Quantitative phase imaging of erythrocytes under microfluidic constriction in a high refractive index medium reveals water content changes.

Changes in the deformability of red blood cells can reveal a range of pathologies. For example, cells which have been stored for transfusion are known to exhibit progressively impaired deformability. Thus, this aspect of red blood cells has been characterized previously using a range of techniques. In this paper, we show a novel approach for examining the biophysical response of the cells with quantitative phase imaging. Specifically, optical volume changes are observed as the cells transit restrictive channels of a microfluidic chip in a high refractive index medium. The optical volume changes indicate an increase of cell's internal density, ostensibly due to water displacement. Here, we characterize these changes over time for red blood cells from two subjects. By storage day 29, a significant decrease in the magnitude of optical volume change in response to mechanical stress was witnessed. The exchange of water with the environment due to mechanical stress is seen to modulate with storage time, suggesting a potential means for studying cell storage.

Shear Modulus Measurement by Quantitative Phase Imaging and Correlation with Atomic Force Microscopy.

Many approaches have been developed to characterize cell elasticity. Among these, atomic force microscopy (AFM) combined with modeling has been widely used to characterize cellular compliance. However, such approaches are often limited by the difficulties associated with using a specific instrument and by the complexity of analyzing the measured data. More recently, quantitative phase imaging (QPI) has been applied to characterize cellular stiffness by using an effective spring constant. This metric was further correlated to mass distribution (disorder strength) within the cell. However, these measurements are difficult to compare to AFM-derived measurements of Young's modulus. Here, we describe, to our knowledge, a new way of analyzing QPI data to directly retrieve the shear modulus. Our approach enables label-free measurement of cellular mechanical properties that can be directly compared to values obtained from other rheological methods. To demonstrate the technique, we measured shear modulus and phase disorder strength using QPI, as well as Young's modulus using AFM, across two breast cancer cell-line populations dosed with three different concentrations of cytochalasin D, an actin-depolymerizing toxin. Comparison of QPI-derived and AFM moduli shows good agreement between the two measures and further agrees with theory. Our results suggest that QPI is a powerful tool for cellular biophysics because it allows for optical quantitative measurements of cell mechanical properties.

Molecular and biophysical analysis of apoptosis using a combined quantitative phase imaging and fluorescence resonance energy transfer microscope.

Apoptotic mechanisms are often dysregulated in cancerous phenotypes. Additionally, many anticancer treatments induce apoptosis and necrosis, and the monitoring of this apoptotic activity can allow researchers to identify therapeutic efficiency. Here, we introduce a microscope which combines quantitative phase imaging (QPI) with the ability to detect molecular events via fluorescence (or Förster) resonance energy transfer (FRET). The system was applied to study cells undergoing apoptosis to correlate the onset of apoptotic enzyme activity as observed using a FRET-based apoptosis sensor with whole cell morphological changes analyzed via QPI. The QPI data showed changes in cell disorder strength during the initiation of apoptotic enzymatic activity.

Response to Comment on "Is the nuclear refractive index lower than cytoplasm? Validation of phase measurements and implications for light scattering technologies": A Comment on "How a phase image of a cell with nucleus refractive index smaller than that of the cytoplasm should look like?", e201800033.

Recently, Maxim A. Yurkin commented on our paper "Is the nuclear refractive index lower than cytoplasm? Validation of phase measurements and implications for light scattering technologies" as well as on a complementary study "Cell nuclei have lower refractive index and mass density than cytoplasm" from Schürmann et al. In his comment, Yurkin concluded that quantitative phase images of cells with nuclei that are less optically dense than the cytoplasm must exhibit a characteristic concavity, the absence of which is evidence against our conclusion of a less-dense nucleus. In this response, we suggest that Yurkin's conclusion is reached through an oversimplification of the spatial refractive index distribution within cells, which does not account for high index inclusions such as the nucleolus. We further cite recent studies in 3-dimensional refractive index imaging, in which the preponderance of studies supports our conclusion. Finally, we comment on the current state of knowledge regarding subcellular refractive index distributions in living cells.

Real-time speckle reduction in optical coherence tomography using the dual window method.

