
Tomographic flow cytometry by digital holography
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ABSTRACT High-throughput single-cell analysis is a challenging task. Label-free tomographic phase microscopy is an excellent candidate to perform this task. However, in-line tomography is
very difficult to implement in practice because it requires a complex set-up for rotating the sample and examining the cell along several directions. We demonstrate that by exploiting the
random rolling of cells while they are flowing along a microfluidic channel, it is possible to obtain in-line phase-contrast tomography, if smart strategies for wavefront analysis are
adopted. In fact, surprisingly, _a priori_ knowledge of the three-dimensional position and orientation of rotating cells is no longer needed because this information can be completely
retrieved through digital holography wavefront numerical analysis. This approach makes continuous-flow cytotomography suitable for practical operation in real-world, single-cell analysis and
with a substantial simplification of the optical system; that is, no mechanical scanning or multi-direction probing is required. A demonstration is given for two completely different
classes of biosamples: red blood cells and diatom algae. An accurate characterization of both types of cells is reported, despite their very different nature and material content, thus
showing that the proposed method can be extended by adopting two alternate strategies of wavefront analysis to many classes of cells. SIMILAR CONTENT BEING VIEWED BY OTHERS LIGHT-FIELD FLOW
CYTOMETRY FOR HIGH-RESOLUTION, VOLUMETRIC AND MULTIPARAMETRIC 3D SINGLE-CELL ANALYSIS Article Open access 04 March 2024 STAIN-FREE IDENTIFICATION OF CELL NUCLEI USING TOMOGRAPHIC PHASE
MICROSCOPY IN FLOW CYTOMETRY Article Open access 10 November 2022 COMPREHENSIVE SINGLE-SHOT BIOPHYSICAL CYTOMETRY USING SIMULTANEOUS QUANTITATIVE PHASE IMAGING AND BRILLOUIN SPECTROSCOPY
Article Open access 31 October 2022 INTRODUCTION To date, one of the most powerful imaging tools for analyzing biological samples is tomography, which is able to furnish complete
characterizations in three-dimensional (3D)1. Various classes of tomographic concepts exist, such as X-ray computed tomography, optical coherence tomography2, 3 and tomographic phase
microscopy (TPM)4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. TPM is based on quantitative phase imaging techniques for the accurate 3D refractive index (RI) mapping of cells, as demonstrated by
Feld’s group at MIT4, or even by white light sources applied to red blood cells (RBCs), as shown by Popescu’s group7, 8. Park and coworkers demonstrated a reconstruction of a 3D RI
distribution employing sparse angle illumination5, 9, 11. Ozcan’s group proposed a lens-free holographic microscope, which enables the imaging of a very large volume (tens of mm2) and the
possibility to perform an optical in-flow tomography by exploiting a pixel super-resolution technique12, 13, 14. Recently, Psaltis’ group proved a smart learning method for building
tomographic structures15, 16. TPM set-ups require the sample to be observed along different directions with respect to the probing beam. Recording is often accomplished by adopting beam
deflection5, 14, 17; however, in this case, some problems arise because the angles are limited to 150°, thus affecting the accuracy of the tomographic reconstruction. Alternatively, direct
mechanical rotation of the sample6 is problematic because mechanically manipulating the biological specimen introduces the risk of altering the sample. Tomographic arrangements occasionally
use holographic optical tweezers18 to rotate the sample without mechanical contact19, 20, 21; unfortunately, they do not allow high-throughput and in-line useful conditions for continuous
in-flow tomography. In another case, Heng _et al._22 adopted an optofluidic microscope, for which the flow was used to provide a scanning mechanism; however, the sample rotation was
considered an undesired effect. In summary, to date, all tomographic methods require a high-precision, opto-mechanical and/or optoelectronic device to acquire a set of many images by probing
the sample along a large number of controlled directions. Moreover, to successfully apply the tomography algorithms, each recorded image must be tagged with the correct cell’s rotation
angle with respect to the probing beam, thus requiring robust and effective control of the angular positions. Overcoming such difficulties represents the main critical issue in all
tomographic approaches and, in practice, limits the implementation of tomography, especially at the microscopic scale, because the current approaches are very cumbersome and unsuitable for
widespread use in the life sciences. Here we present a technological improvement by demonstrating an in-line TPM approach that simplifies the optical set-up, thus opening a route for a
real-world tool in the life sciences for single-cell analysis in continuous flow. Essentially, we show for the first time that the wavefront transmitted by the cells intrinsically carries
