Quantitative Digital Holographic Microscopy Phase Research Articles

Marker-Free Automatic Quantification of Drug-Treated Cardiomyocytes with Digital Holographic Imaging

We use quantitative phase digital holographic microscopy (QP-DHM) to image and quantify the beating movement of cardiomyocytes, derived from induced pluripotent stem cells (iPSCs), in control and d...

Measuring Absolute Cell Volume Using Quantitative-Phase Digital Holographic Microscopy and a Low-Cost, Open-Source, and 3D-Printed Flow Chamber

Although cell volume is a critical cell parameter and acute changes of its regulation mechanisms can reveal pathophysiological states, its accurate measurement remains difficult. On the other hand, custom-made devices are now possible through the application of 3D printing that meet the needs of certain specific applications. Therefore, in this article we describe the development of a low-cost, open-source, and 3D-printed flow chamber optimized to perform non-invasive accurate measurements of important cell biophysical parameters. This measurement development includes the absolute cell volume, mean cell thickness, and whole-cell refractive index using quantitative-phase digital holographic microscopy. Specifically, this advancement makes it possible to develop a flow chamber that preserves cell viability while exhibiting a fast washout process for the two-liquid decoupling procedure - an experimental procedure allowing to separately measure the intracellular refractive index and cell thickness from the quantitative-phase signal - characterized by a homogeneous return of the extracellular refractive index over the entire imaging slit toward the known value of the last perfusion solution. Such a fast washout process has been extensively characterized by monitoring the time required to obtain a stabilization of the quantitative-phase signal following the perfusion of the two liquids. A more rapid quantitative-phase signal stabilization time results in fewer cell changes, including important cell position and shape modifications. These changes likely take place over the washout process time and ultimately result in important artefacts to all cell biophysical measurements. The proposed flow chamber has been used to perform measurements of the whole-cell refractive index, mean cell thickness, and absolute cell volume of three cell types in culture during resting state, and hypo-osmotic and hyper-osmotic challenges. For a typical cell body, these biophysical parameters are measured with a precision of 0.0006, 100 nm, and 50 µm3 respectively. Finally, the design files of the flow chamber will be shared with the scientific community through an open-source model.

Sample and substrate preparation for exploring living neurons in culture with quantitative-phase imaging.

Quantitative-phase imaging (QPI) has recently emerged as a powerful new quantitative microscopy technique suitable for the noninvasive exploration of the structure and dynamics of transparent specimens, including living cells in culture. Indeed, the quantitative-phase signal (QPS), induced by transparent living cells, can be detected with a nanometric axial sensitivity, and contains a wealth of information about both cell morphology and content. However, QPS is also sensitive to various sources of experimental noise. In this chapter, we emphasize how to properly and specifically measure the cell-mediated QPS in a wet-lab environment, when measuring with a digital holographic microscope (DHM). First, we present the substrate-requisite characteristics for properly achieving such cell-mediated QPS measurements at single-cell level. Then, we describe how quantitative-phase digital holographic microscopy (QP-DHM) can be used to numerically process holograms and subsequently reshape wavefronts in association with post-processing algorithms, thereby allowing for highly stable QPS obtainable over extended periods of time. Such stable QPS is a prerequisite for exploring the dynamics of specific cellular processes. We also describe experimental procedures that make it possible to extract important biophysical cellular parameters from QPS including absolute cell volume, transmembrane water permeability, and the movements of water in and out of the cell. To illustrate how QP-DHM can reveal the dynamics of specific cellular processes, we show how the monitoring of transmembrane water movements can be used to resolve the neuronal network dynamics at single-cell level. This is possible because QPS can measure the activity of electroneutral cotransports, including NKCC1 and KCC2, during a neuronal firing mediated by glutamate, the main excitatory neurotransmitter in the brain. Finally, we added a supplemental section, with more technical details, for readers who are interested in troubleshooting live-cell QP-DHM.

Nanoencapsulated capsaicin changes migration behavior and morphology of madin darby canine kidney cell monolayers.

We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystem’s influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) epithelial cells in comparison to the capsaicin-free nanoformulation, free capsaicin, and control cells. For minimally-invasive quantification of cell migration, we applied label-free digital holographic microscopy (DHM) and single-cell tracking. Moreover, quantitative DHM phase images were used as novel stain-free assay to quantify the temporal course of global cellular morphology changes in confluent cell layers. Cytoskeleton alterations and tight junction protein redistributions were complementary analyzed by fluorescence microscopy. Calcium influx measurements were conducted to characterize the influence of the nanoformulations and capsaicin on ion channel activities. We found that both, capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, have a significant impact on directed cell migration and cellular motility. Increase of velocity and directionality of cell migration correlates with changes in the cell layer surface roughness, tight junction integrity and cytoskeleton alterations. Calcium influx into cells occurred only after nanoformulation treatment but not upon addition of free capsaicin. Our results pave the way for further studies on the biological significance of these findings and potential biomedical applications, e.g. as drug and gene carriers.

Accurate quantitative phase digital holographic microscopy with single- and multiple-wavelength telecentric and nontelecentric configurations.