Speckle is an intrinsic noise of interferometric signals which reduces contrast and degrades the quality of optical coherence tomography (OCT) images. Here, we present a frequency compounding speckle reduction technique using the dual window (DW) method. Using the DW method, speckle noise is reduced without the need to acquire multiple frames. A ~25% improvement in the contrast-to-noise ratio (CNR) was achieved using the DW speckle reduction method with only minimal loss (~17%) in axial resolution. We also demonstrate that real-time speckle reduction can be achieved at a B-scan rate of ~21 frames per second using a graphic processing unit (GPU). The DW speckle reduction technique can work on any existing OCT instrument without further system modification or extra components. This makes it applicable both in real-time imaging systems and during post-processing.

Design and implementation of a low-cost, portable OCT system.

Optical coherence tomography (OCT) is a widely used biomedical imaging tool, primarily in ophthalmology to diagnose and stage retinal diseases. In order to increase access for a wider range of applications and in low resource settings, we developed a portable, low-cost OCT system that has comparable imaging performance to commercially available systems. Here, we present the system design and characterization and compare the system performance to other commercially available OCT systems. In addition, future cost reductions and potential additional applications of the low-cost OCT system are discussed.

Cellular shear stiffness reflects progression of arsenic-induced transformation during G1.

Cancer cells consistently exhibit decreased stiffness; however, the onset and progression of this change have not been characterized. To study the development of cell stiffness changes, we evaluated the shear stiffness of populations of cells during transformation to a carcinogenic state. Bronchial epithelial cells were exposed to sodium arsenite to initiate early stages of transformation. Exposed cells were cultured in soft agar to further transformation and select for clonal populations exhibiting anchorage-independent growth. Shear stiffness of various cell populations in G1 was assessed using a novel non-invasive assay that applies shear stress with fluid flow and evaluates nanoscale deformation using quantitative phase imaging (QPI). Arsenic-treated cells exhibited reduced stiffness relative to control cells, while arsenic clonal lines, selected by growth in soft agar, were found to have reduced stiffness relative to control clonal lines, which were cultured in soft agar but did not receive arsenic treatment. The relative standard deviation (RSD) of the stiffness of Arsenic clones was reduced compared with control clones, as well as to the arsenic-exposed cell population. Cell stiffness at the population level exhibits potential to be a novel and sensitive framework for identifying the development of cancerous cells.

Invited Article: Digital refocusing in quantitative phase imaging for flowing red blood cells.

Quantitative phase imaging (QPI) offers high optical path length sensitivity, probing nanoscale features of live cells, but it is typically limited to imaging just few static cells at a time. To enable utility as a biomedical diagnostic modality, higher throughput is needed. To meet this need, methods for imaging cells in flow using QPI are in development. An important need for this application is to enable accurate quantitative analysis. However, this can be complicated when cells shift focal planes during flow. QPI permits digital refocusing since the complex optical field is measured. Here we analyze QPI images of moving red blood cells with an emphasis on choosing a quantitative criterion for digitally refocusing cell images. Of particular interest is the influence of optical absorption which can skew refocusing algorithms. Examples of refocusing of holographic images of flowing red blood cells using different approaches are presented and analyzed.

Structured illumination multimodal 3D-resolved quantitative phase and fluorescence sub-diffraction microscopy.

Sub-diffraction resolution imaging has played a pivotal role in biological research by visualizing key, but previously unresolvable, sub-cellular structures. Unfortunately, applications of far-field sub-diffraction resolution are currently divided between fluorescent and coherent-diffraction regimes, and a multimodal sub-diffraction technique that bridges this gap has not yet been demonstrated. Here we report that structured illumination (SI) allows multimodal sub-diffraction imaging of both coherent quantitative-phase (QP) and fluorescence. Due to SI's conventionally fluorescent applications, we first demonstrate the principle of SI-enabled three-dimensional (3D) QP sub-diffraction imaging with calibration microspheres. Image analysis confirmed enhanced lateral and axial resolutions over diffraction-limited QP imaging, and established striking parallels between coherent SI and conventional optical diffraction tomography. We next introduce an optical system utilizing SI to achieve 3D sub-diffraction, multimodal QP/fluorescent visualization of A549 biological cells fluorescently tagged for F-actin. Our results suggest that SI has a unique utility in studying biological phenomena with significant molecular, biophysical, and biochemical components.

Dual-axis optical coherence tomography for deep tissue imaging.