information beyond the optical path lengths (OPLs) to obtain quantitative phase-contrast maps (QPMs). In fact, we prove that a wavefront transmitted through the cell also contains
information on the value of its angular position with respect to the direction of the probing laser beam. We explain below how the angles can be extracted numerically from the QPMs by
intelligent numerical processing through two different strategies, thus allowing TPM that is suitable for all types of cells by using a single laser beam without mechanical or
electro-optical scanning. The two strategies for angle recovery are conceived on the basis of the inner structure of cells. In particular, the rolling angles of cells with a homogeneous RI
distribution are calculated by exploiting the biolens effect23. Alternatively, rolling angles of cells with inhomogeneous RI are obtained by observing the QPMs’ mirror symmetry around the
axis of rotation. The proposed innovative approach has been tested with two different classes of biological samples having relevant impact in our life, that is, RBCs24, 25 (with homogeneous
RI) and diatoms algae (DAs)26, 27 (with inhomogeneous RI). Note that RBCs and DAs have completely different external shapes and inner structures, although they are all quite simple.
Moreover, RBC and DA are made of diverse materials. Although RBCs and DAs are very different from a biological perspective, they have some features in common. In fact, RBCs and DAs have a
crucial role in the health of the human body and our planet, respectively. Moreover, these materials are characterized by their high abundance: RBCs account for nearly a quarter of the total
number of cells in the human body, and diatoms are responsible for over 40% of the photosynthesis that occurs in the world’s oceans, and without them, the ocean would be unable to support
the amount of life that it does. Furthermore, diatoms are useful tools for monitoring environmental conditions and are commonly used in studies of water quality, and the morphology and
content of RBCs (that is, hemoglobin) are important biomarkers for many severe blood diseases28. An accurate study of the full 3D structure and the content of such diverse types of cells is
of vital importance for human health (regarding RBCs) and for the earth’s ecosystem (regarding diatoms); as a result, a simple diagnostic tool that can operate in a high-throughput mode is
highly desirable to, for example, find and identify rare cells in blood or contaminants in oceans. Here we exploit the random self-rotation of the cells while they are flowing along
microfluidic channels29, 30, 31, thereby avoiding the direct rotation of the cells or the use of laser-beam angular scanning. Furthermore, we demonstrate that _a priori_ knowledge of the
rotation angle in 3D is no longer necessary because it can be accurately retrieved from the intelligent processing of the QPMs. The only requirement is to have one full angular revolution of
the cells inside the field of view of the microscope while they are flowing. We named this proposed approach Rolling-TPM (R-TPM). Moreover, this approach allows fast single-cell analysis in
high-throughput modality because no mechanical or electro-optical angular scanning of the laser beam is required. Regarding RBCs32, 33, we demonstrate their full characterization in terms
of several metrics, such as 3D morphology, corpuscular hemoglobin (CH), volume (_V_) and RI. Moreover, we demonstrate both identification and sorting capabilities for anemia blood disease,
in which some of the RBCs parameters differ slightly from healthy ones. Regarding DA, we instead show the capability to obtain the outer structure and the detailed 3D inner structure of
auto-fluorescent chloroplasts via whole R-TPM imaging without the recurring use of fluorescent imaging. MATERIALS AND METHODS BLOOD PREPARATION AND ISOLATION OF ERYTHROCYTES Human blood
(~4.0 mL) was collected in a 7.2-mg K2 EDTA vacutainer tube (BD, Plymouth, UK) from a healthy volunteer. Blood was centrifuged at room temperature at 2500 r.p.m. for 15 min to separate RBCs
at the bottom of the sterile centrifugation tube from the plasma and buffy coat. After centrifugation, the plasma and buffy coat were discarded, and the RBC pellet (~1.5 mL) was washed with
a saline solution of 0.90% w/v of sodium chloride (NaCl) in sterile water in a 1:1 ratio and re-centrifuged at room temperature at 2500 r.p.m. for 10 min. After the second centrifugation,
the supernatant fraction was removed, and an aliquot of isolated RBCs (~100 μL) was diluted in 10 mL of the saline solution of 0.90% w/v of NaCl in sterile water with a final osmolarity of
308 mOs mL−1 to maintain the osmotic pressure of the RBCs. For the experiments, a final volume of diluted RBCs (~100 μL) was used. Altered RBC shapes were obtained by changing the buffer
osmolarity, and a buffer of 205 and 410 mOs mL−1 was used to perform experiments under hypotonic and hypertonic conditions, respectively. DIATOMS PREPARATION The two strains of diatoms (T.
