The advancement of blood cell research by optical tweezers

Demonstration of the light radiation pressure on a microscopic level by A. Ashkin led to the invention of optical tweezers (OT). Applied in the studies of living systems, OT have become a preferable instrument for the noninvasive study of microobjects, allowing manipulation and measurement of the mechanical properties of molecules, organelles, and cells. In the present paper, we overview OT applications in hemorheological research, placing emphasis on red blood cells but also discussing OT applications for the investigation of the biomechanics of leukocytes and platelets. Blood properties have always served as a primary parameter in medical diagnostics due to the interconnection with the physiological state of an organism. Despite blood testing being a well-established procedure of conventional medicine, there are still many complex processes that must be unraveled to improve our understanding and contribute to future medicine. OT are advancing single-cell research, promising new insights into individual cell characteristics compared to the traditional approaches. We review the fundamental and practical findings revealed in blood research through the optical manipulation, stretching, guiding, immobilization, and inter-/intracellular force measurements of single blood cells

Depolarization increases cellular light transmission

Application of optical methods to human brain tissue in vivo, e.g., measuring oxyhemoglobin and deoxyhemoglobin concentration changes with near-infrared spectroscopy (NIRS), requires the a priori assumption that background optical properties remain unchanged during measurements1,2. However, fundamental knowledge about light scattering by brain cells per se remains sparse; many factors influence light transmission changes through living brain tissue, bringing into question what is being measured. We have observed slow wave-ring spreads of light transmission changes on the rat cerebral cortex during potassium-induced cortical spreading depression (CSD) and ascribed them to squeezing-out of blood from capillaries by swollen brain cells3,4. However, in rat hippocampal slices, where no blood components were involved, similar light transmission changes were observed during K+-induced CSD and ascribed to cell swelling and dendritic beading5,6,7. Here we show that two-dimensional light scattering changes occur through suspensions of osmotically swollen (depolarized) red blood cells, apparently arising from light scattering changes at the less curved, swollen surface of the steep electrochemical gradient coupled with water activity difference across the plasmic membrane. These optical property changes are likely to be relevant to interpretation of photometry or spectroscopy findings of brain tissue in vivo, where neurons are polarizing and depolarizing during brain function

Hemodynamics

Hemodynamics is study of the mechanical and physiologic properties controlling blood pressure and flow through the body. The factors influencing hemodynamics are complex and extensive. In addition to systemic hemodynamic alterations, microvascular alterations are frequently observed in critically ill patients. The book "Hemodynamics: New Diagnostic and Therapeuric Approaches" is formed to present the up-to-date research under the scope of hemodynamics by scientists from different backgrounds

Optical Diagnostics in Human Diseases

Optical technologies provide unique opportunities for the diagnosis of various pathological disorders. The range of biophotonics applications in clinical practice is considerably wide given that the optical properties of biological tissues are subject to significant changes during disease progression. Due to the small size of studied objects (from μm to mm) and despite some minimum restrictions (low-intensity light is used), these technologies have great diagnostic potential both as an additional tool and in cases of separate use, for example, to assess conditions affecting microcirculatory bed and tissue viability. This Special Issue presents topical articles by researchers engaged in the development of new methods and devices for optical non-invasive diagnostics in various fields of medicine. Several studies in this Special Issue demonstrate new information relevant to surgical procedures, especially in oncology and gynecology. Two articles are dedicated to the topical problem of breast cancer early detection, including during surgery. One of the articles is devoted to urology, namely to the problem of chronic or recurrent episodic urethral pain. Several works describe the studies in otolaryngology and dentistry. One of the studies is devoted to diagnosing liver diseases. A number of articles contribute to the studying of the alterations caused by diabetes mellitus and cardiovascular diseases. The results of all the presented articles reflect novel innovative research and emerging ideas in optical non-invasive diagnostics aimed at their wider translation into clinical practice