We have developed dual-axis optical coherence tomography (DA-OCT) which enables deep tissue imaging by using a novel off-axis illumination/detection configuration. DA-OCT offers a 100-fold speed increase compared with its predecessor, multispectral multiple-scattering low coherence interferometry (ms2/LCI), by using a new beam scanning mechanism based on a microelectro-mechanical system (MEMS) mirror. The data acquisition scheme was altered to take advantage of this scanning speed, producing tomographic images at a rate of 4 frames (B-scans) per second. DA-OCT differs from ms2/LCI in that the dual axes intersect at a shallower depth (∼1 mm). This difference, coupled with the faster scanning speed, shifts the detection priority from multiply scattered to ballistic light. The utility of this approach was demonstrated by imaging both ex vivo porcine ear skin and in vivo rat skin from a McFarlane flap model. The enhanced penetration depth provided by the DA-OCT system will be beneficial to various clinical applications in dermatology and surgery.

Is the nuclear refractive index lower than cytoplasm? Validation of phase measurements and implications for light scattering technologies.

The refractive index (RI) of biological materials is a fundamental parameter for the optical characterization of living systems. Numerous light scattering technologies are grounded in a quantitative knowledge of the refractive index at cellular and subcellular scales. Recent work in quantitative phase microscopy (QPM) has called into question the widely held assumption that the index of the cell nucleus is greater than that of the cytoplasm, a result which disagrees with much of the current literature. In this work, we critically examine the measurement of the nuclear and whole-cell refractive index using QPM, validating that nuclear refractive index is lower than that of cytoplasm in four diverse cell lines and their corresponding isolated nuclei. We further examine Mie scattering and phase-wrapping as potential sources of error in these measurements, finding they have minimal impact. Finally, we use simulation to examine the effects of incorrect RI assumptions on nuclear morphology measurements using angle-resolved scattering information. Despite an erroneous assumption of the nuclear refractive index, accurate measurement of nuclear morphology was maintained, suggesting that light scattering modalities remain effective.

Optical Phase Measurements of Disorder Strength Link Microstructure to Cell Stiffness.

There have been sustained efforts on the part of cell biologists to understand the mechanisms by which cells respond to mechanical stimuli. To this end, many rheological tools have been developed to characterize cellular stiffness. However, measurement of cellular viscoelastic properties has been limited in scope by the nature of most microrheological methods, which require direct mechanical contact, applied at the single-cell level. In this article, we describe, to our knowledge, a new analysis approach for quantitative phase imaging that relates refractive index variance to disorder strength, a parameter that is linked to cell stiffness. Significantly, both disorder strength and cell stiffness are measured with the same phase imaging system, presenting a unique alternative for label-free, noncontact, single-shot imaging of cellular rheologic properties. To demonstrate the potential applicability of the technique, we measure phase disorder strength and shear stiffness across five cellular populations with varying mechanical properties and demonstrate an inverse relationship between these two parameters. The existence of this relationship suggests that predictions of cell mechanical properties can be obtained from examining the disorder strength of cell structure using this, to our knowledge, novel, noncontact technique.

Structured illumination microscopy for dual-modality 3D sub-diffraction resolution fluorescence and refractive-index reconstruction.

Though structured illumination (SI) microscopy is a popular imaging technique conventionally associated with fluorescent super-resolution, recent works have suggested its applicability towards sub-diffraction resolution coherent imaging with quantitative endogenous biological contrast. Here, we demonstrate that SI can efficiently integrate together the principles of fluorescent super-resolution and coherent synthetic aperture to achieve 3D dual-modality sub-diffraction resolution, fluorescence and refractive-index (RI) visualizations of biological samples. We experimentally demonstrate this framework by introducing a SI microscope capable of 3D sub-diffraction resolution fluorescence and RI imaging, and verify its biological visualization capabilities by experimentally reconstructing 3D RI/fluorescence visualizations of fluorescent calibration microspheres as well as alveolar basal epithelial adenocarcinoma (A549) and human colorectal adenocarcinmoa (HT-29) cells, fluorescently stained for F-actin. This demonstration may suggest SI as an especially promising imaging technique to enable future biological studies that explore synergistically operating biophysical/biochemical and molecular mechanisms at sub-diffraction resolutions.

