Emerging Tools for Single-Cell Analysis

Index

line scanning approach for, 231 multiphoton multifocal microscopy for, 232

Video scanning conventions, 325 Vidicon, 328–329

Viscous forces, in microfluidics, 96 Visibility, in confocal microscopy, 263

Wagon-wheel effect, 264 Water-cooled lasers, 317 Watts, 307–308

White blood cells, microfabricated devices for, 102, 103f

White-light scanning digital microscopy, 291–306

averaging mode of, 299–300

brightness and color calibration in, 302–303 brightness and focus change in, 297–298 communications of, 300

comparison with camera systems, 303–305 contrast enhancement and suppression in,

298–299, 298f–299f detection system in, 294 future applications of, 306 image size for, 295

operational considerations with, 305–306 oversampling in, 297, 300–302, 301f

359

resolution of, super, 297, 300–302, 302f response function of, 299

scan rate of, 294 scanning spot in, 293–294

signal processing in, 294–296, 295f spot modulation in, 298f, 298–299 summing mode of, 299–300

zoom magnification in, 296–297, 296f–297f, 302

Wide-field illumination, in fluorescent lifetime imaging microscopy, 167–168

Xenon arc lamps, 309–310, 310f

Yield

of cell sorters, 14–15, 23 of rare-cell sorting, 51, 63

Yttrium aluminum garnet (YAG) lasers, 320 Yttrium vanadate (YVO4) lasers, 320

Zappers, 3 Zoom

for confocal microscopy, 245–246, 261, 264 for white-light scanning digital microscopy,

296–297, 296f–297f, 302

Emerging Tools for Single-Cell Analysis: Advances in Optical Measurement Technologies

Edited by Gary Durack, J. Paul Robinson

Copyright © 2000 Wiley-Liss, Inc.

ISBNs: 0-471-31575-3 (Hardback); 0-471-22484-7 (Electronic)

Color Plates

F i g . 7.15. Intensity and lifetime imaging of cellular chloride concentration. The cell is loaded with dihMEQ* and imaged under conditions with 80 MHz and below 4mW laser power at 747 nm using 40x/1.3NA F-Fluar Zeiss objective. The lifetime images are from the modulation measurements. The scale bar is 20 micron.

F i g . 7.17. Lifetime image of cellular chloride concentration from the phase measurement. The above

image displays τphase value of the same measurement shown in Fig. 7.16B′. Very low lifetime features are indicated by white arrows. The lifetime value for these spots is between 2.0–2.5 ns, which is likely due to

NADH (NAD+) autofluorescence in the mitochondria. The fact that τphase measures the low lifetime components is illustrated in Fig. 7.6B, where the phase lifetime stays low at high modulation even with less

than 30% of total intensity for the component with lower lifetime value.

Color Plates

F i g . 9.4. Two-photon imaging of macrophage cell line J774 incubated with FITC–BSA for various periods of time. Macrophage cell line J774 was incubated with FITC–BSA, and the fluorescence intensity (left panel) and fluorescence lifetime (right panel) were monitored at 1h, 5h, and 24h using two-photon fluorescence microscopy with time-resolved fluorescence lifetime imaging. As the probe was degraded within the cell, noticeable increases in fluorescence lifetime and intensity were detected. (Copyright Royal Microscopical Society, 1997. Reprinted with permission.)

Color Plates

F i g . 12.3. Shear force topography (A), feedback error signal (B), and fluorescence (C) images of CHO cells expressing a fusion construct of the EGFR and GFP; ex 488 nm, 160 nW.

A	B
1.7 m	90 kcps
	0 kcps
5 m
shear force topography	2PE fluorescence
C
1 kcps
0 kcps
3PE fluorescence

F i g . 12.7. 2PE and 3PE SNOM images of MCF7 cells. (A) Shear force topography, (B) 2PE fluorescence signal of the MitoTracker Orange-labeled mitochondria, and (C) 3PE fluorescence of the BBI-342- labeled nucleus. Scan parameters: 10 s/line, 128 lines, 256 points/line, excitation: 51 mW at 1064 nm.

Color Plates

F i g . 13.4. Example of a zoomed image. Here, a mosquito head is imaged using the microscopes 10x objective with no zoom (A), zoom at 1.5x (B), and zoom at 3x (C).

F i g . 13.6. Image enhancement features of COSMIC. The normal-mode image of Figure 4 (A), the image in contrast suppression mode (B), and the image in color-inverted mode (C). This mode is particularly useful to identify thin structures in cells where the color inversion highlights the objects.

A	B

F i g . 13.8. Superresolution effect is demonstrated with an image of the diatom Pleurasigma angulatum. The holes in the diatoms are approximately 0.25 m in diameter and hexagonal in shape. The full-color images were taken in white light. Obtaining such high-resolution images is very difficult with most other systems. Figure 13.8 (A) was taken with a 40x (0.75NA) objective. Figure 13.8 (B) was taken with a 100x (1.3NA oil) objective. However, the sample is in air under the cover slip, so the maximum usable NA for the 100x objective is less than 1.0. The condensor had an NA of 1.4 (oil).