rotula CCMP 3264 and S. marinoi CCMP 2092) were purchased from the National Center for Marine Algae and Microbiota (Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA). Microalgae
were cultured in 75 cm2 flasks in sterile f/2 medium at 20.0±1.0 °C. The artificial light illumination (100 μmol photons m−2 s−1) was provided by daylight fluorescent tubes with a 14:10 h
light:dark photoperiod. Then, 5-mL subsamples of each culture were collected with serological pipettes under the fume hood and transferred to 15-mL falcons. Highly concentrated cultures were
diluted with filtered (0.22 μm) sterile seawater and maintained at room temperature until the analyses/measurements were performed. HOLOGRAPHIC RECORDING AND RECONSTRUCTIONS We built an
optofluidic platform (depicted in Figure 1) to provide high-resolution images of both flowing and rotating samples. The DH modulus is made with a 400 mW fiber-coupled laser at 532 nm acting
as the source for a Mach-Zehnder interferometer, whose main beam (in green) is directed into a customized inverted microscope equipped with a water-immersion 60 × objective, numerical
aperture 1.20 microscope objective, thus allowing bright-field imaging of the cells. The reference beam (also in green) is recombined with the first beam by a beam splitter generating
interference fringe patterns (the digital holograms) of the samples that are recorded by a 2048 × 2048 CCD camera (USB 3.0 u-eye, from IDS), recording at 75 fps in full frame. The optical
lateral resolution is ~360 nm, and the axial resolution is ~170 nm34. In our case, different from Cotte _et al._34, we are limited by the minimum detectable angle of rotation, that is, ~1°
for this optical set-up. The holographic recordings and reconstructions33 occur as follows. First, through a digital holography apparatus, several out-of-focus digital holograms of cells
(obtained while the cells are tumbling in the microfluidic channel) are acquired for different angular positions of the cell. Next, for each acquired hologram, the corresponding QPI is
numerically calculated via the angular spectrum technique35, which is a standard propagation algorithm that reconstructs the complex wavefield in terms of both amplitude and phase diffracted
by the object at a certain distance from the hologram plane. The channel (by _Microfluidic Chip Shop_) is made of PMMA polymer and has dimensions of 1000 μm × 200 μm (width × height). At
the bottom, the cover lid is 140 μm thick, which allows the use of the oil-immersion objective. The sample is injected into the channel by a syringe and capillary tubes furnished by the
Microfluidic Chip Shop. For the experiments with diatoms, we added a fluorescence modulus (Figure 1a). Light from a fluorescence lamp (X-cite series 120 pc Lumen Dynamics) is directed onto
the sample (blue path); a combination of excitation (GFP) and emission (Tritc) filters, which are suitable for detecting the diatom’s chlorophyll, is used, together with a dichroic mirror.
