Microfluidic Impedance Cytometry: Measuring Single Cells at High Speed

AbstractLight-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 μm) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

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Abstract 15707: Fluorescence Voltage Recordings From Stem Cell-Derived Cardiomyocytes Enable High-Throughput Screening for Cardiotoxicity

Circulation ◽

10.1161/circ.130.suppl_2.15707 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Jordan S Leyton-Mange ◽

Robert W Mills ◽

Min-Young Jang ◽

Xaio Ling ◽

Patrick T Ellinor ◽

...

Keyword(s):

Stem Cell ◽

High Throughput ◽

High Throughput Screening ◽

High Speed ◽

Single Cells ◽

Drug Application ◽

Negative Control ◽

Optical Measurements ◽

Drug Induced ◽

Dose Dependent

Introduction: The lack of high quality predictive models for drug-induced QT prolongation continues to be a significant problem in pharmaceutical development. While human pluripotent stem cell derived-cardiomyocytes (hPSC-CMs) hold promise to be a valuable tool for drug discovery, efforts have been frustrated by the labor-intensive nature of electrophysiological recordings and the heterogeneity of hPSC-CMs populations. Methods: Using lentivirus, we introduced the genetically encoded fluorescent voltage reporter, A242-Arclight, into hPSC-CM monolayers in multi-well plates. An inverted fluorescence microscope was fit with an environmentally controlled enclosure and automated stage. High speed imaging with a Photometrics Evolve 128 EMCCD camera was performed at baseline and after administration of test compounds. Optical traces were processed using a custom program and composite AP durations, APD80, were compared before and after drug application (Figures A & B). Results: Baseline APD80 values displayed high degree of consistency between wells: 483±59 msec. High-throughput data acquisition demonstrated dose dependent APD80 increases from all QT-prolonging agents tested as well as dose dependent APD80 decrease from pinacidil. In contrast, negative control compounds caused no significant changes in APD80. Results from a representative plate are shown (Figure C). Conclusions: Optical measurements provide rapid recordings of drug-induced AP duration changes, and offer a strategy to non-invasively screen hPSC-CMs in high-throughput. Recording from cell monolayers as opposed to single cells and using paired comparisons may be beneficial in addressing the heterogeneity amongst hPSC-CM preparations.

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A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis

Microorganisms ◽

10.3390/microorganisms9040680 ◽

2021 ◽

Vol 9 (4) ◽

pp. 680

Author(s):

Christopher T. Evans ◽

Sara J. Baldock ◽

John G. Hardy ◽

Oliver Payton ◽

Loren Picco ◽

...

Keyword(s):

Atomic Force Microscopy ◽

High Resolution ◽

High Speed ◽

Single Cells ◽

Sample Surface ◽

Live Cells ◽

Contact Mode ◽

Force Microscopy ◽

Atomic Force ◽

Afm Analysis

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

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An optofluidic system with integrated microlens arrays for parallel imaging flow cytometry

Lab on a Chip ◽

10.1039/c8lc00593a ◽

2018 ◽

Vol 18 (23) ◽

pp. 3631-3637 ◽

Cited By ~ 11

Author(s):

Gregor Holzner ◽

Ying Du ◽

Xiaobao Cao ◽

Jaebum Choo ◽

Andrew J. deMello ◽

...

Keyword(s):

Flow Cytometry ◽

High Speed ◽

Parallel Imaging ◽

Single Cells ◽

Rapid Analysis ◽

High Speed Imaging ◽

Microlens Arrays ◽

Imaging Flow Cytometry

In recent years, high-speed imaging has become increasingly effective for the rapid analysis of single cells in flowing environments.

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ANALYTICAL AND NUMERICAL MODELING METHODS FOR IMPEDANCE ANALYSIS OF SINGLE CELLS ON-CHIP

NANO ◽

10.1142/s1793292008000800 ◽

2008 ◽

Vol 03 (01) ◽

pp. 55-63 ◽

Cited By ~ 38

Author(s):

TAO SUN ◽

NICOLAS G. GREEN ◽

HYWEL MORGAN

Keyword(s):

Numerical Modeling ◽

High Speed ◽

Single Cell Analysis ◽

Single Cells ◽

Equivalent Circuit Model ◽

Microfluidic Channel ◽

Electrical Impedance Spectroscopy ◽

Static Case ◽

Immobilized Cell ◽

Modeling Methods

Electrical impedance spectroscopy (EIS) is a noninvasive method for characterizing the dielectric properties of biological particles. The technique can differentiate between cell types and provide information on cell properties through measurement of the permittivity and conductivity of the cell membrane and cytoplasm. In terms of lab-on-a-chip (LOC) technology, cells pass sequentially through the microfluidic channel at high speed and are analyzed individually, rather than as traditionally done on a mixture of particles in suspension. This paper describes the analytical and numerical modeling methods for EIS of single cell analysis in a microfluidic cytometer. The presented modeling methods include Maxwell's mixture theory, equivalent circuit model and finite element method. The difference and advantages of these methods have been discussed. The modeling work has covered the static case — an immobilized cell in suspension and the dynamic case — a moving cell in the channel.

