Patent Application
20110207207
1. Field of the Invention
The present invention relates to apparatus for measuring and sorting cells within a microfluidic structure using broadband coherent anti-Stokes Raman scattering (CARS).
2. Description of the Related Art
The general idea of flow cytometry is to perform rapid measurements on cells flowing in a hydro-dynamically focused single stream. These measurements are typically optical (i.e. light scattering, fluorescence) because of the inherent sensitivity and fast data rates. The data rate determines the number of cells that can be measured in a given time. In current cytometers, typical rates of ˜5,000 to 10,000 cells/s are used to obtain good statistics. The disadvantages of flow cytometry are its size and non-portability, the requirement for a sterile lab environment, the need for large sample volumes, and its limited chemical analysis capabilities.
Raman spectroscopy measures the vibrational spectrum of biomolecules and can distinguish between different cell types, viruses, and other pathogens. In Raman scattering, the fundamental laser (pump) is scattered by a vibrational mode, producing a lower energy (Stokes) or higher energy (anti-Stokes) photon.
The Raman spectrum provides a measure of the vibrational mode density that can be translated to biochemical content. Traditional Raman spectra of a variety of biomolecules and cells have been measured, showing the capability of this technique to identify different cells types, bacteria, and viruses. Raman spectroscopy can also distinguish between healthy and unhealthy cells, and cancerous and non-malignant cells. Thus, Raman spectroscopy is a powerful technique for label-free identification and characterization. Unfortunately, despite these advantages, this technique has limited use for biomedical applications due to the long acquisition times required for the measurement. In the Raman process, a narrow band laser illuminates the sample and a portion of the incident photons are scattered by interactions with molecular vibrations, resulting in a shift to higher (anti-Stokes) or lower frequency (Stokes) photons. The signal intensity is very weak because of the extremely low scattering cross-section (˜10−30 cm2/molecule).
CARS is a nonlinear optical process that selectively and coherently excites vibrational resonances of biomolecules to rapidly obtain the Raman (vibrational) spectrum. Compared to traditional Raman scattering, the CARS process increases the detection sensitivity by 107 to allow rapid data acquisition. With the associated decrease in measurement times, CARS has been applied in biomedical microscopy to image live cells at video rates without extrinsic fluorescence dye labeling. Two photons (pump and Stokes) excite a specific vibrational resonance coherently. A third photon (probe) subsequently measures the density of the vibrational resonance. The density of the vibrational resonance is proportional to number of emitted anti-Stokes photons that are energy shifted by that vibrational mode. A traditional CARS setup uses two synchronized picosecond lasers or a single picosecond laser with an optical parametric amplifier to generate the two laser beams with the difference in frequencies matched to one particular vibrational resonance. The disadvantage of this approach is its difficulty in frequency tuning needed to measure a full spectrum (as opposed to a single vibrational mode). Mechanical movement or temperature control, such as rotation or heating of an optical crystal, is needed to tune the optical frequencies of the two lasers, resulting in misalignment or very slow frequency tuning rate of the system. Therefore, this two-laser approach is not suitable for flow cytometry and sorting because measuring a single Raman resonance is not adequate to separate a wide range of cell types. Instead a full spectrum inside the Raman fingerprint region (300 cm−1 to 1800 cm−1) or several different Raman signature frequencies are needed in order to characterize the chemical composition of a cell. Another advantage of acquiring the full spectrum is that by taking the ratio of different spectral regions, it is possible to identify different cell types without calibration because a ratiometric measurement is not dependent upon the overall magnitude of the signal.
In contrast to CARS with two narrow linewidth lasers, in broadband CARS, a broadband Stokes beam excites many modes simultaneously, which allows the entire vibrational spectrum to be measured in a single shot. In broadband CARS, since the broadband laser beam is used to excite many modes, a higher laser intensity is needed to measure the output signal resulting in a non-resonance background. Fortunately, this background can be eliminated and the Raman spectrum retrieved using a variety of phase and polarization pulse shaping methods.
A micro-fluidic cell sorter utilizes a coherent broadband laser to implement broadband CARS flow cytometry. A preferred embodiment combines multiplex Coherent Anti-Stokes Raman Scattering (CARS) spectroscopy using a broadband coherent laser source with a microfluidic device with hydrodynamic focused channel for label free cell characterization, quantification, and sorting.