FLUID DYNAMIC CONDITIONS FOR CELLS TUMBLING After the preparation is completed, the cells are injected into a microfluidic channel by a syringe pump. In our experiment, the deformation of
the RBCs is negligible. Theoretical studies and numerical simulations by G Gompper and coworkers describe the different ways that an RBC moves inside a microchannel, depending on the
confinement and flow strength36. In our experiment, with a very small confinement and flow strength, we observe the tumbling condition; that is, each RBC undergoes a rotation and can be
approximated by a rigid body. By estimating the number of cells accomplishing a complete rotation (360°) in the field of view, we observe more than 150 rolling cells per minute. This
throughput is several times larger than that obtained with classical TPM methods9. TOMOGRAPHIC RECONSTRUCTION We use optical projection tomography37, where the inputs of the filtered
back-propagation algorithm are the aligned-oriented QPMs, and the rotation angles are around the _x_-axis, _θ_. The number of QPMs for each imaged cell in the field of view is ~200 for our
camera with a frame rate of 75 fps. If the number of recordings decreases, then the resolution becomes worse, but the final shape reconstructions can be overlapped up to a lower recording
limit of ~80 holograms. The calculation of the slices, which corresponds to the RI distribution of the sample along the planes orthogonal to the plane _x_–_y_, is performed through the
inverse Radon transform. More specifically, for a given coordinate of the rotating axis in the plane _x_–_y_, the corresponding values of the QPMs along the orthogonal direction to the
_x_-axis are collected for all rotation angles and are used together to calculate the corresponding slices by using the inverse Radon transform. Finally, these slices are joined together and
processed to obtain a tomographic representation of the cell, that is, the 3D RI distribution. All numerical processing is performed off-line. By considering ~200 images, the angle recovery
step requires a computational time of 11.2 s for RBCs and 16.8 s for DAs, and the projection tomography step requires 1.8 and 4.2 s for RBCs and DAs, respectively. Video-rate processing in
tomographic phase microscopy has been recently demonstrated by using Nvidia’s CUDA C platform38. An informal high-level description (that is, pseudo-code) of the operating principle of the
whole tomographic reconstruction process, including the holographic 3D tracking step, rotating angle calculation using the two proposed methods and tomographic reconstruction algorithm, is
reported in the Supplementary Information. RESULTS AND DISCUSSION The working conditions and the adopted optical system are depicted in Figure 1a. Cells tumble while flowing along a
microfluidic chip probed by a single fixed laser beam. A second beam is used as a reference to generate interference fringes on the CCD camera. More details regarding the optical set-up are
given in Materials and methods. Hundreds of cells per minute have been analyzed; for each one, the hologram’s sequence is recorded, and the corresponding QPMs is retrieved. First, 3D
holographic tracking is performed to re-align each cell with respect to its center of mass39, 40, 41. Next, the rotation angles are numerically estimated using two strategies, the choice of
which depends on the type of the cell under analysis. In Figure 1b, we report the conceptual block diagram summarizing the main steps of the two proposed strategies. In particular, the cells
are classified according to their RI distribution (homogeneous and inhomogeneous). By this taxonomy, almost all classes of cells can be tagged, and the performance of the two proposed
algorithms depends on their bio-physical features. To demonstrate the effectiveness of the two proposed strategies, we focus on RBCs as cells with homogeneous RI and DAs as cells with a more
complex inner structure, that is, with inhomogeneous RI. Specifically, RBCs can be modeled as optofluidic microlenses23; thus, Zernike polynomials are used to quantify their aberrations.
The flow direction occurs along the _y_-axis (see the reference coordinates system in Figure 1a), thus allowing the contribution of rotation angle _ψ_ to be neglected. The other two
orientations (_θ_ and _ϕ_) are calculated by aberration analyses using Zernike fitting and are computed for each QPM. We reveal here that the trend of the focus shift term _C_4 can be fitted
by the square cosine function of the rotation angle _θ_ and that the orientation _ϕ_ can be retrieved from the tilt terms _C_1 and _C_2. In Equation (1), R is the spatial coordinates
vector, _i_(_θ_) denotes the _i_th QPM and corresponds to a specific rotating angle _θ_, _Z__k_ is the Zernike basis functions and _C__k_ is the corresponding Zernike coefficients. Each QPM
is re-oriented to the first one of an angle equal to −_ϕ_. For cells that have complex inner structures, such as DAs, we exploit an alternate intelligent image processing strategy. In fact,
such cells have a detectable RI inner structure. It can be argued that a spatial symmetry exists in the reconstructed QPMs as a function of rotation angle. In particular, each QPM is
compared with all others in the reconstruction sequence by searching for a twin-mirror QPM that maximizes their spatial correlation coefficient (SCC) because in these two directions, the
OPLs should be equal for each pair of mirror pixels. Specifically, when the SCC of two QPMs is >0.95, if _θ_ is in the range [0°, 90°], then we assign _θ_ and 180°−_θ_ as the rotating
angles, whereas if _θ_ is in the range [180°, 270°], then we assign _θ_ and 540°−_θ_ as the rotating angles. In Equation (2), _i_ and _j_ with _j_>_i_ are the _i_th and _j_th QPMs,
respectively. Because not all QPMs may have a corresponding mirror QPM, we tag the remaining QPMs by assuming uniform angular rotation. Finally, we use the optical projection tomography
method to calculate the 3D RI distribution of the sample32, where the inputs of the filtered back-propagation algorithm are the aligned-oriented QPMs and the rotation angles around the
_x_-axis, _θ_. Complete proofs of both mathematical relationships for retrieving the angles are reported in the Supplementary Information together with the holographic 3D tracking algorithm.
