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Optical chromatography is a technique for the separation of particles that capitalizes on the balance between optic and fluidic forces. When microscopic particles in a fluid flow encounter a laser beam propagating in the opposite direction, they are trapped axially along the beam. They are then optically pushed upstream from the laser focal point to rest at a point where the optic and fluidic forces on the particle balance. Because optical and fluid forces are sensitive to differences in the physical and chemical properties of a particle, both coarse and fine separations are possible. We describe how an optical chromatography beam directed into a tailored flow environment, has been adapted to operate as an optical filter for the concentration/bioenrichment of colloidal and biological samples. In this work, the demonstrated ability to concentrate spores of the biowarfare agent, Bacillus anthracis, may have significant impact in the biodefense arena. Application of these techniques and further design of fluidic and optical environments will allow for more specific identification, concentration and separation of many more microscopic particle and biological suspensions.
©2007 Optical Society of America
1. Introduction
Light from a laser can be used to exert significant forces on microscopic particles resulting from the transfer of photon momentum to matter. Through careful application of optical forces, one can trap particles in a liquid, manipulate them, and sort them based upon a variety of criteria.[1–4] There are two general separation approaches being pursued: the first is the use of optical tweezers and/or arrays of optical traps for spatially sorting microscopic particles and the second is the use of optical chromatography to separate small groups of particles.
The optical tweezers technique uses one or more laser beams that are highly focused into a solution containing microscopic particles. The momentum transfer resulting from the highly convergent rays (using a microscope objective) generates a net restoring force which retains particles at the focal point of the laser. Through translation of the surrounding medium or movement of the laser beam, particle translation and manipulation can be achieved. This method has been used to separate particles based on their appearance including size, shape, or other features, and potentially using fluorescent labels.[5, 6] Recently more sophisticated and automated optical techniques have been developed to separate microscopic objects.[7–9]. In this type of work, arrays of optical traps in a fluid flow were used to preferentially transport microscopic objects, which experience a greater optical force away from those which experience a lesser force. Other techniques involving novel beams have been demonstrated.[10, 11]
Optical chromatography[12–17] relies on a mildly focused laser beam to propel particles along its axis of propagation. The beam is aimed directly against a fluid flow; the balance of fluid drag force and optical pressure results in the stable trapping of particles within the beam. Particles of larger size or refractive index experience greater optical pressure and are thus propelled further than smaller or lower refractive index particles. This results in unique positions (separation) along the beam axis for particles of different size and composition (refractive index). Using this technique it has been shown that particles and microorganisms can be separated by size[12, 18], refractive index[16], shape and morphology[19], and fluid drag characteristics[19].
An important application of optically based separations is for microscopic samples of biological origin encompassing such diverse materials as pollen, yeast, erythrocytes, bacteria, and viruses, to name only a few. Of specific interest to the U.S. Department of Defense (DoD) is the possibility of using optical chromatography for biological warfare agent (BW) detection. Of particular concern is Bacillus anthracis, the causative agent of the deadly mammalian disease, anthrax. Detection and characterization based upon physical and biochemical properties alone would enable a new generation of rapid and portable biological detection capability. Our laboratory is working to develop methods for separating a wide variety of microbiological samples[18–20]. Applications include the coarse filtering of bacteria from viruses, separating bacterial cells from spores, and possibly distinct bacterial species from one another. Recently, this method has been used to separate spores of different Bacillus species based on their optical and fluidic properties[19].
In this paper we demonstrate a new method based on optical chromatography for the complete separation of injected groups of particles. In traditional optical chromatography, the majority of injected particles flow past the laser beam due to the large size of the channels used and the small focal point (200-1000 μm diameter channel, and a 10-20 μm laser beam waist).[12, 16] Typically, only a small number of particles (10-50 out of an injection of many thousands of particles) are trapped and separated, and are thought to be representative of the bulk mixture. Until the work described in this paper, optical chromatography could only be considered an analytical technique. With the ability to completely interrogate the injected sample, this new method has the potential to impact both analytical and preparative separations.
2. Experimental
In optical chromatography, a laser is mildly focused by a long focal length lens (100 mm) into a fluid channel containing the microscopic samples rather than a high numerical aperture objective (NA > 1.3), as used in optical trapping. The system consisted of a CW laser focused by a 1 inch diameter plano-convex 100 mm focal length lens into a microfluidic system. The laser used in this work was a 1064nm ytterbium fiber laser (IPG Photonics, Oxford, MA, USA). The fiber laser was aligned with the microfluidic flowcell using a custom-made adaptor fitting a 1 inch diameter lens tube system (Thorlabs, Inc., Newton, NJ, USA). The entire assembly was mounted on a linear translation stage with 1” of travel (Thorlabs, Inc., Newton, NJ, USA). The microfluidic network was mounted on a 5 axis positioner (New Focus, San Jose, CA) and the entire aligned optic and fluidic system was placed underneath a microscope (Lumam, LOMO America, Prospect Heights, IL), mounted on a custom x-y-z translation platform. This platform allowed for movement of the microscope used for observation and image data collection around the laser / microfluidic system.
