Quantitative Phase Signal Research Articles

Simultaneous measurements of a specimen quantitative-phase signal and its surrounding medium refractive index using quantitative-phase imaging.

This Letter demonstrates a method to simultaneously measure the quantitative-phase signal (QPS) of the observed specimen and the refractive index of its surrounding medium (nm) in a time-resolved manner using a micro-structured coverslip. Such coverslips, easily integrated into perfused live-cell imaging chambers, allow to use various quantitative-phase imaging techniques to achieve this dual measurement. Since QPS is crucially dependent on nm, the measurement of the latter paves the way for its manipulation in a controlled manner leading to a QPS contrast modulation for appealing applications, including visualizing the interior of cells.

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.

Implementation of simultaneous quantitative phase with Raman imaging

We present a technical overview of a multimodal system combining Raman microspectroscopy and quantitative phase microscopy (QPM), which allows two independent and simultaneous measurements of both the local molecular content and dynamic sample morphology. We present in detail the setup implementation and measurement procedure, and show how different features of QPM can be used to ensure optimal Raman measurement conditions and matched fields of view, through off-line calibration procedures such as digital propagation of the measured complex field and analysis of the system’s optical aberrations which can then be employed for numerical compensation and calibration. We present measurements on live cells, where images based both on the quantitative phase signal and on the Raman molecular contrast can simultaneously be retrieved and compared. The dynamic measurements obtained from QPM also enable the monitoring of the cell morphology during the laser scanning of the Raman measurement, making it possible to identify the movements which may occur during the measurement. PACS codes: 87.64.-t; 87.64.kp; 42.40.Kw

Cell volume regulation monitored with combined epifluorescence and digital holographic microscopy.

Quantitative phase imaging emerged recently as a valuable tool for cell observation, by enabling label-free imaging through the intrinsic phase-contrast provided by transparent living cells, thus greatly simplifying observation protocols. The quantitative phase signal, unlike the one provided by the widely used phase-contrast microscope, can be related to relevant biological indicators including dry mass, cell volume regulation or transmembrane water movements. Here, we present quantitative phase imaging coupled with live fluorescence, making it possible to follow the phase signal in time to monitor the cell volume regulation, an early indicator of cell viability, along with specific information such as intracellular Ca2+ imaging with Fura-2 ratiometric fluorescence.

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.

Exploring Neural Cell Dynamics with Digital Holographic Microscopy

In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological information provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach.

Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABAA Receptor

Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABAA mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABAA receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABAA receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.