The results of R-TPM, as applied to RBCs, are reported in Figure 2 and Supplementary Movie 1. Four interesting cases are revealed. In particular, we examined both healthy (Figure 2a and 2b)
and pathological (Figure 2c and 2d) RBCs, such as iron deficiency anemia and thalassemia, which are two highly diffused blood disorders. Specifically, some of the QPMs with the
corresponding measured angles are illustrated on the top row of each panel of Figure 2. The inner plots show the rotating angle recovery approach, which was obtained from Equation (1). The
final cytotomography results are displayed on the bottom side together with a picture of the correspondent central slices (_z_=0 and _y_=0 planes). In addition, the red boxes in the insets
report a plastic model obtained by a 3D printer for educational purposes. In particular, we report the complete 3D RI distribution, and Supplementary Movie 1 display slice-by-slice the
tomogram reconstructions and the inner RI map corresponding to the hemoglobin distribution. Figure 2a shows an abnormal RBC detected in the healthy sample, and a one-side concavity is
present resembling the shape typically observed in hereditary stomatocytosis. Figure 2b shows one of the RBCs analyzed for the healthy sample in hypertonic solution, where the shape
modification, as induced by the medium, provides the typical burr shape. We compare real cases with the simulated one (Supplementary Information) by means of the healthiness parameter, _H_,
that is, the correlation coefficient between the measured and ideal 3D RI distributions. We assume that for 0.9⩽_H_⩽1, a RBC can be considered normal; otherwise, the deviation from the
discocyte shape is not negligible. _H_ values are reported for all of the cases presented in Figure 2 together with other global morphometric parameters, namely, the average RI, bio-volume
(_V_) and CH (see Supplementary Information for details). To verify the accuracy of the proposed approach, we have tested the R-QPM for two highly diffused blood disorders. The first sample,
as shown in Figure 2c, is from a patient affected with iron refractory iron deficiency anemia (IRIDA) caused by mutations in the TMPRSS6 gene (L63Pfs13-W590R in compound heterozygosity)42.
The second sample, in Figure 2d, is from a patient affected with alpha-thalassemia caused by a heterozygous deletional event of both in-cis HBA1 genes (—CAMPANIA in heterozygosity)43. CBC
reveals that MCVs are equal to 62.6 and 67.5 fl and that MCHs are 18.5 and 21 pg for the two patients, respectively. (Analysis performed by DAI.Med.Lab AOU Federico II University, Naples,
Italy). In the case of anemia caused by an inherited defect in iron metabolism and thalassemia, the CH and _V_ values are in good agreement with the analysis reported in the literature and,
by comparison, with the CBC from patients with a similar genotype42, 43, 44. Under our experimental conditions, the deformations of RBCs are negligible compared to the rotations they
undergo, and the approximation of a rigid body is achieved36, 45 (see Materials and methods for details). The second class of cells we investigated are DAs. These cells have a much more
complex inner structure26, 27. As a test case, we analyzed the _Skeletonema marinoi_ and the _Thalassiosira rotula_ diatoms, as shown in Figure 3 and Supplementary Movies 5 and 6. Figure 3a
and 3e shows two mirror QPMs recovered by SCC maximization. The retrieved 3D RI distributions of diatoms are depicted in Figure 3b and 3f, where the external shape, labeled in green, is used
to identify the whole occupied volume. The fascinating result for DAs is that by choosing the proper RI threshold, the tomographic algorithm reveals the real shape and dimensions of the
chloroplasts and their location (Figure 3c and 3g), in perfect agreement with the corresponding fluorescence images (Figure 3d and 3h). In other words, the fluorescence acquisition or
labeling of cells is, in principle, no longer necessary for R-TPM. The corresponding calculated volumes are _V_tot=183.2 μm3 and _V_chlor=38.7 μm3 for the _Skeletonema_, _V_tot=1105.8 μm3
and _V_chlor=100.1 μm3 for _Thalassiosira_. Note that the chloroplasts (which correspond to the fluorescence part of the cell) represent only a fraction of the entire volume. The tomographic
reconstruction allows us to exactly identify their location and dimensions. This achievement is a key point as variations in chloroplast shape and location may be used in diagnostics at the
