On-Chip Fabry-Pérot Microcavity for Refractive Index Cytometry and Deformability Characterization of Single Cells

Une identification correcte et précise du phénotype et des fonctions cellulaires est fondamentale pour le diagnostic de plusieurs pathologies ainsi qu’à la compréhension de phénomènes biologiques tels que la croissance, les réponses immunitaires et l’évolution de maladies. Conséquemment, le développement de technologies de pointe offrant une mesure multiparamétrique à haut débit est capital. À cet égard, la cytométrie en flux est l’étalon de référence due à sa grande spécificité, sa grande sensibilité et ses débits élevés. Ces performances sont atteintes grâce à l’évaluation précise du taux d’émission de fluorophores, conjugués à des anticorps, ciblant certains traits cellulaires spécifiques. Néanmoins, sans ce précieux étiquetage, les propriétés physiques caractérisées par la cytométrie sont limitées à la taille et la granularité des cellules. Bien que la cytométrie en flux soit fondamentalement un détecteur optique, elle ne tire pas avantage de l’indice de réfraction, un paramètre reflétant la composition interne de la cellule. Dans la littérature, l’indice de réfraction cellulaire a été utilisé comme paramètre phénotypique discriminant pour la détection de nombreux cancers, d’infections, de la malaria ou encore de l’anémie. Également, les structures fluidiques de la cytométrie sont conçues afin d’empêcher une déformation cellulaire de se produire. Cependant, les preuves que la déformabilité est un indicateur de plusieurs pathologies et d’état de santé cellulaire sont manifestes. Pour ces raisons, l’étude de l’indice de réfraction et de la déformabilité cellulaire en tant que paramètres discriminants est une avenue prometteuse pour l’identification de phénotypes cellulaires. En conséquence, de nombreux biodétecteurs qui exploitent l’une ou l’autre de ces propriétés cellulaires ont émergé au cours des dernières années. D’une part, les dispositifs microfluidiques sont des candidats idéaux pour la caractérisation mécanique de cellules individuelles. En effet, la taille des structures microfluidiques permet un contrôle rigoureux de l’écoulement ainsi que de ses attributs. D’autre part, les dispositifs microphotoniques excellent dans la détection de faibles variations d’indice de réfraction, ce qui est critique pour un phénotypage cellulaire correcte. Par conséquent, l’intégration de composants microfluidiques et microphotoniques à l’intérieur d’un dispositif unique permet d’exploiter ces propriétés cellulaires d’intérêt. Néanmoins, les dispositifs capables d’atteindre une faible limite de détection de l’indice de réfraction tels que les détecteurs à champ évanescent souffrent de faibles profondeurs de pénétration. Ces dispositifs sont donc plus adéquats pour la détection de fluides ou de molécules. De manière opposée, les détecteurs interférométriques tels que les Fabry- Pérots sont sensibles aux éléments présents à l’intérieur de leurs cavités, lesquelles peuvent mesurer jusqu’à plusieurs dizaines de micromètres.----------Abstract Accurate identification of cellular phenotype and function is fundamental to the diagnostic of many pathologies as well as to the comprehension of biological phenomena such as growth, immune responses and diseases development. Consequently, development of state-of-theart technologies offering high-throughput and multiparametric single cell measurement is crucial. Therein, flow cytometry has become the gold standard due to its high specificity and sensitivity while reaching a high-throughput. Its marked performance is a result of its ability to precisely evaluate expression levels of antibody-fluorophore complexes targeting specific cellular features. However, without this precious fluorescence labelling, characterized physical properties are limited to the size and granularity. Despite flow cytometry fundamentally being an optical sensor, it does not take full advantage of the refractive index (RI), a valuable labelfree measurand which reflects the internal composition of a cell. Notably, the cellular RI has proven to be a discriminant phenotypic parameter for various cancer, infections, malaria and anemia. Moreover, flow cytometry is designed to prevent cellular deformation but there is growing evidence that deformability is an indicator of many pathologies, cell health and state. Therefore, cellular RI and deformability are promising avenues to discriminate and identify cellular phenotypes. Novel biosensors exploiting these cellular properties have emerged in the last few years. On one hand, microfluidic devices are ideal candidates to characterize single cells mechanical properties at large rates due to their small structures and controllable flow characteristics. On the other hand, microphotonic devices can detect very small RI variations, critical for an accurate cellular phenotyping. Hence, the integration of microfluidic and microphotonic components on a single device can harness these promising cellular physical properties. However, devices achieving very small RI limit of detection (LOD) such as evanescent field sensors suffer from very short penetration depths and thus are better suited for fluid or single molecule detection. In opposition, interference sensors such as Fabry-Pérots are sensitive to the medium inside their cavity, which can be several tens of micrometers in length, and thus are ideally suited for whole-cell measurement. Still, most of these volume sensors suffer from large LOD or require out-of-plane setups not appropriate for an integrated solution. Such a complex integration of high-throughput, sensitivity and large penetration depth on-chip is an ongoing challenge. Besides, simultaneous characterization of whole-cell RI and deformability has never been reported in the literature

