Patented optics and off-the-shelf vision components help scientists measure particles more easily.
By Andrew Wilson, Editor
When a marine vessel enters port it may hold water from another part of the world, which might contain foreign organisms such asred tide—a microscopic algae Karenia brevis that produces a toxin that paralyzes fish so they cannot breathe. To properly analyze water samples, many research facilities and laboratories use cytometers to measure the physical or chemical characteristics of any cells or particles in a fluid sample. Using flow cytometry allows researchers to make measurements as the cells or particles pass through the measuring apparatus. In some systems, using electrical or mechanical methods to divert and collect cells with specific size and/or charge can extend this capability.
While doing research on how to correct lens aberrations at the Thayer School of Engineering at Dartmouth College (Hanover, NH, USA), Christian Sieracki developed a depth-of-field (DOF) enhancer: a phased array that elongates the point-spread function (PSF) of light into a narrow pencil of light with a larger DOF. After joining The Bigelow Laboratory for Ocean Sciences (West Boothbay Harbor, ME, USA) in 1997, Sieracki recognized the potential of the device in flow cytometry. With a grant from the National Science Foundation (Arlington, VA, USA), Sieracki developed a more-robust version of the flow cytometer and left to found Fluid Imaging Technologies (Edgecomb, ME, USA) to commercially develop a product now known as the Flow Cytometer and Microscope (FlowCAM; see Fig. 1).
FIGURE 1. The Fluid Imaging Technologies Flow Cytometer and Microscope combines the benefits of conventional flow cytometers with microscopes. In this way, the user is not only presented with a dot plot or scattergram that relates to the cell's size and chlorophyll content but also an image of the cell itself.
"What makes the FlowCAM unique," says Kent Peterson, CEO of Fluid Imaging Technologies, "is that it combines the benefits of conventional flow cytometers with microscopes. In this way, the user is not only presented with a dot plot or scattergram that relates to the cell's size and chlorophyll content, but also an image of the cell itself." The FlowCAM can be operated on discreet sample batches or on an in-line, continuous basis.
While products from manufacturers such as Becton, Dickinson and Company (Franklin Lakes, NJ, USA), Beckman Coulter (Miami, FL, USA), DakoCytomation (Glostrup, Denmark), Partec USA (Münster, Germany), and CompuCyte (Cambridge, MA, USA) are mostly used in medical cell analysis, Fluid Imaging Technologies has decided to initially target water-analysis markets, as well as to pioneer process and quality-control applications in the pharmaceutical, chemical, bioterrorism, cosmetic, and petroleum industries. FlowCAM technology will benefit these industries by providing a proprietary tool to have real-time particle counting, sizing, and imaging in one cost-effective data stream.
In the design of standard flow cytometers, fluid-containing particles or cells are introduced into a narrow tube or injector that is coaxial with a cell-free sheath through which a particle-free fluid is flowing. Outside fluid flow draws the sample through the flow cytometer, where it is illuminated and scattered by laser light. The FlowCAM simplifies this process by using only a single capillary tube from Vitrocom (Mountain Lakes, NJ, USA), which allows greater volumes of fluid to be analyzed per unit of time.
If the particle is small, then the amount of scattered light will also be small, resulting in a low-level output at the photodetector. If the particle is large, then the signal will be correspondingly larger. By correlating the strength of the signal at the output of the photodetector, particle size can be computed. In currently available cytometers, the nozzle is typically limited to 60 µm because most cytometers are built to analyze blood and not water. This limits the diameter of any particle that can pass through to an absolute maximum of 60 µm.
In most instruments, maximum flow is limited to 50 ml/min. Although this is useful in blood-cell analysis, where limited flow does not present a problem, such instruments cannot rapidly process hundreds of milliliters of water. Also, because conventional cytometers only measure the light scattered form the particles, only so much information can be discerned from the collected data.
The design of the FlowCAM incorporates the benefits of traditional flow cytometers such as those from Becton, Dickinson and Company and others and adds a DOF enhancer and imaging capability. In this way, the cytometer can perform traditional applications with the benefits of adding images of the particles or cells, all correlated in a PC-based database.
FIGURE 2. To examine fluids, a pump on the output/exhaust side of the flow chamber draws the sample at tens of milliliters per minute into the chamber. A laser is fanned onto the flow chamber illuminating particles that scatter light and fluoresce. If either fluorescence detector receives a strong enough signal, indicating the presence of a particle of interest, a flash LED is fired for a very short interval. This light is then imaged with the objective onto the CCD camera.
In the FlowCAM design, a pump on the output/exhaust side of the flow chamber draws the sample at tens of milliliters per minute into the chamber (see Fig. 2). Once inside the chamber, a green 15-mW, 532-nm solid-state laser from Edmund Industrial Optics (Barrington, NJ, USA) illuminates the sample. "Because the particles to be detected are chlorophyll and phytoplankton, a green laser is used to make these fluoresce. If we wanted to examine blood samples, then a blue laser would be used to make the cells fluoresce," says Sieracki.
