Sequential Array Cytometry: Multi-Parameter Imaging with a Single Fluorescent Channel

Abstract

Abstract:Heterogeneity within the human population and within diseased tissues necessitates a personalized medicine approach to diagnostics and the treatment of diseases. Functional assays at the single-cell level can contribute to uncovering heterogeneity and ultimately assist in improved treatment decisions based on the presence of outlier cells. We aim to develop a platform for high-throughput, single-cell-based assays using well-characterized hydrodynamic cell isolation arrays which allow for precise cell and fluid handling. Here, we demonstrate the ability to extract spatial and temporal information about several intracellular components using a single fluorescent channel, eliminating the problem of overlapping fluorescence emission spectra. Integrated with imaging technologies such as wide field-of-view lens-free fluorescent imaging, fiber-optic array scanning technology, and microlens arrays, use of a single fluorescent channel will reduce the cost of reagents and optical components. Specifically, we sequentially stain hydrodynamically trapped cells with three biochemical labels all sharing the same fluorescence excitation and emission spectrum. These markers allow us to analyze the amount of DNA, and compare nucleus-to-cytoplasm ratio, as well as glycosylation of surface proteins. By imaging cells in real-time we enable measurements of temporal localization of cellular components and intracellular reaction kinetics, the latter is used as a measurement of multi-drug resistance. Demonstrating the efficacy of this single-cell analysis platform is the first step in designing and implementing more complete assays, aimed toward improving diagnosis and personalized treatments to complex diseases.

Integrating Immunology and Microfluidics for Single Immune Cell Analysis

“…Later in 2009, Faley et al presented their design of a microfluidic chip with multiple traps that was used to study signaling dynamics of isolated, individual, hematopoietic stem cells ( 99 ). Besides these studies, several groups have used hydrodynamic cell trap arrays for multiple applications in biology and chemistry ( 100 – 102 ). Of all these groups, the Voldman laboratory has extensively used a modified version of the hydrodynamic cell trap design for specifically studying immune cell interactions at single-cell level ( 103 – 106 ).…”

A microfluidic device enabling high-efficiency single cell trapping

“…Subsequently, this group extended the design to extract intracellular spatial-temporal information via fluorescent imaging, confirming its capability of high-throughput single cell analysis. 26 Very recently, Zhang et al 27 improved the design by positioning cell trap sites on the side of the channel walls. Though it recorded success in trapping cells with predefined patterns, this approach is based on stochastic flow and cannot work with rare cell samples.…”

A label-free microfluidic assay to quantitatively study antibiotic diffusion through lipid membranes

“…The small sample amounts required, high throughput capabilities and controlled microenvironments involved offer many advantages for the development of drug assays 5,6,7,8,9,10 . In this work, we develop a novel microfluidic technique to quantitatively examine the passive diffusion of antibiotics across lipid membranes.…”