The importance of single-mode behavior in silicon-on-insulator rib waveguides with very large cross section for resonant sensing applications

Control of light properties through propagation in large cross section optical rib waveguides is of utmost importance to obtain the desired behavior, especially with resonant cavities. We have simulated, fabricated, and experimentally tested optical rib waveguides to evaluate the advantages of single-mode properties against multimode. Modal analysis of very large cross section rib waveguides showed that only particular dimensions allow a single-mode behavior. Furthermore, on-chip Fabry-Perot cavities were coupled to rib waveguides to assess impacts for a typical resonator. Experimental results are in good agreement with simulation guidelines, effectively highlighting the importance of single-mode behavior for resonant sensing applications

High-throughput volume refractive index distribution measurement through mechanical deformation of single cells

This paper reports a high-throughput microphotonic biosensor measuring volume refractive index distribution through mechanical deformation of single cells. Preliminary results suggest that different cell states can be distinguished. This feature could readily add novel parameters for cell analysis without resorting to nucleic acid dies

High-throughput refractive index-based microphotonic sensor for enhanced cellular discrimination

This paper presents a novel microphotonic sensor based on silicon technologies for high-throughput single cell measurements. It employs a highly sensitive Fabry-Pérot resonant cavity to extract cellular refractive index information. The integrated large cross-section rib waveguides provide a single-mode like behavior important for resonant cell sensing. Differentiated myeloid cells derived from a promyelocytic leukemia cell line were injected in a microchannel, sheathlessly focused using inertial forces and analyzed while flowing through the resonant cavity volume. Results were compared against a commercial flow cytometer and showed a substantial improvement on cellular discrimination. Thus, this sensor has the ability to discriminate cell populations, usually identified using fluorescent parameters, without any dyes and can reach measurement rate as high as 2000 cells per second. By harnessing the cell's effective volume refractive index, our device offers complementary measurements readily improving actual technologies and thus providing crucial information for research and clinical professionals

Immature and mature bone marrow-derived dendritic cells exhibit distinct intracellular mechanical properties

ABSTRACT: Dendritic cells (DCs) patrol the organism at an immature stage to detect the presence of pathogens. Once activated, these mature DCs reach the lymph nodes to activate antigen-specific T lymphocytes and thus initiate an adaptative immune response to control the pathogen. The migration of both immature and mature DCs is a key process for their optimal function. DC migration requires transit through narrow constrictions that is allowed by their high local and global deformation capabilities. In addition to cytoplasmic changes, the nucleus mechanical properties also have a major impact for cellular migration and motility. Yet, nucleus intracellular mobility of dendritic cells or its variation upon maturation have not been investigated. Our study defines the biophysical phenotypic variations of dendritic cells upon maturation using interferometric deformability cytometry. This method characterizes different cellular mechanical properties, such as elongation and nucleus offset, by assessing the refractive index spatial distribution of shear-induced deformed cells. By using these parameters, our data suggest that in vitro bone marrow derived dendritic cell (BMDC) maturation induces cell stiffening and reduces nucleus mobility, allowing to distinguish immature and mature dendritic cells. Overall, our method provides insights on intracellular mechanical properties of two dendritic cell states

Immature and mature bone marrow-derived dendritic cells exhibit distinct intracellular mechanical properties

Abstract Dendritic cells (DCs) patrol the organism at an immature stage to detect the presence of pathogens. Once activated, these mature DCs reach the lymph nodes to activate antigen-specific T lymphocytes and thus initiate an adaptative immune response to control the pathogen. The migration of both immature and mature DCs is a key process for their optimal function. DC migration requires transit through narrow constrictions that is allowed by their high local and global deformation capabilities. In addition to cytoplasmic changes, the nucleus mechanical properties also have a major impact for cellular migration and motility. Yet, nucleus intracellular mobility of dendritic cells or its variation upon maturation have not been investigated. Our study defines the biophysical phenotypic variations of dendritic cells upon maturation using interferometric deformability cytometry. This method characterizes different cellular mechanical properties, such as elongation and nucleus offset, by assessing the refractive index spatial distribution of shear-induced deformed cells. By using these parameters, our data suggest that in vitro bone marrow derived dendritic cell (BMDC) maturation induces cell stiffening and reduces nucleus mobility, allowing to distinguish immature and mature dendritic cells. Overall, our method provides insights on intracellular mechanical properties of two dendritic cell states