Emerging Tools for Single-Cell Analysis: Advances in Optical Measurement Technologies

Edited by Gary Durack, J. Paul Robinson Copyright © 2000 Wiley-Liss, Inc.

ISBNs: 0-471-31575-3 (Hardback); 0-471-22484-7 (Electronic)

1

Cell-Sorting Technology

Gary Durack

University of Illinois at Urbana-Champaign, Urbana, Illinois

INTRODUCTION

Flow-cytometry-based cell sorting was first introduced to the research community more than 20 years ago. It is a technology that has been widely applied in many areas of life science research, serving as a critical tool for those working in fields such as genetics, immunology, molecular biology, and environmental science. Recent advances in underlying technologies, especially digital electronics, have made possible a new generation of high-speed cell-sorting devices. Instruments capable of processing more than 100,000 cells per second are becoming commercially available. Laboratories throughout the world are beginning to find new research and clinical applications for these high-speed cell sorters. On another technology front, rapid advances in MicroElectro-Mechanical Systems (MEMS) design and fabrication techniques are producing biochips that can sequentially process cells or other small particles (Masuda et al., 1989; Washizu et al., 1990). These micro–laboratory devices have the potential to revolutionize many cell measurement and processing techniques. The first five chapters of this text describe the state-of-the-art for these rapidly evolving technologies. They cover both theoretical considerations and practical application issues that should be of interest to both instrument designers and end users. This chapter provides a review of cell-sorting technology and serves as an introduction to those not already well acquainted with the field. It is followed by chapters covering

1

2	Cell-Sorting Technology

high-speed cell sorting (van den Engh), rare-event detection (Leary), clinical applications of high-speed cell sorting (Leemhuis), and MEMS-based cell-handling systems (Beebe). Taken together, this suite of chapters offers a comprehensive presentation of the recent advances in cell-sorting instrumentation and provides some insight into emerging technologies in this important area.

Cell Sorting Versus Cell Separation

A number of excellent texts have been published that exhaustively cover the physical principles that govern cell sorting (Melamed et al., 1990; Shapiro, 1995; Vandilla et al., 1985). The purpose of this chapter is not to duplicate those texts, but rather to provide a concise, comprehensive review of cell-sorting technology for those not already familiar with the field. It will also serve as a reference for the discussion of the emerging high-speed cell sorting, rare-event detection, and MEMS cell-handling technologies that appear in the subsequent chapters.

Unlike bulk cell separation techniques such as immuno-panning or magnetic column separation, flow-cytometry-based cell-sorting instruments measure, classify, and then sort individual cells or particles serially at rates of several thousand cells per second. This rapid “one-by-one” processing of single cells has made flow cytometry a unique and valuable tool for extracting highly pure subpopulations of cells from otherwise heterogeneous cell suspensions. Cells targeted for sorting are usually labeled in some manner with a fluorescent material. The fluorescent probes bound to a cell emit fluorescent light as the cell passes through a tightly focused, high-intensity light beam (usually a laser). A computer records emission intensities for each cell. These data are then used to classify each cell for specific sorting operations. Flow-cytometry-based cell sorting has been successfully applied to hundreds of cell types, cell constituents, and microorganisms as well as to many types of inorganic particles of comparable size.

Types of Cell Sorters

There are two basic types of cell sorters in wide use today: the droplet cell sorter and the fluid-switching cell sorter. The droplet cell sorter, first reported by Fulwyler (1965), utilizes microdroplets as “containers” to transport selected cells to a collection vessel. The microdroplets are formed by coupling ultrasonic energy to a jetting stream. Droplets containing cells selected for sorting are then electrostatically steered to the desired location, according to a technique first developed by Sweet (1965) for high-speed data recording. A droplet cell sorter can process selected cells at rates of tens of thousands of cells per second, limited primarily by the frequency of droplet generation and the time required for illumination. The weakness of a droplet sorter lies in the stability of the droplet generation system, which can be significantly disrupted by even temporary obstructions in the orifice of the droplet generator. Droplet sorters may also produce aerosols that can present intolerable biohazards when working with pathogenic material (Schmid et al., 1997; Merrill, 1981).

The second type of flow-cytometry-based cell sorter is the fluid-switching cell sorter. Most fluid-switching cell sorters utilize a piezoelectric device to drive a

Droplet Cell Sorters

3

mechanical system that diverts a segment of the flowing sample stream into a collection vessel. These systems were also developed in the late 1970s and early 1980s (Duhnen et al., 1983; Goehde and Shumann, 1987), and several excellent systems are commercially available today. Because they are closed systems, fluid-switching sorters fare better in both their stability of operation and their biosafety characteristics. They also typically employ flow channels with a greater cross-sectional area, which makes them less susceptible to blockage. Compared to droplet cell sorters, fluid-switching cell sorters have a lower maximum cell-sorting rate due to the cycle time of the mechanical system used to divert the sample stream. This cycle time, the time between initial sample diversion and restoration of stable nonsorted flow, is typically significantly greater than the period of a droplet generator on a droplet cell sorter. This longer cycle time limits fluid-switching cell sorters to processing rates of several hundred cells per second. For the same reason, the stream segment switched by a fluid cell sorter is usually at least 10 times the volume of a single microdrop from a droplet generator. This results in a correspondingly lower concentration of cells in the fluidswitching sorter collection vessel as compared to a droplet cell sorter collection vessel.