genus level. Moreover, diatom chloroplasts are the main targets of some water contaminants, such as fluoranthene, which is one of the principal constituent of PAH-contaminated aquatic
systems, and copper. It has been demonstrated that in the presence of these elements, chloroplasts show signs of structural rupture or even disintegration. Consequently, disturbances in the
chloroplasts’ integrity could lead to an inhibition of photosynthesis, thus leading to a reduction in the amount of energy that is available to the cells. Due to structural damage, the
function of the chloroplast may be affected; hence, algal cells will not be able to produce sufficient energy for growth and reproduction27. As a result, the abundance of diatoms is commonly
considered a valid indicator of ecosystem health. CONCLUSIONS In conclusion, the R-TPM approach is a single-cell analysis tool that allows the study of hundreds of cells and supplies as
output their complete morphologic classification in 3D via the self-rotation of cells in microfluidic channels. Technological improvement obtained in the practical implementation of
tomography is challenging because the wavefront analysis of the sample along different directions intrinsically carries not only QPM information on the OPL of the cell but also information
about the rotation angles. An intelligent numerical process can easily extract the data of angles, thus allowing accurate phase-contrast cyto-tomography. This avoids cumbersome calibration
and the use of any mechanical and/or optoelectronic device to control the rotation, thereby enormously simplifying the experimental system compared with the current state of the art4, 5, 8,
21. By exploiting this concept, we have shown that it is possible to furnish a full 3D RI distribution for many types of cells flowing along a microchannel with a relatively simple internal
RI distribution. Similar algorithms are under study to adapt the technique to more general cases, such as inhomogeneous RI cells with little symmetry. The technique has been proved for cells
with different shapes and characteristics, such as RBCs and DA. The R-TPM is simple, quick and reliable. A route for full screening at the single-cell level can be a quite challenging
achievement by means of the proposed approach. Applications are foreseen, for example, in the isolation and characterization of ‘foreign’ cells in the blood stream and in revealing specific
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10: 4258–4267. Article ADS Google Scholar Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * CNR-ISASI, Istituto di Scienze Applicate e Sistemi Intelligenti ‘E.
Caianiello’, Francesco Merola, Pasquale Memmolo, Lisa Miccio, Roberto Savoia, Martina Mugnano & Pietro Ferraro * CNR—Consiglio Nazionale delle Ricerche, Pozzuoli, 80078, Italy Francesco
Merola, Pasquale Memmolo, Lisa Miccio, Roberto Savoia, Martina Mugnano & Pietro Ferraro * CNR-ICB, Istituto di Chimica Biomolecolare, Pozzuoli, 80078, Italy Angelo Fontana, Giuliana
D'Ippolito & Angela Sardo * Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II & CEINGE—Advanced Biotechnologies, Napoli, 80145, Italy
Achille Iolascon & Antonella Gambale Authors * Francesco Merola View author publications You can also search for this author inPubMed Google Scholar * Pasquale Memmolo View author
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Angelo Fontana View author publications You can also search for this author inPubMed Google Scholar * Giuliana D'Ippolito View author publications You can also search for this author
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can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Pietro Ferraro. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of
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Miccio, L. _et al._ Tomographic flow cytometry by digital holography. _Light Sci Appl_ 6, e16241 (2017). https://doi.org/10.1038/lsa.2016.241 Download citation * Received: 04 May 2016 *
Revised: 03 October 2016 * Accepted: 10 October 2016 * Published: 17 October 2016 * Issue Date: April 2017 * DOI: https://doi.org/10.1038/lsa.2016.241 SHARE THIS ARTICLE Anyone you share the
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Nature SharedIt content-sharing initiative KEYWORDS * microfluidics * red blood cells * tomographic microscopy * wavefront analysis