WO2012102833A2

WO2012102833A2 - Systems and methods for stimulated raman scattering flow-cytometry

Info

Publication number: WO2012102833A2
Application number: PCT/US2012/020027
Authority: WO
Inventors: Xiaoliang Sunney Xie; Dan FU; Christian Freudiger
Original assignee: President & Fellows Of Harvard College
Priority date: 2011-01-24
Filing date: 2012-01-03
Publication date: 2012-08-02
Also published as: WO2012102833A3

Abstract

A flow cytometry system is disclosed that includes a light source, a spectral shaper, a modulator system, an optics system, an optical detector, a flow conveyance system and a processor. The light source system is for providing a first train of pulses including a first broadband range of frequency components, and a second train of pulses including a second optical frequency. The spectral shaper is for spectrally modifying an optical properly of at least some frequency components of the broadband range of frequency components. The modulator system is for modulating a property of at least one of the shaped first train of pulses and the second train of pulses at a modulation frequency. The optics system is for directing and focusing the shaped first train of pulses and the second train of pulses as modulated toward a common focal volume. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of a train of pulses of interest transmitted or reflected through the common focal volume. The flow conveyance system is for providing flow path of a fluid through the common focal volume. The processor is for detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the train of pulses of interest, and for providing an output signal for the flow cytometry system.

Description

SYSTEMS AND METHODS FOR

STIMULATED RAMAN SCATTERING FLOW-CYTOMETRY PRIORITY

The present application claims priority to U.S. Provisional Patent Application Ser. No. 61/435,597 filed January 24, 201 1 , the entire disclosure of which is hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT

This invention was made with United States government support under grant no. 1R01EB010244-01 awarded by the National Institutes of Health. The United States government has certain rights to the invention. BACKGROUND

The invention generally relates to flow cytometry systems and methods. Flow cytometty is a widely used technique for counting and sorting micro-particles such as cells. Generally, fluidic flow passes the sample through a laser diagnostic system, and processing electronics are used for functional analysis and sorting. The power of flow cytometiy relies on the fact that many physical or chemical parameters may be measured simultaneously and rapidly. The multi-parametric approach allows separation of particles that are almost indistinguishable based on a single physical/chemical parameter alone.

Fluorescence activated cell sorting (FACS) provides a method for sorting cells based on DNA or anti-body staining. See, for example, U.S. Patent Applications Publications Nos. 2010/0032584, 2009/0201 501 , 2009/012231 1 , 2008/0069300 and 2005/0225745. To acquire multiple parameters simultaneously, typically many lasers and fluorescence detectors are used. Fluorescence flow cytometry systems however, require extrinsic labeling of the sample, which inherently perturb the sample and could lead to uncertainty in data analysis. Further, certain chemical species cannot be labeled reliably (e.g., lipids).

While Raman based approaches eliminated the need for labeling, the signal of spontaneous Raman spectroscopy is very weak. Thus particles sorting based on spontaneous Raman scattering is too s!ow to be useful for measuring fast flowing samples. See for example, U.S. Patent No. 7,333,197.

Coherent-anti-Stokes Raman scattering (CARS) significantly improves the speed of measurement and has been demonstrated for flow cytometry applications. See for example, "Microfluidic CARS cytometry," by H. W. Wang, N. Bao, T. T. Le, C. Lu, and J. X. Cheng, Optics Express 16, 5782-5789 (2008), and "Multiplex coherent anti-Stokes Raman scattering (MCARS) for chemically sensitive, label-free flow cytometry," by C. H. Camp, S. Yeg anarayanan, A. A. Eftekhar, H. Sridhar, and A. Adibi, Optics Express 17, 22879-22889 (2009). Flow cytometry systems employing CARS however, still have the problems of 1 ) a strong non-resonant background, which is independent of the presence of the target molecules; 2) a nonlinear dependence of the signal on the concentration of the target species; and 3) excitation spectral which are distorted from Raman line-shape. As such CARS has not been found by the applicants to be unsuitable for use in flow cytometry systems in certain analytical chemistry applications.

An improved method and system for providing more reliable higher resolution flow cytometry without requiring the use of fluorescent agents. SUMMARY

In accordance with an embodiment, the invention provides a flow cytometry system that includes a light source, a spectral shaper, a modulator system, an optics system, an optical detector, a flow conveyance system and a processor. The light source system is for providing a first train of pulses including a first broadband range of frequency components, and a second train of pulses including a second optica! frequency such that a set of differences between the first broadband range of frequency components and the second optical frequency is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses. The spectral shaper is for spectrally modifying an optical property of at least some frequency components of the broadband range of frequency components such that the broadband range of frequency components is shaped producing a shaped first train of pulses to specifically probe a spectral feature of interest from a sample, and to reduce information from features that are not of interest from the sample. The modulator system is for modulating a property of at least one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses. The optics system is for directing and focusing the shaped First train of pulses and the second train of pulses as modulated toward a common focal volume. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of a train of pulses of interest transmitted or reflected through the common focal volume. The flow conveyance system is for providing flow path of a fluid through the common focal volume. The processor is for detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the train of pulses of interest due to the non- linear interaction of the shaped first train of pulses with the second train of pulses as modulated in the common focal volume, and for providing an output signal for the flow cytometry system.

