In hematology and immunology, flow cytometry is a well-established diagnostic tool, used to count and differentiate blood cells. Passing in single file through an interaction region, the cells are distinguished with respect to light scatter, impedance change, and fluorescence intensity if stained by monoclonal antibodies.1,2 Of several approaches suggested over the past decade,3–6 integrating a sample preparation3,4 prior to fabricating microfluidic systems for one-time use seems especially promising. Such low-cost disposable microfabricated flow cytometers (see Figure 1) would have point-of-care uses, applications in emergency medicine, and also facilitate delivery of services in developing countries.
Figure 1. Microfabricated flow cytometer. Tubes on top of device carry fluid. A fiber-coupled light source (not visible) delivers 488nm blue light to the interaction region. Some of the light continues, unscattered, into the axial light loss (ALL) fiber. Another fiber gathers the forward light scattering (FLS) emissions. These fibers are visible because blue light leaks from the cladding. Light is also scattered at 42° in side scattering (SSC), and at 90° downward.
Figure 2. Structure of the microfabricated flow cytometer with cascaded hydrodynamic focusing. Grooves for mounting source and detection fibers are indicated. ALL: axial light loss. FLS: forward light scattering. SSC: side scattering. OLS: orthogonal light scattering. Fluorescence and OLS are collected with an external objective with numerical aperture 0.5. Platinum electrodes are mounted outside the microstructure and connected by conducting liquid channels.
Two-dimensional positioning of particles within the fluidic channel by hydrodynamic focusing is critical to flow cytometric analysis. However, lithography-based manufacturing techniques are limited to structures of one specific channel height or, when using multilayer lithography, to a small number (e.g., three) of different heights.6 Although these latter structures have been shown to allow two-dimensional hydrodynamic focusing and high-throughput particle detection, supplying sheath fluid through several input ports while maintaining defined pressure differences remains a drawback.
To overcome these restrictions and to develop microfluidic structures for practical applications, we used a different approach: hot embossing, with an embossing tool fabricated by an ultra-precision milling technique. Complex three-dimensional structures can be readily fabricated. Also multistage hydrodynamic focusing can be achieved using a single inlet port for the sheath fluid.
Figure 2 shows the layout of a microfluidic structure for flow cytometric analyses. Hot embossing in polycarbonate plates of 1.5mm thickness was used to produce the microchips. Optical fibers were inserted into the corresponding grooves and subsequently top and bottom plates were welded by laser radiation.
Cascaded two-stage hydrodynamic focusing reduces velocity gradients between sheath and sample flows. Typical values when operating the microstructured flow cytometer ranged from 1000–1500μL/min for the sheath flow, which corresponded to particle velocities of about 3m/s. Maximum volume throughput for the sample fluid to maintain stable hydrodynamic focusing amounted to 40μL/min. The width of the focused sample stream, derived from microscopic fluorescence images when using fluorescent dye solutions as sample fluid, was 5–7μm in both lateral directions.
A polarization-maintaining mono-mode fiber delivered the light emitted by an argon-ion laser (488nm) and a HeNe laser (633nm) to the interaction region. The spot size of the laser beam at the position of the hydrodynamically focused sample stream was 29μm. Home-built photomultiplier units that allowed for spectral separation and selection detected light-scattering signals through multimode optical fibers (100μm core diameter). The fiber used for forward light scatter (FLS) collects light in directions corresponding to polar angles between 2.5° and 10°. A 40× microscope objective collected orthogonal light scattering (OLS) and fluorescence signals. A multi-channel pulse height analyzer recorded pulse heights, as part of a MoFlo™ cell sorter (Cytomation).
Figure 3. Two dimensional scatter plot for diluted whole blood (count rate 1.8kHz).
The microstructure we investigated can differentiate red blood cells and blood platelets by light scattering, as demonstrated in Figure 3. The FLS intensities were measured simultaneously for each cell at 488nm and 633nm, employing logarithmic amplification. The sample used was fresh blood, treated with anticoagulant and diluted to a volume fraction of about 1:100 by adding isotonic solution. The number of events (i.e. cells) is visualized in the scatter plot in false colors. The three discernable clusters correspond to red blood cells, platelets, and noise. Apart from this measurement, the microfluidic structure can also be applied to detect subpopulations of leukocytes stained with fluorescently-labeled monoclonal antibodies and can measure impedance signals of individual particles.
The three-dimensional structure also features cascaded hydrodynamic focusing and yields stable hydrodynamic focusing at flow rates varying over three orders of magnitude. Blood cells can be efficiently differentiated at high flow rates in the kilohertz region. Further improvement with respect to device mounting and handling, stability, and pulse height resolution will be achieved by using integrated waveguides instead of optical fibers and integrated lenses to efficiently collect fluorescence.
This research was partially supported by the Investitionsbank Berlin and the European Regional Development Fund (ERDF).
Andreas Kummrow, Hülya Yildirim, Marcin Frankowski, Jörg Neukammer
AG 8.32, Flow Cytometry and Microscopy
Janko Theisen, Christopher Sprenger, Martin Schmidt
Institut für Konstruktion, Mikro- und Medizintechnik
Technische Universität Berlin