The micro-flowcell was constructed entirely of soda lime glass plates, with wet-etched channels (50 μm depth and 100 μm width), to deliver the fluid and sample to the separation channel and subsequently carry it out of the microfluidic device to waste. The microfluidic and optical pathways can be seen in Fig. 1(A): The flow moves into the device and across to the laser separation channel where it meets the laser beam. At this point the laser beam fills the channel and thus all particles in the fluid must experience optical pressure. After passing through the laser separation region, fluid moves out of the device where it can be collected for additional analysis. The glass plates were drilled for fluid input and output of the etched channels, bonded together and the holes were fitted with fluidic connectors (Nanoport, Upchurch Scientific, Inc.) to couple both fluid and sample introduction tubing. The separation channel was 50 μm in diameter and 500 μm in length and the beam was focused such that it completely filled the channel. This opto-fluidic approach results in a high photon density within the separation region where particles must experience the laser radiation force. Given a certain power required for the sample in question, particles will be retained. The entire volume of the microfluidic device was less than 600 nL. A separate channel and associated fluidic connector were placed near the separation region and used for sample injections. Fluids were controlled with two syringe pumps (NE-1000, New Era Pump Systems, Inc, Farmingdale, NY): one for fluid flow and the other for sample injection. The flow rates used in the experiment were 20 and 50 nL/minute and the injection size was 30 nL - 50 nL. For all of the experiments, a second channel with a larger inner diameter (100 μm) was included in the device downstream of the separation channel to better image the particles after concentration. The larger channel produced clearer images and slower flow to aid automated image processing. The polymer particle concentrations were so high that accurate counts were not possible and so an image processing technique was used to estimate concentration (described below). In the case of Bacillus anthracis (B.a.) spores, their concentration was dilute enough that accurate counts were possible.
Fig.1. Optical chromatography glass micro-flowcell. A) Construction of the microfluidic device showing the pathway for fluid (shown in gray) into and out of the separation channel, and the laser beam focused through the channel. B) Illustration of a separation in which sample particles are constrained to the focal point of the beam by the size of the separation channel that is filled by the laser beam.
The polystyrene (PS) beads used in these experiments were 2 microns in diameter (Polysciences, Inc., Warrington, PA) dispersed in water at a concentration of 4×107 particles/mL. Bacillus anthracis Sterne strain 34F2 (nonpathogenic, vaccine strain) was obtained from Colorado Serum Co., Denver, CO. To obtain spores, overnight cultures of B.anthracis were grown on trypticase soy agar (TSA) plates (Difco, BD, Franklin Lakes, NJ) at 37°C. A few colonies of each strain were resuspended in PBS buffer pH 7.0 and plated on 2×SG sporulation agar plates followed by incubation at 37°C. The spores were collected as soon as the culture reached over 95% of phase bright spores, usually after four days, and resuspended and rinsed in 2 ml of cold sterile milliQ water. The spores were further diluted in water before injection into the system to a concentration of 6×107 spores/mL.
Data collection and analysis were performed using ImagePro Plus version 6.0 (Media Cybernetics, Inc., Silver Spring, MD). Relative PS particle concentrations were assessed using automated software routines for measuring greyscale levels in the flowcell (particles appear dark on a light background, thus yielding a higher greyscale “concentration” value).Another method based upon automated contrast thresholding algorithms available in the software package was used to count the B.a. spores. This provided a reproducible method for achieving accurate particle counts in the injected and laser concentrated bands.
3. Results
Initial results obtained using the optical chromatography laser filter system indicate that concentration of a continuous stream or an entire injected band are possible. Fig. 2 shows the process of laser retention and sample concentration. In Fig. 2(A), a liquid containing 2 μm polystyrene particles was introduced as a constant flow (i.e. not a discreet injection) at a flow rate of 3.0 μL/hr. The objective of the experiment was to “clean up” the particle laden stream and in the process produce a concentrated band of 2 μm particles. With the 0.9 W 1064 nm laser beam focused into the 50 μm channel in Fig. 2(B), no particles were visible in the channel as they were all trapped outside the 50 μm channel. After three minutes of particle retention, the beam was turned off in Fig. 2(C), and the concentrated band of particles was released (note the concentrated band of particles along the center line of the channel).Additional data for a similar experiment (laser power = 1.0 W, fluid flow = 3.0 μL/hr) showing a six minute laser retention period are given in Fig. 3. Initially, in Fig. 3(A), particles are present at a dilute concentration in the device (few particles visible in field of view) and then the laser is switched on at around 1 minute. With the laser on, the particle concentration decreases to near zero (relative concentration was estimated from automated analysis of greyscale levels in the video images). At six minutes the laser was switched off and a concentrated band was released, as indicated by the peak and the image in Fig. 3(C).After the concentrated band passed through the flowcell, the stock concentration was again seen in the flowcell, Fig. 3(D).
