Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Summary

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

Abstract

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture – information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Introduction

Optical imaging presents a powerful tool and cell-based assay to (almost) non-invasively visualize the detailed spatial distribution of many cellular/subcellular components, thus uncovering a multitude of morphological, biophysical, and biomolecular signatures of cells. However, this ability to extract high-content information from single cells has generally been compromised when an enormous and heterogeneous population of cells had to be investigated. This marks a common trade-off in cell-based assays between measurement throughput and content. A notable example is that adding imaging capability to flow cytometry has resulted in a down-scaling of throughput by at least 1-2 orders of magnitude compared to that of the classical non-imaging flow cytometers. Although it could offer complex morphological single-cell analysis that is not possible with standard flow cytometers¹, imaging flow cytometry generally lacks sufficient throughput to identify cellular heterogeneity with high statistical confidence. This is necessary for new discoveries in biology and for gaining an understanding of the pathogenesis of diseases. The key technical challenge lies in the inherent speed limit imposed by the common optical imaging strategies: laser-beam scanning, (e.g., by galvanometric mirrors), and/or image sensors (e.g., charge-coupled device (CCD) and complementary metal-oxide semiconductor (CMOS)). The laser scanning speed is intrinsically restricted by the mechanical inertia of the scanning mirrors, whereas the frame rate of CCD or CMOS is limited by the fundamental trade-off between imaging speed and sensitivity (i.e., increasing the frame rate leads to reduced signal detection sensitivity, and vice versa).

Based on an all-optical, ultrafast image-encoding mechanism, optical time-stretch imaging has been demonstrated as an attractive platform for high-throughput imaging flow cytometers, without need of the conventional image sensors or mechanical laser scanning^2,3. Detailed descriptions of the working principle of time-stretch imaging can be found in references^4,5,6,7. In brief, it consists of two interchangeable mapping steps: (i) spectral encoding (wavelength-space mapping), in which the spatial coordinates of the imaged specimen are mapped to different wavelengths across the spectrum of the light-pulsed beam^8,9, and (ii) a dispersive Fourier transformation (wavelength-time mapping)⁹, in which the wavelength components of individual laser pulses are transformed (stretched) via group velocity dispersion (GVD) into temporal (wavelength-swept) waveforms (Figure 1). An important feature of time-stretch imaging is optical amplification, which plays a critical role in combatting the loss of sensitivity due to ultrafast photodetection and GVD loss, thus enhancing the image signal-to-noise ratio (SNR) without being contaminated by the photodetector noise⁹. Since each laser pulse encodes a line-scan of the imaged specimen, which is orthogonal to the unidirectional flow of the cells, an effective line-scan rate is determined by the laser repetition rate, which is typically beyond 10 MHz. This ultrafast operation enables blur-free, single-cell image capture at a throughput of 10,000-100,000 cells/s (i.e., 10-100 times higher than conventional imaging flow cytometry). As a result, time-stretch imaging could find unique applications in high-throughput, single-cell, image-based screening, especially when there is a need for identifying unknown heterogeneity or rare/aberrant cells within a sizable population (thousands to millions of cells), such as rare cancer cell screening¹⁰ or micro-algae classification¹¹.