Not widely used but worthy of note is the photodamage technique for cell sorting, often referred to as the cell “zapper.” In this case a gated, high-energy laser pulse is used to render the cells not selected for sorting nonviable by inflicting photodamage to DNA (Herweijer and Stokdijk, 1988). Photosensitivity may be intrinsically or selectively induced by adding an appropriate DNA-specific fluorescent probe. This technique was recently described by Los Alamos National Laboratory (Martin et al., 1998) as a means for high-speed chromosome selection. The photodamage method has a much faster cycle time (<1 µs) than the period of a droplet generator. The weakness of this system lies in the expense of the gated pulsed laser system and in the need to calibrate the “death pulse” for the biological target. It also leaves a significant amount of cellular debris in the sorted fraction. At this time photodamage cell sorters are not commercially available.

DROPLET CELL SORTERS

Brief History

A schematic of a typical laser-based droplet cell sorter appears in Figure 1.1. The forerunner of this laser-based cell sorter was a Coulter volume instrument described by Fulwyler (1965), who elegantly combined fundamental flow cytometry with a rapid graph recorder technology developed by Sweet (1965) to produce an instrument that could sort cells based on variations in their volume. Like modern droplet sorters, Fulwyler’s sorter measured, classified, and then sorted individual cells by electrostatically steering microdroplets. Bonner et al. (1972) reported on a droplet sorting device that had been constructed in the Herzenberg laboratory and that utilized a modified Crosland-Taylor (1953) coaxial flow system, a 300-mW laser, and fluorescence measurement technology. This instrument incorporated all the major components of today’s modern droplet cell sorters. The Stanford Fluorescence Activated

4	Cell-Sorting Technology

F i g . 1.1. A typical laser-based droplet cell sorter.

Cell Sorter (FACS II) described by Herzenberg et al. (1976) clearly demonstrated the function and utility of droplet cell-sorting technology. Since the FACS II there have been a multitude of technological advancements in optics, electronics, and fluid handling; however, modern droplet cell sorters continue to be built around the fundamental designs developed for these early instruments. It is interesting to note and somewhat ironic that the very first droplet cell sorter provided Coulter volume measurement, yet today, no commercial supplier of droplet cell sorters offers this valuable measurement capability.

Components of Droplet Sorters

Figure 1.1 shows the basic components of a droplet cell sorter. The fluidic design is based on a variation of the Crosland-Taylor coaxial flow system. Figure 1.2 shows a cross section of this coaxial system. The outer stream, or sheath stream, usually consists of phosphate-buffered saline. The inner stream, or core stream, consists of the single-cell suspension to be sorted. The diameter of the core stream is determined by

Droplet Cell Sorters

5

the effective pressure differential between the sheath and the core. The conical geometry of the nozzle causes the sheath stream to undergo acceleration as it converges toward the exit orifice. The single-file stream of cells is introduced by the needle into the center of the sheath flow at the base of this inverted cone (point A in Fig. 1.1). At this point the sheath velocity is low due to the greater cross section of the channel. As flow continues down toward the exit orifice, the converging sheath focuses (narrows) the sample stream, thus accelerating the cells. This acceleration helps to separate the cells spatially along the axis of flow, and the converging sheath confines the cells to a narrow, predictable path at the center of the stream. As long as nonturbulent flow is maintained, the cells will pass through the laser interrogation point at close to constant velocity, reliably positioned near the center of the flow axis. This system of hydrodynamic focusing and coaxial flow serves three primary purposes. First, it creates spatial separation among the individual cells. Second, it aligns the cells at the center of the sheath stream’s cross section. Third, it causes the cells to traverse the distance between the measurement and droplet break-off points at very close to a constant velocity, an absolutely essential requirement for accurate cell sorting.

The vehicles that transport the cells to their respective collection vessels are the microdroplets, which ultimately break away from the stream at point B (Fig. 1.1). The droplet formation is based on principles first described by Lord Rayleigh (1879). Stable droplet generation is accomplished by coupling acoustic energy to the jetting stream. Typically, a piezoelectric device, driven by a sinusoidal signal, is used to transfer single-frequency acoustic waves to the exit nozzle tip, where they cause lowlevel modulation of the stream velocity. This velocity modulation induces periodic variations in the stream diameter. Another approach, often used for sorting large particles (Harkins and Galbraith, 1987), is to actually modulate the stream pressure, and thus its velocity, directly. Either method results in stream undulations that become more defined as the distance from the exit orifice increases. Eventually, the undula-

F i g . 1.2. Cross section of the stream for a typical droplet sorter. The ratio of the stream diameter to the core diameter is usually greater than 5 : 1.