In accordance with another embodiment, the invention provides a method of performing flow cytometry using frequency modulation that includes the steps of: providing a first train of pulses at including a first broadband range of optical frequency components; providing a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second optical frequency is resonant witli a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses; spectrally modifying an optical propeily of at least some frequency components of the first broadband range of frequency components to provide a shaped first train of pulses that is shaped to specifically probe a spectral feature of interest from a sample, and to reduce information from features that are not of interest from the sample; modulating an optical property of one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses and providing the other of the shaped first train of pulses and the second train of pulses as a non- modulated train of pulses; directing and focusing the modulated train of pulses and the non-modulated train of pulses toward a common focal volume; providing flow path of a fluid through the common focal volume; detecting an integrated intensity of substantially all optical frequency components of the other of the modulated train of pulses and the non-modulated train of pulses transmitted or reflected through the common focal volume by blocking the modulated train of pulses; detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the non-modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non-modulated train of pulses in the common focal volume; and providing the detected modulation as the signal for flow cytometry.

In accordance with a further embodiment, the invention provides a method of performing flow cytometry comprising the steps of: a) providing a first train of pulses at including a first broadband range of optical frequency components; b) providing a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second frequency component is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses; c) spectrally modifying an optical property of at least some frequency components of the first broadband range of frequency components such that the first train of pulses is shaped to provide a shaped first train of pulses to specifically probe a spectral feature of interest from a sample; d) modulating a property of one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses and to provide the other of the shaped first train of pulses and the second train of pulses as a non-modulated train of pulses; e) directing and focusing the modulated train of pulses and the non- modulated train of pulses toward a common focal volume; f) providing flow path of a fluid through the common focal volume; g) detecting an integiated intensity of substantially all optical frequency components of the non-modulated train of pulses at a modulation frequency transmitted or reflected through the common focal volume by blocking the modulated train of pulses; h) detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non- modulated train of pulses in the common focal volume; i) further spectrally modulating an optical property of at least some frequency components of the first broadband range of frequency components such that the first train of pulses is negatively shaped to provide to provide a negatively shaped first train of pulses to specifically probe a spectral feature from a sample that interferes with the spectral feature of interest from the sample; j) modulating a property of one of the negatively shaped first train of pulses and the second train of pulses at a modulation frequency to provide a further modulated train of pulses to provide the other of the shaped first train of pulses and the second train of pulses as a non-further modulated train of pulses; k) directing and focusing the further modulated train of pulses and non-further modulated train of pulses toward a common focal volume; 1) detecting an modulation of an integrated intensity of substantially all optical frequency components of non- further-modulated train of pulses and the further modulated train of pulses at a modulation frequency transmitted or reflected through the common focal volume by blocking the further modulated train of pulses; m) subtracting the modulation of the integrated intensity of substantially all of the optical frequency components obtained from the modulation of the integrated intensity of substantially all of the further modulated train of pulses due to the non-linear interaction of the further modulated train of pulses and the non-further modulated train of pulses in the common focal volume to obtain a difference signal; and n) providing an output signal for flow cytometry.

In accordance with a further embodiment, the invention provides a flow cytometry system that includes a light source, a spectral modulator, an optics system, an optical detector, a flow conveyance system, and a processor. The light source system is for providing a first train of pulses including a first broadband range of frequency components, and a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second optical frequency is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses. The spectral modulator is for independently modulating the optical properties of the frequency components of the first train at independent electrical frequencies and phases. The optics system is for directing and focusing the shaped first train of pulses and the second train of pulses as modulated toward a common focal volume. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of a train of pulses of interest transmitted or reflected through the common focal volume. The flow conveyance system is for providing flow path of a fluid through the common focal volume. The processor is for analyzing the output of the optical detector to provide the independent signals of the modulations at the independent electrical frequencies and phases due to the non-linear interaction of the first train of pulses with the second train of pulses in the common focal volume, and for providing an output signal for the flow cytometry system

BRIEF DESCRIPTION OF THE DRAWINGS

The following description may be further understood with reference to the accompanying drawings in which: FIG. 1 shows an illustrative diagrammatic view of flow cytometry system in accordance witli an embodiment of the invention; and

FIG. 2 shows an illustrative diagrammatic view of a flow cytometry system in accordance with another embodiment of the invention involving epi-detection.