Time-stretch imaging predominantly relies on bright-field (BF) image capture, from which the image contrast is generated through light scattering and absorption from the cells^2,3,9,10,11. Such label-free, single-cell imaging capabilities could bypass the detrimental effects associated with the fluorescent labels, such as cytotoxicity and photobleaching, and yet provide valuable information for single-cell analysis based on the cellular and sub-cellular texture and morphology. These label-free parameters are proven to be effective for the deep image classification of cells, especially when an enormous cell population is available^11,12. However, in many occasions, BF imaging fails to provide sufficient contrast to reveal the detailed morphology of the label-free transparent cells. Different label-free, phase-contrast, time-stretch imaging modalities have been developed for enhancing the imaging contrast at ultrafast frame rates^13,14,15. Among these techniques, asymmetric-detection time-stretch optical microscopy (ATOM) was developed to reveal the phase-gradient (differential-interference-contrast-(DIC)-like) contrast based on a concept similar to Schlieren photography, enabling the label-free, high-contrast imaging of single cells at an ultrahigh microfluidic speed (up to 10 m/s)¹⁶. This effect can be readily generated through oblique detection or illumination by partially blocking the image-encoded beam path or tilting the beam before photodetection. Another advantage of ATOM is its ability to simultaneously acquire two phase-gradient contrasts along opposite orientations. Intensity subtraction and summation of two opposite-contrast images yield the differential phase-gradient contrast and the absorption contrast, respectively, from the same line-scan. This work presents a detailed protocol describing the implementation of ATOM, including the establishment of the optical setup, the sample preparation, and the data acquisition and visualization. Specifically, this work demonstrates the ATOM operation with single-cell imaging of human blood cells, cancer cells, and phytoplankton (microalgae). This highlights the applicability of ATOM to imaging flow cytometry, not only in the biomedical arena, but also in marine and biofuel research^17,18.

Protocol

1. Sample Preparation Sample preparation (adherent cells; MCF-7 cells) Take out the cell culture dish from the incubator and drain the culture medium. Rinse the cells on a dish with 1x phosphate-buffered saline (PBS) to remove excessive culture medium. Add 3 mL of a solution of 0.25% trypsin to the culture dish (diameter of 100 mm) and put it in a 37 °C, 5% CO2 incubator for 4 min. NOTE: The trypsin dissolves the adhesive protein of th…

Representative Results

This work illustrates two single-cell imaging demonstrations by ATOM: one with mammalian cells (human peripheral blood mononuclear cells (PBMC) and breast cancer cells (MCF-7)) and another with phytoplanktons (Scenedesmus and Chlamydomonas). The first experiment was motivated by the growing interest in liquid biopsy for the detection, enumeration, and characterization of circulating tumor cells (CTCs) in the blood23. The ability to…

Discussion

There are several technical details that require special attention during the ATOM system setup. First, it is essential to note that asymmetric/oblique spectrally-encoded illumination could introduce residual phase-gradient components (i.e., the shadowing effect) in the absorption contrast and influence the enhancement of phase-gradient contrast in ATOM. Therefore, this effect of oblique illumination should be minimized. Second, it should be emphasized that the time-multiplexing or time-interleaving schemeinvolv…

Materials

Nikon Plan Fluorite Physiology Objectives 40X	Nikon	MRF07420	Objective lens (Obj2)
Olympus Plan Fluorite Objective, 0.75 NA, 0.51 mm WD, 40X	Olympus	RMS40X-PF	Objective lens (Obj1)
N-BK7 Plano-Convex Lenses	Thorlabs	LA1145-C	For relaying spectral shower
FC/APC Fiber Collimation Package	Thorlabs	F220APC-1064	For outputing and collecting laser pulses
Pellicle beamsplitter	Thorlabs	BP145B3	For making beam replica
Protected silver mirror	Thorlabs	PF10-03-P01	For reflecting light
800-1650nm 12GHz single mode DC-coupled NIR Photoreceiver	Newport	1544-B	For converting light into electrical signal
Infiniium High-Performance Oscilloscope	Agilent	DSOX91604A	To save the light-converted electrical signals
HI1060 optical fiber	Corning	HI1060	Optical fiber for time-stretch
YTTERBIUM DOPED FIBER AMPLIFIER	Keopsys	KPS-STD-BT-YFA-37-BO-SM-111-FA-FA	Optical in-line amplifier
Holographic grating	Wasatch Photonics	020305-6	Grating
Infuse/Withdraw Syringe Pumps	Harvard Apparatus	PHD 2000	Syringe pump for sample loading in micro-fluidic channels