Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots^® nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.

Microvascular endothelial cells are an essential part of many biological barriers, such as the blood–brain barrier (BBB) and the endothelium of the arteries and veins. A reversible opening strategy to increase the permeability of drugs across the BBB could lead to improved therapies due to enhanced drug bioavailability. Vanilloids, such as capsaicin, are known to reversibly open tight junctions of epithelial and endothelial cells. In this study, we used several in vitro assays with the murine endothelial capillary brain cells (line cEND) as a BBB model to characterize the interaction between capsaicin and endothelial tight junctions.

Ulcerative colitis (UC) is characterized by chronic inflammation of the colorectum. Histological remission has emerged as a potential future treatment goal; however, the histopathological assessment of intestinal inflammation in UC remains challenging with a multitude of available scoring systems and the need for a pathologist with expertise in inflammatory bowel disease (IBD). In previous studies, quantitative phase imaging (QPI) including digital holographic microscopy (DHM) was successfully applied as an objective method for stain-free quantification of the degree of inflammation in tissue sections. Here, we evaluated the application of DHM for the quantitative assessment of histopathological inflammation in patients with UC. In our study, endoscopically obtained colonic and rectal mucosal biopsy samples from 21 patients with UC were analyzed by capturing DHM-based QPI images that were subsequently evaluated using the subepithelial refractive index (RI). The retrieved RI data were correlated with established histological scoring systems including the Nancy index (NI) as well as with endoscopic and clinical findings. As a primary endpoint, we found a significant correlation between the DHM-based retrieved RI and the NI (R² = 0.251, p < 0.001). Furthermore, RI values correlated with the Mayo endoscopic subscore (MES; R² = 0.176, p < 0.001). An area under the receiver operating characteristics (ROC) curve of 0.820 confirms the subepithelial RI as a reliable parameter to distinguish biopsies with histologically active UC from biopsies without evidence of active disease as determined by conventional histopathological examination. An RI higher than 1.3488 was found to be the most sensitive and specific cut-off value to identify histologically active UC (sensitivity of 84% and specificity of 72%). In conclusion, our data demonstrate DHM to be a reliable tool for the quantitative assessment of mucosal inflammation in patients with UC.

SignificanceDigital holographic microscopy (DHM) is a label-free microscopy technique that provides time-resolved quantitative phase imaging (QPI) by measuring the optical path delay of light induced by transparent biological samples. DHM has been utilized for various biomedical applications, such as cancer research and sperm cell assessment, as well as for in vitro drug or toxicity testing. Its lensless version, digital lensless holographic microscopy (DLHM), is an emerging technology that offers size-reduced, lightweight, and cost-effective imaging systems. These features make DLHM applicable, for example, in limited resource laboratories, remote areas, and point-of-care applications.AimIn addition to the abovementioned advantages, in-line arrangements for DLHM also include the limitation of the twin-image presence, which can restrict accurate QPI. We therefore propose a compact lensless common-path interferometric off-axis approach that is capable of quantitative imaging of fast-moving biological specimens, such as living cells in flow.ApproachWe suggest lensless spatially multiplexed interferometric microscopy (LESSMIM) as a lens-free variant of the previously reported spatially multiplexed interferometric microscopy (SMIM) concept. LESSMIM comprises a common-path interferometric architecture that is based on a single diffraction grating to achieve digital off-axis holography. From a series of single-shot off-axis holograms, twin-image free and time-resolved QPI is achieved by commonly used methods for Fourier filtering-based reconstruction, aberration compensation, and numerical propagation.ResultsInitially, the LESSMIM concept is experimentally demonstrated by results from a resolution test chart and investigations on temporal stability. Then, the accuracy of QPI and capabilities for imaging of living adherent cell cultures is characterized. Finally, utilizing a microfluidic channel, the cytometry of suspended cells in flow is evaluated.ConclusionsLESSMIM overcomes several limitations of in-line DLHM and provides fast time-resolved QPI in a compact optical arrangement. In summary, LESSMIM represents a promising technique with potential biomedical applications for fast imaging such as in imaging flow cytometry or sperm cell analysis.

We present a single-capture multimodal bright-field (BF) and quantitative phase imaging (QPI) approach that enables the analysis of large, connected, or extended samples, such as confluent cell layers or tissue sections. The proposed imaging concept integrates a fiber-optic Mach–Zehnder interferometer-based off-axis digital holographic microscopy (DHM) with an inverted commercial optical BF microscope. Utilizing 8-bit grayscale dynamic range multiplexing, we simultaneously capture both BF images and digital holograms, which are then demultiplexed numerically via Fourier filtering, phase aberration compensation, and weighted image subtraction procedures. Compared to previous BF-DHM systems, our system avoids synchronization challenges caused by multiple image recording devices, improves acquisition speed, and enhances versatility for fast imaging of large, connected, and rapidly moving samples. Initially, we perform a systematic characterization of the system’s multimodal imaging performance by optimizing numerical as well as coherent and incoherent illumination parameters. Subsequently, the application capabilities are evaluated by multimodal imaging of living cells. The results highlight the potential of single-capture BF-DHM for fast biomedical imaging.