In this work, we investigate, both theoretically and experimentally, single-wavelength and multiwavelength digital holographic microscopy (DHM) using telecentric and nontelecentric configurations in transmission and reflection modes. A single-wavelength telecentric imaging system in DHM was originally proposed to circumvent the residual parabolic phase distortion due to the microscope objective (MO) in standard nontelecentric DHM configurations. However, telecentric configurations cannot compensate for higher order phase aberrations. As an extension to the telecentric and nontelecentric arrangements in single-wavelength DHM (SW-DHM), we propose multiple-wavelength telecentric DHM (MW-TDHM) in reflection and transmission modes. The advantages of MW-TDHM configurations are to extend the vertical measurement range without phase ambiguity and optically remove the parabolic phase distortion caused by the MO in traditional MW-DHM. These configurations eliminate the need for a second reference hologram to subtract the two-phase maps and make digital automatic aberration compensation easier to apply compared to nontelecentric configurations. We also discuss a reconstruction algorithm that eliminates the zero-order and virtual images using spatial filtering and another algorithm that minimizes the intensity of fluctuations using apodization. In addition, we employ two polynomial models using 2D surface fitting to compensate digitally for chromatic aberration (in the multiwavelength case) and for higher order phase aberrations. A custom-developed user-friendly graphical user interface is employed to automate the reconstruction processes for all configurations. Finally, TDHM is used to visualize cells from the highly invasive MDA-MB-231 cultured breast cancer cells.

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders.

Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed.

Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening

Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.

Shift-variant digital holographic microscopy: inaccuracies in quantitative phase imaging

Inaccuracies introduced in quantitative phase digital holographic microscopy by the use of nontelecentric imaging systems are analyzed. Computer modeling of the experimental result shows that even negligible errors in the radius and center of curvature of the numerical compensation needed to get rid of the remaining quadratic phase factor introduce errors in the phase measurements; these errors depend on the position of the object in the field-of-view. However, when a telecentric imaging system is utilized for the recording of the holograms, the numerical modeling and experimental results show the shift-invariant behavior of the quantitative-phase digital holographic microscope.

Digital Holographic Microscopy for Assessment of Experimental Colitis and Monitoring of Intestinal Wound Healing in vitro

Purpose: Intestinal epithelial barrier function is crucial for pathogenesis of inflammatory bowel disease and for evaluation of potential anti-inflammatory drugs in vitro. Digital holographic microscopy (DHM) enables marker-free quantitative phase contrast imaging and provides assessment of cell thickness and tissue density by measuring optical path length delay (OPD). Aim of this study was to evaluate DHM for assessment of experimental colitis in vivo and wound healing in vitro. Methods: Experimental colitis was induced by dextran sodium sulfate in CL57/B6 WT mice. Cryostat colonic sections (5 μm) of healthy (n=7) and colitic mice (n=9) were examined via DHM. To quantify tissue density, segmental OPD analysis of colonic wall (75 measurements/animal) was performed according to anatomic structure (epithelium (e), submucosa (sm) and stroma (st)). DHM was used for time lapse monitoring of wound healing in scratch assay of human intestinal fibroblasts and intestinal epithelial cells (Caco-2 cells) by automated measurements over time (digital holograms were recorded every 3 min). Results: Average optical OPD varied significantly within the different intestinal wall-layers in all mice (e: 1.50 ± 0.30, sm: 1.22 ± 0.59, st: 1.80 ± 0.60 rad; p < 0.001). In contrast, OPD in colitic mice (n=9) was significantly decreased reflecting a significant loss to density in all parts of the colonic wall compared to healthy mice (e: 1.62 ± 0.27 vs. 1.41 ± 0.29; p < 0.001, sm: 1.77 ± 0.42 vs. 0.80 ± 0.29; p < 0.001, st: 1.84 ± 0,53 vs. 1.76 ± 0.66 rad; p < 0.05). Wound assays via DHM from living Caco-2-cells and human intestinal fibroblasts allow quantification of proliferation over time (Caco-2: T1(12h)=34.2%, T2(24h)=53.2% and fibroblasts: T1=(24h)=15.0%, T2=(48h)=30.4% growth in plane axis) with simultaneous morphology imaging (Figure 1).Figure: [1622] Figure 1 Wound assay: time-dependent quantitative DHM phase contrast images of living Caco-2-cells (a-c) and fibroblasts (d-f) coded to 256 gray levels. The wound borders are marked with arrows at different time intervals (T0,T1,T2).Conclusion: DHM allows differentiation between healthy and inflamed intestines and correctly detects density changes of colonic wall during experimental colitis, reflecting loss of epithelial barrier function. In addition, DHM may serve as a valuable tool for quantitative live monitoring of wound healing in vitro for evaluation of novel potential drugs in different cell types.

Application of partially coherent light in live cell imaging with digital holographic microscopy

The main drawbacks of laser-based digital holographic microscopy (DHM) are coherent noise and disturbances due to parasitic reflections in the experimental setup. To overcome some of these problems, investigations on the performance of partially coherent light in DHM-based live cell imaging were carried out. First, the impact of different partially coherent light sources on the formation and the reconstruction of digital off-axis holograms was analyzed. Therefore, the achieved lateral resolution and the accuracy for the detection of optical path length changes were compared to results obtained with laser light. In additional experiments, we investigated if partial coherent light can be utilized in DHM for quantitative phase contrast imaging of living cells in incident light configuration by using light from specifically selected reflective sample interfaces. The obtained results show the applicability of partially coherent light in off-axis DHM and its use to for the reduction of disturbances due to coherence effects. Furthermore, it is demonstrated that quantitative DHM phase contrast imaging of phase objects can be performed with experimental arrangements in which an illumination of the sample in transmission due to geometric conditions is not possible.

Automated three-dimensional tracking of living cells by digital holographic microscopy

Digital holographic microscopy (DHM) enables a quantitative multifocus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms enables subsequent automated focus realignment by numerical propagation of the digital holographically reconstructed object wave. In combination with a calibrated optical imaging system, the obtained propagation data quantify axial displacements of the investigated sample. The evaluation of quantitative DHM phase contrast images also enables an effective determination of lateral cell displacements. Thus, 3-D displacement data are provided. Results from investigations on sedimenting red blood cells and HT-1080 fibrosarcoma cells in a collagen tissue model demonstrate that DHM enables marker-free automated quantitative dynamic 3-D cell tracking without mechanical focus adjustment.