Fig. 2. A) Stock concentration stream of 2 μm polymer particles in the channel, B) laser ON and retaining all polymer particles (no particles in the channel), C) concentrated stream of 2 μm polymer particles released when the laser was switched off. Laser power was 0.9 W at 1064 nm, fluid flow rate was 3.0 μL/hr.
Fig. 3. Six minute laser concentration of 2 Fig. 3 μm polystyrene particles. A) Sample of polymer particles in system with laser off, B) laser ON – flowcell is free of polymer particles with the laser retaining them, C) laser OFF – flowcell is filled with concentrated stream of particles previously retained by the laser, D) return to original dilute mixture of polymer particles. Data were background subtracted.
For a continuous sample input of fixed particle concentration, the amount of concentration attainable depends on the length of time the laser irradiates the flowcell. This can be seen in Fig. 4 where the laser was left on for 0 (no laser - baseline), 2, 4, 6, 8, and 10 minutes (Supplemental Movie 1). With each subsequent two minute increase in laser collection time the concentration of particles increased. With respect to the data one can see the peak increase in height and width as the laser collection period increased. What is not obvious in the plotted data is the spatial concentration of particles along the center of the flow channel.Spatial concentration of particles is defined as the extent to which particles are co-located in one region (center of the channel) by the action of laser radiation pressure. The image data clearly show a tightly clustered group of particles within the channel, whereas the plotted data are the result of the entire channel analysis. The effect of laser concentration time on the resulting particle bands is summarized in Table 1. The spatial width (thickness of the particle band in the center of the channel) increases from 17 μm to 28 μm within the 100 μm channel.Without the laser, the particles are distributed uniformly throughout the channel (100 μm width). At the longer laser concentration times, more particles necessarily occupy a larger width of the channel upon exit. The temporal band width (length of the particle band in the channel) increases almost three-fold as a function of the laser concentration time. This is due to the particles exiting the laser separation channel in a somewhat sequential fashion (versus a tight knit group) thus creating a longer temporal band width with increasing numbers.
Table 1. Time based laser concentration of particles
Fig. 4. Laser concentration of 2 micron PS particles. A) Baseline / no laser, B) 2 minute laser concentration of sample, C) 4 minutes of laser concentration, D) 6 minutes of laser concentration, E) 8 minutes of laser concentration, and F) 10 minutes of laser concentration.Images were taken from the peak region for each laser concentration time. (Supplemental Movie 1) [Media 1]
Concentration of an important biological sample has been accomplished using the optical chromatography laser filter system. Spores of Bacillus anthracis have been bioenriched using the laser filter system. The graph of particle counts versus time in Fig. 5, shows the effect of the laser on an injected band of B.a. spores. Without the use of the laser, the injected band of particles quickly passes through the imaging flow channel with a broad peak between 2 and 4 minutes. With the laser operating at 2.5 W, and a flow rate of 1.2 μL/hr, the majority of the injected spores were retained and a spatially and temporally concentrated band was detected after the laser beam was blocked at 10.5 minutes (Supplemental Movie 2). Using automated image analysis software, the number of particles in both experiments was counted. With the laser off, 178 particles passed through the system and were detected in the imaging flowcell.In the next experiment with the laser on, 152 spores were retained by the laser and only 8 spores were unretained by the laser which equals a retention efficiency of 95 %. In addition, excellent sample concentration was achieved. If one compares a hypothetical fraction of spores contained in the laser concentrated peak (0.22 min baseline to baseline) to the same period for the injected sample peak (no laser), there is a substantial increase in spore concentration. There were 21 spores / nL in the laser concentrated peak versus 4 spores / nL in the injected peak (no laser): a five-fold laser concentration factor. Such a concentration factor could have substantial benefit for spectroscopic applications (fluorescence[21], Raman[22], etc.) where signals from small numbers or even single particles / organisms are routinely measured. Furthermore these applications may be implemented on-chip thus simplifying the analytical process.
Fig. 5. Injection of (□) B. anthracis spores in water with the laser off, and (◯) B.anthracis spores with the laser on. The laser beam was blocked at 10 min, releasing a concentrated band of B.a. spores. (Supplemental Movie 2) [Media 2]
4. Conclusions
The use of an optical chromatography based system adapted for complete sample interrogation has been shown. Previous work using optical chromatography was limited by the inability to interrogate all injected particles. Concentration of particles from continuous input streams and discreet injections have been demonstrated using a new experimental design. The carrier fluid and suspended particles are forced through a flow channel which is filled by the mildly focused optical chromatography laser beam. The ability to interrogate all injected particles represents a significant achievement and advance in optical chromatography based systems. This capability will drastically facilitate applications in both detection and sample preparation using this technology. Currently work is in progress to increase the number of particles / microorganisms that can be optically retained and separated. This will facilitate coupling with many more complementary techniques.
Acknowledgments
The authors would like to acknowledge the Naval Research Laboratory (NRL) and the Defense Threat Reduction Agency (DTRA) for support of this research.
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