A microfluidic cell sorter has a microfluidic structure with a sample input channel leading into an observation region, two buffer channels configured to hydrodynamically focus a sample target cell within the observation region, and at least two output channels. Apparatus directs the target cell into a selected output channel based on a cell sorting control signal. A CARS pulse source generates CARS sources, which are directed to the target cell within the observation region. A detector detects CARS illumination scattered from the target signal and generates a spectrum signal based on the detected illumination. A processor identifies the target cell based on the illumination signal and generates the cell sorting control signal based on the identity of the target cell.
In one embodiment, back-pressure is applied to the non-selected output channels so that the target cell exits the selected output channel. The microfluidic structure might comprise a silicon structure anodically bonded between two pyrex slides.
The CARS source might comprise a broadband laser source which provides the pump, probe and Stokes frequencies as well as a pulse shaper. The broadband laser source could be a femtosecond laser and a photonic crystal fiber to broaden the optical spectrum of the pulse. The photonic crystal can also amplify the intensity of the probe frequency. Or, the broadband laser source could comprise a fiber laser.
In one embodiment, the pulse shaper delays the probe wavelength to improve the CARS output signal.
Similarly, a microfluidic flow cytometer for examining cells in an input sample includes a microfluidic structure including a sample input channel leading into an observation region and two buffer input channels configured to hydrodynamically focus a continuous stream of cells in a single file line within the observation region. A coherent anti-Stokes Raman scattering (CARS) pulse source generates CARS pulses, and apparatus for detecting the presence of each of a series of target cells as they enter the observation region triggers the CARS pulse source accordingly. The CARS pulses are directed into the target cells within the observation region. Then, a detector detects CARS illumination scattered from each target cell and generates a spectrum signal based upon the detected illumination. A processor analyzes each target cell based upon its associated spectrum signal.
The present invention can measure the spectrum of a cell in around 1-10 ms, resulting in a data rate on the order of 100-1000 cells/second, fast enough for flow cytometry applications in real time.
The following reference numbers are used in the figures:
The microfluidic device of
Sample reservoir 110 provides sample fluid 202 containing cells 204 to the cell sorter via input channel 116. Buffer reservoirs 106, 108 provide buffer 206 (via two buffer channels 118) to the microfluidic device for the purpose of hydro-dynamically focusing the sample stream 202 at focusing junction 112. This permits only one target cell 208 at a time to pass through the observation region 214. Observation region 214 includes a light scattering measurement location 210, where cell 208 is detected in order to trigger CARS measurement, and CARS measurement location 212, where the scattered output CARS illumination from the sample is detected. After cell 208 passes through observation region 208 it is sorted into target output reservoir 102 or waste output 104 via output channels 114 A and B.
In a preferred embodiment, the channel widths range from 100-400 microns. The channel heights are 25-30 microns.
Alternative methods for creating fluid flow in microfluidic devices include electroosmotic flow and integrated miniature fluidic pumps.
Next, the substrate is placed in the reactive ion etch and the channels are etched into the silicon wafer completely removing all the silicon down to the pyrex substrate, as shown in
Alternative methods for manufacturing microfluidic devices with optical transparency include: reactive ion etching in glass or laser machining in glass and glass/glass bonding. Use of SU-8 photoresist as the layer containing the channels may replace the silicon wafer and adhered to the glass with epoxy.
At time 508, cell 208 is sorted into channel 114B. In this embodiment, the sorting is accomplished by creating back pressure 536 at the other two exit channels 114A and 114C.
Alternative methods for switching cells to the target exit channel(s) include electro-osmotic switching by switching an applied voltage to the target exit channel, switching using fluidic pumps, optical trapping by laser fields, and dielectric trapping by integrated electrodes with applied voltages.
There are several approaches to solve this problem.