Application of Luminescent Materials to Optical Sensing

Development of sensors for detection of various chemical and biological species is an important and ever-growing field. In particular, optical-based sensors enable a remote, rapid method for continuous or on-demand monitoring. Monitoring humidity is important across many applications, such as humidity control within moisture-sensitive environments and in medical, semiconductor, and food science fields. Following a study of photobleaching, defect-related emission of zinc oxide nanoparticles was monitored as a function of relative humidity. An important next step is its application to monitoring toxic gases, as air pollution has been identified as a major health concern. Of importance for the biomedical field is monitoring key blood analytes for human health. Monitoring blood pH is critical for specific patient groups, such as those suffering from diabetic ketoacidosis and congenital lactic acidosis. A pH-sensitive fluorophore was loaded within red blood cells for use as a continuous blood analyte monitor. Future work will focus on glucose, as current estimates show that one out of every three children born in 2000 will develop diabetes in his or her lifetime – thus, the global impact of this disease is immense. Results from ZnO studies indicate that photobleaching is related to the surface area to volume ratio. ZnO nanoparticles display a linear response to humidity with a sensitivity of 0.008417 RH^-1 and 0.01898 RH^-1 for nitrogen and air environments, respectively. Owing to reversibility and high sensitivity, ZnO nanoparticles have great potential as optical-based environmental sensors. Results from dye-loaded ghost studies indicate that fluorescence intensity of intracellular dyes report on extracellular pH. Resealed ghosts loaded with a fluorescein isothiocyanate-glycylglycine conjugate reversibly track pH with a resolution down to 0.014 pH unit. For use in vivo, the development of an NIR pH-sensitive dye was paramount. Unfortunately, all NIR dyes tested exhibited poor pH sensitivity while displaying sensitivity to external factors (e.g., temperature, concentration, proteins). However, circulation kinetics of resealed ghosts were easily monitored once injected in vivo with an optical fiber-based system. Although the cells were rapidly removed from circulation, the loaded ghosts resulted in higher signal than would be expected for free dye alone. Once optimized, the resealed ghosts could serve as a long-term, continuous, circulating biosensor for the management of diseases

Plasma Volume Hematocrit (PVH): “Big Data” Applied to Physiology Enabled by a New Algorithm

This work describes the ongoing analysis of blood noninvasively in vivo along with the in vitro validation of the algorithm. The blood is taken as two components, red blood cells and plasma, both of which cause elastic emission (from Mie and Rayleigh scattering) and inelastic emission (from fluorescence and Raman emission). The algorithm describes the linear dependence of the volume fractions of both red blood cells and plasma with both the elastic and inelastic emissions where the two equations are independent. These equations are used to calculate the Hematocrit which is defined as the volume fraction of red blood cells in the total volume of blood. We believe that monitoring changes in the Hematocrit with sufficient sensitivity could give information about many physiological parameters including an early indication for internal hemorrhaging. The stability of the baseline was analyzed in 10 test subjects across 29 experiments including over 8 million frames of data to give the smallest physiological increment of ±0.033 Hematocrit units. Compared to the medical standard blood draw method, with a standard deviation of ±2.0 Hematocrit units, our device is 60 times more sensitive to changes in the Hematocrit. Repeating patterns in the Hematocrit can be analyzed by a Fourier transform to give respiration rate and pulse rate earning the title of “big data.” Changes in the Hematocrit were also observed in dialysis patients (where the blood is manually cleaned due to kidney failure) and in a rat model where large portions of the blood can be removed and reintroduced. Blood loss and addition of fluid reveal changes in the Hematocrit that are distinguishable from the baseline. The algorithm was validated by a well-defined in vitro system modeling the blood components. The model demonstrates that an optically thin sample in the linear range produces a good fit by the algorithm. Finally, the blood was analyzed in vitro to demonstrate that the red blood cells and plasma show linearity within the physiological ranges observed in vivo. At 830 nm excitation, the same wavelength used in vivo, volume fractions of red blood cells and plasma at the physiological range demonstrate linearity. All of the experiments and analysis appear to give evidence supporting the measuring of changes in the Hematocrit noninvasively in vivo on a medically useful timescale

Quantification of red blood cell adhesion using holographic optical tweezers and single cell force spectroscopy

In dependence on the ambient conditions, red blood cells tend to form aggregates. In this work two different adhesion phenomena of red blood cells were investigated. In order to investigate these phenomena, holographic optical tweezers, microfluidics and single cell force spectroscopy were used. The first investigated adhesion process takes place when activated platelets release a messenger that triggers the red blood cells to aggregate. The second adhesion process involves macromolecules that exert an osmotic pressure onto the red blood cells. These macromolecules trigger the cells to form aggregates that look similar to a stack of coins. Both phenomena could be investigated statistically as well as quantitatively.Rote Blutzellen neigen in Abhängigkeit der Umgebungsbedingungen dazu Aggregate zu bilden. In dieser Arbeit wurden zwei verschiedene Adhäsions-Phänomene roter Blutzellen untersucht und quantifiziert. Hierbei kamen Instrumente wie die optische Pinzette, Mikrofluidiken und Einzelzell-Spektroskopie zum Einsatz. Das erste untersuchte Adhäsionsphänomen findet während der Blutgerinnung statt, wenn aktivierte Blutplättchen Botenstoffe aussenden, die die roten Blutzellen zur Aggregation stimulieren. Das zweite untersuchte Adhäsionsphänomen tritt im statischen Blut oder bei geringen Scherraten auf. In diesen Fällen neigen rote Blutzellen dazu lineare Aggregate zu bilden die dem Abbild von Geldrollen ähneln. Diese unspezifisch auftretende Adhäsion erfolgt infolge eines osmotischen Drucks umgebender Makro-Moleküle. Beide Adhäsions-Phänomene konnten sowohl statistisch untersucht, als auch mittels der Einzelzell-Spektroskopie quantifiziert werden

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