The drawings are shown for illustrative purposes only.

DETAILED DESCRIPTION

The present invention involves the use of stimulated Raman scattering (SRS) to extract intrinsic chemical signals from the particles based on their vibrational spectroscopy. In comparison to CARS, SRS removes all of the mentioned constraints. In a previous application SRS has been used in microscopy (see "Label- Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy," by C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, Science 322, 1857-1861 (2008), the disclosure of which is hereby incorporated by reference in its entirety) where two synchronized laser beams are focused onto the sample and the focal point is raster scanned to acquire an image based on the stimulated Raman contrast.

In the typical implementation two narrowband beams are used and the difference frequency is tuned to match a particular vibrational frequency of the target molecule. It can be conceived that such an implementation can be used for flow sample and the laser focal spot is parked to continuously acquire Raman spectroscopy information from the sample. However, such an approach will only provide information of a single Raman band and will not be useful to count or sort complex samples. A more advanced method involving spectrally tailored-excitation can be used to measure the concentration of a target molecule in the presence of interfering species based on more complete Raman spectra (see "Highly specific label-free molecular imaging with spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy," by C. W. Freudiger, W. Min, G. R. Holtom, B. Xu, M. Dantus, and X. Sunney Xie, Nat Photon advance online publication (201 1 ), the disclosure of which is hereby incorporated by reference in its entirety). Nonetheless, the single parameter output still limits the application of it to separation of complicated samples such as biological cells.

Unlike multiplex CARS spectroscopy, where multiple band output can be simultaneously acquired by employing a spectrograph, SRS employs a modulation transfer theme and requires a !ock-in detection and therefore only works with a single- element photo-detector. In order to overcome this problem, a frequency-multiplexing method is disclosed in which a narrowband and broadband laser are used to excite multiple vibrations of the sample simultaneously. The femtosecond laser is amplitude modulated with a modulator that applies different RP modulation frequencies to different spectral components. In doing so, the Raman information is encoded in the modulation RF frequency instead of the color of the laser. On the detection side, the same single-element photo -detector is employed and its output will be digitized. The Fourier-transform retrieves the modulation transfer information, wherein each frequency component corresponds to a particular Raman band. Multi-parameters can be obtained simultaneously without the use of a spectrometer. In a different implementation, the spectral components of the femtosecond laser may be dispersed on to a smart photo-detector-array and each element is demodulated separately.

Stimulated Raman scattering (SRS) is therefore used to extract intrinsic chemical signals from the particles based on their vibrational spectroscopy in a flow cytometry system. SRS allows signal amplification of the weak spontaneous Raman signal by up to 100,000x, thus allowing for fast cell sorting (up to 1 ,000,000 cell/s). Compared to coherent anti-Stokes Raman scattering (CARS), which allows similar signal amplification and has been used for flow-cytometry, SRS lias the advantage, that it has better sensitivity, a linear concentration dependence, spectra identical to spontaneous Raman, and is free from artifacts. In particular, the systems employs multi-color (i.e., multi Raman bands) detection schemes based on spectral imaging (see U.S. Patent Application Publication No. 2010/0188496, the disclosure of which is hereby incorporated by reference in its entirety), Fourier-transform spectroscopy, and lock- in detector arrays. The use of a high-frequency (>lMHz) modulation transfer scheme is also provided for improved sensitivity as previously used for SRS microscopy. Flow-cytomety may also be used in vivo (i.e., the flow is provided by a blood-vessel) for cell-counting applications.

Figure 1 , for example, shows a flow-cytometiy system in accordance with an embodiment of the invention. The system includes a picoseconds (narrowband) optical parametric oscillator 16, a synchronization unit 18, a femtosecond (broadband) Ti:SA laser 20, and a modulator 22 (e.g., an amplitude modulator). The pump-beam is modulated at frequency f(>\ MHz) with the amplitude modulator 22, and the pump and Stokes beams are provided to a flow cytometry system. The pump and Stokes beams that are transmitted or reflected through the flow cytometry focal volume are filtered by an optical filter 26 to block the modulated pump beam and the filtered Stokes beam 30 is detected by a photodetector such as a photodiode 28. In various embodiments, the first train of pulses may be femtosecond pulses and the second train of pulses may be picosecod pulses or femtosecond pulses.

