Biophysical (mechanical and electrical) properties of living cells have been proven to play important roles in the regulation of various biological activities at the molecular and cellular level, and can serve as promising label-free markers of cells' physiological states. In the past two decades, a number of research tools have been developed for understanding the association between the biophysical property changes of biological cells and human diseases; however, technical challenges of realizing high-throughput, robust and easy-to-perform measurements on single-cell biophysical properties have yet to be solved. In this paper, we review emerging tools enabled by microfluidic technologies for single-cell biophysical characterization. Different techniques are compared. The technical details, advantages, and limitations of various microfluidic devices are discussed.

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Recent advances in microfluidic techniques for single-cell biophysical characterization

A critical review is presented on the use of linear and nonlinear Raman microspectroscopy in biomedical diagnostics of bacteria, cells, and tissues. This contribution is combined with an overview of the achievements of our research group. Linear Raman spectroscopy offers a wealth of chemical and molecular information. Its routine clinical application poses a challenge due to relatively weak signal intensities and confounding overlapping effects. Nonlinear variants of Raman spectroscopy such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) have been recognized as tools for rapid image acquisition. Imaging applications benefit from the fact that contrast is based on the chemical composition and molecular structures in a label-free and nondestructive manner. Although not label-free, surface enhanced Raman scattering (SERS) has also been recognized as a complementary biomedical tool to increase sensitivity. The current state of the art is evaluated, illustrative examples are given, future developments are pointed out, and important reviews and references from the current literature are selected. The topics are identification of bacteria and single cells, imaging of single cells, Raman activated cell sorting, diagnosis of tissue sections, fiber optic Raman spectroscopy, and progress in coherent Raman scattering in tissue diagnosis. The roles of networks-such as Raman4clinics and CLIRSPEC on a European level-and early adopters in the translation, dissemination, and validation of new methods are discussed.

The many facets of Raman spectroscopy for biomedical analysis.

Single cell analysis is an emerging field requiring a high level interdisciplinary collaboration to provide detailed insights into the complex organisation, function and heterogeneity of life. This review is addressed to life science researchers as well as researchers developing novel technologies. It covers all aspects of the characterisation of single cells (with a special focus on mammalian cells) from morphology to genetics and different omics-techniques to physiological, mechanical and electrical methods. In recent years, tremendous advances have been achieved in all fields of single cell analysis: (1) improved spatial and temporal resolution of imaging techniques to enable the tracking of single molecule dynamics within single cells; (2) increased throughput to reveal unexpected heterogeneity between different individual cells raising the question what characterizes a cell type and what is just natural biological variation; and (3) emerging multimodal approaches trying to bring together information from complementary techniques paving the way for a deeper understanding of the complexity of biological processes. This review also covers the first successful translations of single cell analysis methods to diagnostic applications in the field of tumour research (especially circulating tumour cells), regenerative medicine, drug discovery and immunology.

Making a big thing of a small cell – recent advances in single cell analysis

The bioimpedance spectroscopy (BIS) technique is potentially a useful tool to differentiate malignancy based on the variation of electrical properties presented by different tissues and cells. The different tissues and cells present variant electrical resistance and reactance when excited at different frequencies. The main purpose of this area of research is to use impedance measurements over a low-frequency bandwidth ranging from 1 kHz to 3 MHz to 1) differentiate the pathological stages of cancer cells under laboratory conditions and 2) permit the extraction of electrical parameters related to cellular information for further analysis. This provides evidence to form the basis of bioimpedance measurement at the cellular level and aids the potential future development of rapid diagnostics from biopsy materials. Three cell lines, representing normal breast epithelia and different pathological stages of breast cancer, have been measured using a standard impedance analyzer driving a four-electrode chamber filled with different cell suspensions. We identify the specific BIS profile for each cell type and determine whether these can be differentiated. In addition, the electrical parameters, e.g., the intracellular conductivity, membrane capacitance/capacity, characteristic frequency, are extracted by the use of equivalent circuit models and physical models to provide details of the cell electric signatures for further analysis of cancer cell pathology.

Bioimpedance Analysis for the Characterization of Breast Cancer Cells in Suspension

A peripheral nerve stimulator can be used to stimulate a peripheral nerve to treat essential tremor, Parkinson tremor, and other forms of tremor. The peripheral nerve stimulator can be either a noninvasive surface stimulator or an implanted stimulator. Stimulation can be electrical, mechanical, or chemical. Stimulation can be delivered using either an open loop system or a closed loop system with feedback.