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Encapsulated Cell Dynamics in Droplet Microfluidic Devices with Sheath Flow

Micromachines ◽

10.3390/mi12070839 ◽

2021 ◽

Vol 12 (7) ◽

pp. 839

Author(s):

Peter E. Beshay ◽

Ali M. Ibrahim ◽

Stefanie S. Jeffrey ◽

Roger T. Howe ◽

Yasser H. Anis

Keyword(s):

Flow Field ◽

High Speed ◽

Single Cells ◽

Microfluidic Channel ◽

Internal Flow ◽

Shear Stresses ◽

Rotational Flow ◽

Label Free ◽

Cell Dynamics ◽

Sheath Flow

In this paper we study the dynamics of single cells encapsulated in water-in-oil emulsions in a microchannel. The flow field of a microfluidic channel is coupled to the internal flow field of a droplet through viscous traction at the interface, resulting in a rotational flow field inside the droplet. An encapsulated single cell being subjected to this flow field responds by undergoing multiple orbits, spins, and deformations that depend on its physical properties. Monitoring the cell dynamics, using a high-speed camera, can lead to the development of new label-free methods for the detection of rare cells, based on their biomechanical properties. A sheath flow microchannel was proposed to strengthen the rotational flow field inside droplets flowing in Poiseuille flow conditions. A numerical model was developed to investigate the effect of various parameters on the rotational flow field inside a droplet. The multi-phase flow model required the tracking of the fluid–fluid interface, which deforms over time due to the applied shear stresses. Experiments confirmed the significant effect of the sheath flow rate on the cell dynamics, where the speed of cell orbiting was doubled. Doubling the cell speed can double the amount of extracted biomechanical information from the encapsulated cell, while it remains within the field of view of the camera used.

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Microfluidic impedance cytometry device with N-shaped electrodes for lateral position measurement of single cells/particles

Lab on a Chip ◽

10.1039/c9lc00819e ◽

2019 ◽

Vol 19 (21) ◽

pp. 3609-3617 ◽

Cited By ~ 7

Author(s):

Dahou Yang ◽

Ye Ai

Keyword(s):

Single Cells ◽

Lateral Position ◽

Position Measurement ◽

Continuous Flows ◽

Impedance Cytometry

In this paper, we present an N-shaped electrode-based microfluidic impedance cytometry for the measurement of the lateral position of single cells and particles in continuous flows.

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Biophysical mechanism of ultrafast helical twisting contraction in the giant unicellular ciliate Spirostomum ambiguum

10.1101/854836 ◽

2019 ◽

Cited By ~ 1

Author(s):

L. X. Xu ◽

M. S. Bhamla

Keyword(s):

Physical Model ◽

High Speed ◽

Structural Changes ◽

Single Cells ◽

Cellular Level ◽

Cell Biophysics ◽

High Speed Imaging ◽

Simple Physical Model ◽

Biophysical Mechanism ◽

Spirostomum Ambiguum

The biophysical mechanism of cytoskeletal structures has been fundamental to understanding of cellular dynamics. Here, we present a mechanism for the ultrafast contraction exhibited by the unicellular ciliate Spirostomum ambiguum. Powered by a Ca2+ binding myoneme mesh architecture, Spirostomum is able to twist its two ends in the same direction and fully contract to 75% of its body length within five milliseconds, followed by a slow elongation mechanism driven by the uncoiling of the microtubules. To elucidate the principles of this rapid contraction and slow elongation cycle, we used high-speed imaging to examine the same-direction coiling of the two ends of the cell and immunofluorescence techniques to visualize and quantify the structural changes in the myoneme mesh, microtubule arrays, and the cell membrane. Lastly, we provide support for our hypotheses using a simple physical model that captures key features of Spirostomum’s ultrafast twisting contraction.SIGNIFICANCEUltrafast movements are ubiquitous in nature, and some of the most fascinating ultrafast biophysical systems are found on the cellular level. Quantitative studies and models are key to understand the biophysics of these fast movements. In this work, we study Spirostomum’s ultrafast contraction by using high-speed imaging, labeling relevant cytoskeletal structures, and building a physical model to provide a biophysical mechanism especially of the helical same direction twisting of this extremely large single cell organism. Deeper understanding of how single cells can execute extreme shape changes hold potential for advancing basic cell biophysics and also inspire new cellular inspired actuators for engineering applications.

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