Imaging deformation of adherent cells due to shear stress using quantitative phase imaging.

We present a platform for detecting cellular deformations from mechanical stimuli, such as fluid shear stress, using rapid quantitative phase imaging. Rapid quantitative phase imaging was used to analyze changes in the optical path length of adherent skin cancer cells during mechanical displacement. Both the whole-cell phase displacement and the resultant shift of the cellular center of mass were calculated over the duration of the stimulus. Whole-cell phase displacement images were found to match expectation. Furthermore, center-of-mass shifts of adherent cells were found to resemble that of a one-dimensional Kelvin-Voigt (KV) viscoelastic solid. Cellular steady-state displacements from step fluid shear stimuli were found to be linearly related to the shear stress. Shear stiffness constants for cells exposed to a cytoskeletal disrupting toxin were found to be significantly lower than unexposed cells. This novel technique allows for elastographic analysis of whole-cell effective shear stiffness without the use of an exogenous force applicator, a specialized culture substrate, or tracking net perimeter movement of the cell.

Spatial frequency-domain multiplexed microscopy for simultaneous, single-camera, one-shot, fluorescent, and quantitative-phase imaging.

Multimodal imaging is a crucial tool when imaging biological phenomena that cannot be comprehensively captured by a single modality. Here, we introduce a theoretical framework for spatial-frequency-multiplexed microscopy via off-axis interference as a novel wide-field imaging technique that enables true simultaneous multimodal and multichannel wide-field imaging. We experimentally demonstrate this technique for single-camera, simultaneous two-channel fluorescence and one-channel quantitative-phase imaging for fluorescent microspheres and fixed cells stained for F-actin and nuclear fluorescence.

Quantitative phase imaging with molecular sensitivity using photoacoustic microscopy with a miniature ring transducer.

We present a dual-modality system for both structural and molecular cell imaging based on coregistered quantitative phase imaging (QPI) and photoacoustic microscopy (PAM). The QPI system was based on off-axis holography, whereas the PAM system comprised a sinusoidally modulated optical source for excitation and a narrow-band low profile and low-cost ring ultrasonic transducer for detection. This approach facilitated a simple confocal alignment of the excitation beams of both modalities and the ultrasonic detector. This system was demonstrated by imaging endogenous molecules in red blood cells (RBCs) as well as by imaging exogenous molecular labels on cancer cells using gold nanoparticles (GNPs) functionalized to target epidermal growth factor receptor. QPI provided high resolution imaging of the cellular structures while PAM provided molecular contrast. This dual-modality microscopy method can potentially be implemented as a compact and low cost cellular diagnostic assay.

Wavelet transform fast inverse light scattering analysis for size determination of spherical scatterers.

We present a fast approach for size determination of spherical scatterers using the continuous wavelet transform of the angular light scattering profile to address the computational limitations of previously developed sizing techniques. The potential accuracy, speed, and robustness of the algorithm were determined in simulated models of scattering by polystyrene beads and cells. The algorithm was tested experimentally on angular light scattering data from polystyrene bead phantoms and MCF-7 breast cancer cells using a 2D a/LCI system. Theoretical sizing of simulated profiles of beads and cells produced strong fits between calculated and actual size (r(2) = 0.9969 and r(2) = 0.9979 respectively), and experimental size determinations were accurate to within one micron.

Fast wide-field photothermal and quantitative phase cell imaging with optical lock-in detection.

We present a fast, wide-field holography system for detecting photothermally excited gold nanospheres with combined quantitative phase imaging. An interferometric photothermal optical lock-in approach (POLI) is shown to improve SNR for detecting nanoparticles (NPs) on multiple substrates, including a monolayer of NPs on a silanized coverslip, and NPs bound to live cells. Furthermore, the set up allowed for co-registered quantitative phase imaging (QPI) to be acquired in an off-axis holographic set-up. An SNR of 103 was obtained for NP-tagging of epidermal growth factor receptor (EGFR) in live cells with a 3 second acquisition, while an SNR of 47 was seen for 20 ms acquisition. An analysis of improvements in SNR due to averaging multiple frames is presented, which suggest that residual photothermal signal can be a limiting factor. The combination of techniques allows for high resolution imaging of cell structure via QPI with the ability to identify receptor expression via POLI.