Devices and methods for controlling tremor

Chip-based impedance measurement on single cells for monitoring sub-toxic effects on cell membranes.

Circulating tumor cells (CTCs) from blood of cancer patients are valuable prognostic markers and enable monitoring responses to therapy. The extremely low number of CTCs makes their isolation and characterization a major technological challenge. For label-free cell identification a novel combination of Raman spectroscopy with a microhole array platform is described that is expected to support high-throughput and multiplex analyses. Raman spectra were registered from regularly arranged cells on the chip with low background noise from the silicon nitride chip membrane. A classification model was trained to distinguish leukocytes from myeloblasts (OCI-AML3) and breast cancer cells (MCF-7 and BT-20). The model was validated by Raman spectra of a mixed cell population. The high spectral quality, low destructivity and high classification accuracy suggests that this approach is promising for Raman activated cell sorting.

Raman-Spectroscopy Based Cell Identification on a Microhole Array Chip

The invention relates to a method, and to a device wearable on the body, for reducing tremor in a body part. The device (30) comprises a sensor for generating a signal that correlates with a tremor in the body part; a regulator designed to determine a manipulated variable as a function of the signal from the sensor; and a mass displacement device comprising at least one mass element, which mass displacement device is designed to move the at least one mass element relative to the body part as a function of the manipulated variable, in such a way that the movement of the at least one mass element exerts a counterforce on the body part, which counteracts a movement of the body part induced by tremor.

Device and method for reducing tremor

Highly-sensitive analysis systems based on cellular multi-parameter are needed in the diagnostics. Therefore we improved our previously developed chip platform for another additional analysis method, the fluorescence in situ hybridization. Fluorescence in situ hybridization (FISH) is a technique used in the diagnostics to determine the localization and the presence or absence of specific DNA sequence. To improve this labor- and cost-intensive method, we reduced the assay consumption by a factor of 5 compared to the standard protocol. Microhole chips were used for making the cells well addressable. The chips were fabricated by semiconductor technology on the basis of a Silicon wafer with a thin deposited silicon nitride layer (Si 3 N 4 ). Human retina pigment epithelia (ARPE-19) cells were arrayed on 5-μm holes of a 35×35 microhole-array by a gently negative differential pressure of around 5 mbar. After 3 hours of incubation the cells were attached to the chip and the FISH protocol was applied to the positioned cells. A LabView software was developed to simplify the analysis. The software automatically counts the number of dots (positive labeled chromosome regions) as well as the distance between adjacent dots. Our developed platform reduces the assay consumption and the labor time. Furthermore, during the 3 hours of incubation non-invasive or minimal-invasive methods like Raman- and impedance-spectroscopy can be applied.

Towards a cellular multi-parameter analysis platform: Fluorescence in situ hybridization (FISH) on microhole-array chips

Highly productive cells are required for the manufacture of biopharmaceutical proteins, but the best-performing cells can be difficult to isolate from a large and heterogeneous background. Although this problem has been addressed by gently positioning individual cells on microhole arrays, such a procedure is typically monitored by microscope which limits the size of the array to the field view of the microscope and also requires the use of direct illumination, which bleaches the sensitive fluorescent reagents needed to identify the most productive cells. We have circumvented these problems by developing an impedance-based method for cell positioning, and by defining a relationship between impedance and occupancy we are also able to monitor the progress of the cell positioning process. We identified cells secreting large amounts of a model antibody by mixing the cells with semi-solid medium and a light-sensitive antibody detection reagent, and then using impedance controlled positioning to capture cells on the microholes in absence of direct light. Following a 1-day incubation, antibody secretion could be determined qualitatively using fluorescence imaging and a simple image processing algorithm. For the first time, we have shown that antibody secretion from individual cells on a microhole array can be classified and used to identify the most productive cells. Because this procedure does not rely on optical analysis it could easily be scaled up to allow the screening of millions of cells at a time.

Christian M. Kurz

Papers

Chip-based impedance measurement on single cells for monitoring sub-toxic effects on cell membranes.

Raman-Spectroscopy Based Cell Identification on a Microhole Array Chip

Device and method for reducing tremor

Towards a cellular multi-parameter analysis platform: Fluorescence in situ hybridization (FISH) on microhole-array chips

Impedance-controlled cell entrapment using microhole-array chips allows the isolation and identification of single, highly productive cells