Raman spectra of cells can be obtained with a single broadband coherent laser source through optical pulse shaping. A broadband coherent ultrafast laser 602 is used as the excitation light source and subsequently the amplitude, the phase and/or the polarization of the pulse (any or a combination), pulse width (compression and spreading) are pulse shaped. Raman spectroscopy of biological samples typically covers the “Raman fingerprint” region ranging from 0 to 1800 cm−1. In microscopy, this range can be extended to above 3000 cm−1 to reach to some higher vibrational frequencies, such as the CH2 vibrational stretch of lipids at 2840 cm−1. In order to cover such a wide frequency range, a broad optical spectrum is required. For example, for Ti:Sa femtosecond lasers with center wavelength at 800 nm, a broad spectrum from 700 nm to 900 nm is needed to cover Raman frequencies above 3000 cm−1. Such a broad optical spectrum is typically not available from a femtosecond or picosecond laser source. Therefore, a photonic crystal fiber 604 (such as SCG-800 of Crystal fiber) can be used to generate a super-continuum spectrum from ultrafast pulses through nonlinear optical processes. As an alternative, a super-continuum fiber laser source (such as the SC500-FC of Fianium) can also be used directly as the broadband light source. The photonic crystal fiber is not a necessity in all embodiments of the design. For example, if only the Raman fingerprint region from 0 to 1800 cm−1 is needed to be covered, a careful designed Ti:Sa oscillator can provide enough bandwidth ranging from 750 to 850 nm without other external elements.
Besides broadening an optical spectrum, the photonic crystal fiber 604 can also be used to tailor the optical spectrum to enhance the intensities of the probe wavelengths, as shown in
The broadband laser source is subsequently sent to an optical pulse shaper for intensity, phase and/or polarization shaping. In one embodiment, the optical pulse shaper is based on the standard 4-f pulse shaper geometry with a pair of dispersive grating 802, 816 (or dispersive prisms) combining with curved mirrors/lenses 804, 814 to spatially distribute the optical spectrum at the conjugate plane of the setup. A spatial light modulator (SLM) 806, such as SLM-128 of CRI, is used to modulate the phases and polarizations of each individual frequency across the optical spectrum. The SLM is not limited to use liquid-crystal based technology and other technology such as acousto-optic spatial light modulation can also be used to implement the optical pulse shaper. The amplitude shaping can also be implemented by inserting an opaque mask at the conjugate plane of the pulse shaper to block out some unwanted optical frequencies.
In one embodiment of the implementation of the multiplex CARS for cell sorting, the full CARS spectrum covering the whole Raman fingerprint region is obtained. In this case, the pulse shaper is used to compensate for high order material dispersion acquired by the pulse when the pulse propagates through the optical setup to generate a transform limited pulse at the sample. This allows all optical frequencies arrive at the sample at the same time in order to excite all vibrational frequencies through different frequency combinations across the whole optical spectrum of the pulse. However, a potential drawback of this approach is that the high optical intensity of the broadband source with the full optical spectrum incident on the cells could cause optical damage to the cells.
In such scenario, an alternative scanning approach can be used. The pulse shaper can be configured to allow only two (or a few) discreet frequencies to transmit through the SLM at a time. Since an SLM operates through either electro-optical or electroacousto effects and no mechanical movements are involved, rapid frequencies scanning is possible through sweeping the pump frequencies against the Stokes frequencies across the whole optical spectrum. Since more optical intensity can be concentrated at just these two frequencies to enhance the CARS signal, cell damage can be greatly reduced.
In addition, sometimes only a small Raman region or only several specific Raman frequencies are needed, because it has been determined that this small range of Raman spectrum will be adequate to differentiate cells. A small portion of the optical frequencies or these specific frequencies can also be “cut out” through intensity modulation by the pulse shaper to avoid putting in excessive optical energy into the cells.
The shaped optical pulse is now ready to send to the microfluidic device 500 to generate the CARS spectrum of the cell for cell sorting. A chromatic aberration corrected microscope objective is used to focus the pulses to the cells. The generated anti-Stokes frequencies are then collected either in the forward or epi direction for analysis.
In the embodiment of
The generated CARS signal 528 is finally sent to a detector 530 for data acquisition. Depending on the scheme to remove the non-resonance background, a one-dimensional spectrometer, a two-dimensional spectrometer, or a signal element detector, such as a photo-multiplier tube, can be used to acquire the data. In the embodiment of
In the present invention, a galvo mirror 818 is used to dynamically guide pulses 526. This is useful for two reasons. First, if a longer acquisition is desired, the pulses can follow the target cell as it travels along the microfluidic device. Second, the pulses may be scanned across the width of the cell. Or, both may be accomplished at once.
Those skilled in the art will appreciate that the elements can be mixed and matched in a variety of ways, and that other elements not specifically shown and described could perform these functions. It will be appreciated by one skilled in the art that there are many possible variations on these designs that fall within the scope of the present invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2009/062419 | 10/28/2009 | WO | 00 | 4/27/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61197548 | Oct 2008 | US |