To block spurious emissions at other wavelengths, the laser passes though a 532-nm filter from Chroma Technology (Rockingham, VT, USA), a dichroic mirror, and an objective lens from Olympus (Melville, NY, USA), which fans the laser onto the flow chamber. After a particle passes though the flow chamber, it scatters light and fluoresces. When it does this, the fluorescing light travels back through a 540-nm emission filter and the custom-built DOF enhancer. This light then passes through the system to a 590-nm long-pass filter. The shorter-wavelength light (yellow or green) is reflected by the long-pass filter to the 500- to 590-nm fluorescence photomultiplier detector from Hamamatsu (Bridgewater, NJ, USA). Longer-wavelength light (red) passes through a long-pass filter to the 590- to 700-nm fluorescence detector, also from Hamamatsu.
If either fluorescence detector receives a strong enough signal, indicating the presence of a particle of interest, a flash LED is fired for a very short interval. This light is then imaged with the objective onto the CCD camera. Here, the DOF enhancer stretches out the focus of the objective into a continuum of foci, keeping the entire 0.3-mm flow in focus (see "DOF enhancer holds the key to cytometric imaging" [below]).
After the LED is fired, images are captured by a XC-7500n camera from Sony Electronics (Park Ridge, NJ, USA), which is interfaced to a PC using a Meteor II frame grabber from Matrox (Dorval, QC, Canada). To synchronize the measurement of fluorescent events and image capture, Fluid Imaging developed a PCI add-in board that reads the signals from the PMTs and generates trigger signals for the frame grabber and camera. In operation, an event, triggered by the PMTs, is used as an input to the frame grabber that initializes the camera. This image is then coordinated with the event so that fluorescence information can be correlated with an image of the particle.
To program the cytometer to recognize specific particles, Flow Imaging used the Matrox Imaging Library (MIL). C++ was used to develop the code, and image-processing routines for pattern recognition were called from the MIL. "Thus," says Sieracki, "the instrument can be trained to search for specific types of cells, patterns, or species in the fluid." The company considered using other image-processing packages for the task, but found that every time a new camera was required, a new imaging module had to be purchased for the frame grabber.
In operation, the system analyzes either discrete samples of several milliliters in volume or continuous flows of 10 ml per minute. Cells or particles with chlorophyll or phycoerythrin between 10 and 1000 µm (1 mm) in size are analyzed and imaged. The cytometer automatically draws water through an intake tube, which is usually pumped from a shoreline or dock. Inside a shelter, the water is sampled from a jug or flask.
Every cell measured has data describing 12 different characteristics, including time of passage through the laser beam, cell size, and chlorophyll and phycoerythrin content. "Someday," says Peterson, "the FlowCAM may be used as an early warning system to detect the presence of harmful algae or bioterrorist events."
Each cell is shown as a dot whose location is dependent on the cell's size and chlorophyll content (upper) or phycoerythrin content (lower). Within the sample, FlowCAM detects, counts, and images cells and particles that exhibit chlorophyll or phycoerythrin fluorescence. Cells with similar size and phycoerythrin or chlorophyll content tend to form groups on the plot. In the interactive scattergram, areas are dynamically linked to the original images used to generate the scattergram (see Fig. 3).
FIGURE 3. Within the fluid sample, FlowCAM detects, counts, and images cells and particles that exhibit chlorophyll fluorescence. Cells with similar size and chlorophyll content tend to form groups on an interactive scattergram, where areas can be dynamically linked to the original images.
"With FlowCAM," says Peterson, "thousands of cells can be counted in just 10 s, no sample preparation is required, images of each cell are stored on computer disk, and data on each cell are stored automatically." In addition, since the cytometer can continuously monitor water from a site for days at a time, it can detect the movement of organisms into or out of a region. The device automatically counts cells per liter of water, allowing it to be used to determine the severity of blooms of organisms and how much water toxicity may be affected. Stored digital images can be accessed at any time for identification.
DOF enhancer holds key to cytometric imaging
Although many microscopic lenses are useful in cytometer applications, their limited depth of field and lateral resolution means that they are unsuitable for imaging large volumes of liquid simultaneously. Because of this, Christian Sieracki, president of Fluid Imaging, developed a specialized optical element while performing research at the Thayer School of Engineering at Dartmouth College (Hanover, NH, USA).
Using an optical element constructed with the same photolithography and etching technologies used to fabricate integrated circuits, the so-called depth-of-field (DOF) enhancer increases the focus by a factor of four. This is accomplished by etching the glass in a series of concentric rings in a pattern at quarter-wavelength intervals.
In effect, this DOF enhancer acts as a phased array that elongates the point spread function of the light into a narrow pencil of light that can be used to illuminate a long, thin area of the specimen. A full description of this DOF enhancer can be found inAn Experimental and Computational Study of Binary Optical Elements for Aberration Correction in Three-Dimensional Fluorescence Microscopy, Christian Sieracki Ph.D. thesis, Thayer School of Engineering, Dartmouth College. AW
Becton, Dickinson and Company www.bd.com
Beckman Coulter www.beckmancoulter.com
Bigelow Laboratory for Ocean Sciences www.bigelow.org
Chroma Technology Corp. www.chroma.com
DakoCytomation Denmark A/S www.dakocytomation.com
Dartmouth College www.dartmouth.edu
Edmund Industrial Optics www.edmundoptics.com
Fluid Imaging Technologies www.fluidimaging.com
Matrox Electronic Systems www.matrox.com/imaging
National Science Foundation www.nsf.gov
Partec GmbH www.partec.de
Sony Electronics Inc. www.sony.com/videocameras