivotal process for immunological studies, diagnosis of hematologic disorders, and the facilitation of immunotherapy. Despite the ubiquity of density gradient centrifugation in WBC isolation, its influence on WBC functionality remains inadequately understood. This research employs holotomography to explore the effects of two distinct WBC separation techniques, namely conventional centrifugation and microfluidic separation, on the functionality of the isolated cells. We utilize three-dimensional refractive index distribution and time-lapse dynamics to analyze individual WBCs in-depth, focusing on their morphology, motility, and phagocytic capabilities. Our observations highlight that centrifugal processes negatively impact WBC motility and phagocytic capacity, whereas microfluidic separation yields a more favorable outcome in preserving WBC functionality. These findings emphasize the potential of microfluidic separation techniques as a viable alternative to traditional centrifugation for WBC isolation, potentially enabling more precise analyses in immunology research and improving the accuracy of hematologic disorder diagnoses.