The modulation of the detected intensity of the Stokes beam due to the nonlinear interaction with the sample is extracted with an electronic processing unit such as a lock-in amplifier. Excitation spectroscopy is performed by shaping the broadband pulse by an amplitude or polarization pulse shaper 31 that consists of a dispersive element 32 that disperses the individual frequency components of the broadband beam onto the different elements of a multiplex amplitude or polarization shaper such as a spatial light modulator 36. Such a device may

in reflection mode (as shown) or in transmission mode. Typically, a lens 34 is positioned in a way to refocus the reflected beam such that an un-chirped, spectrally homogenous beam is provided to the spatial light modulator 36. In various embodiments, the settings on the pulse shaper 31 may also be changed or modulated during imaging to provide either a modulation of the pulse train or to provide different sets of pulse shapes for probing multiple species within a sample.

In line with the high-frequency modulation scheme presented above and necessaiy for high-sensitivity SRS detection, amplitude modulation is performed with an additional electro-optical or acoustic-optical modulator 22. Alternatively, it is possible that pulse-shaping of the broadband beam and amplitude modulation for high-frequency detection may be performed by the same device such as an acousto- optic tunable filter (AOTF). Pump and Stokes beam may be combined with a dichroic beam-combiner. If the pump-beam is modulated for stimulated Raman gain (SRG) as discussed further below, all frequency components of the Stokes beam are collected with a single photodiode and the SRG for a particular excitation pattern is extracted with electronic processing systems such as a lock-in detector.

The flow cytometry system includes a flow conveyance system 40 that carries fluid from a sample volume 42 by way of a pump 44 (e.g., a syringe flow system) through an interrogation focal volume 46 that is provided by an objective lens 48. Illumination from the focal volume 46 is directed by a condenser 50 onto a photo- detector 28 (e.g., a photo diode or a smart photo- detector array). The output of the photo -detector is provided to a lock-in amplifier 52 that is coupled to a signal processor and control electronics unit 54. The unit 54 drives the modulator 22 and also controls the operation of a flow switching unit 56 in the flow path of the fluid, to direct the fluid to one of two different reserves 58, 60 as shown. In particular, if certain samples 62 are found to be in the fluid stream, the switching unit 56 directs such samples 62 (e.g., cancer cells) to the reserve 58 instead of the reserve 60 as shown.

In the pulse shaper 31 , an excitation mask may be generated from a broadband laser spectra and the dispersive element 32 (grating or prism) together with the multiplex amplitude shaper 36 (such as a spatial light modulator SLM or printed transmission/reflection mask) that can individually control the intensity of eveiy frequency components of the broadband excitation pulse to provide a shaped train of pulses.

Such an approach may improve specificity by implementation of a background subtraction scheme for interfering species. Instead of illuminating the sample with one excitation mask, the signal for two masks is measured. A first mask 1 contains mainly the frequency components of the target molecule and a second mask 2 contains mainly the frequency components of interfering species. Because of the spectral interference of the target molecule with interfering or other species in certain applications, the signal for mask 1 can never be chosen to only contain contributions from the target species but will always excite signal from the interfering or other species. It is however, always possible to design the two excitation masks in a way, that the difference between the intensities for the two masks is independent of the concentration of the interfering or other species. In accordance with further embodiments, the two more species may be probed separately wherein for each probing information from the non-probed species is reduced.

The difference between the signal from mask 1 and mask 2 may either be taken on a pixel by pixel basis for an image from the interrogation volume 46 or an image with mask 1 and mask 2 may be taken first and the subtraction may be performed in the post-processing.

In accordance with further embodiments, pulse shaping may be achieved using a multiplex electro-optic modulator, a multiplex electro-acoustic modulator, an acousto-optic tunable filter, or a Dazzler system as sold by Fastlite Societe a responsabilite limitee of Saint-Aubin France. The broadband beam is not required to have all frequency components within a range present in the beam, but instead may be composed of a plurality of center frequency components, as long as sufficient frequency components are present in the broadband beam that may be shaped for probing a sample as desired. The spectral range of the broadband beam may be, for example, at least 15.0 nm, or at least 5.0 run, or at least 1.0 nm, or even at least 0.5 nm in certain embodiments.

The masks may be designed such that the signal from an isolated peak of an interfering species is scaled in such a way that after subtractioir this only leaves the pure signal from the target species, which is independent of the concentration of component B. The two masks may also be chosen to maximize the signal from the peak and subtract the flat background. An objective is to design positive excitation spectral shapes. In a mixture of for example, A, B and C with unknown concentrations c of each one, two positive excitation spectral shapes Ι₊ (Αω) and

I_ (A >) (i.e., masks) may be designed such that the difference signal AS from these two excitation masks can selectively predict the concentration of molecule A without getting interference from molecules B and C. For a given excitation spectral shape Ι(Αω), the obtained absorption signal S will be described by the following

S cc

For two excitation spectral shapes 7₊(Δω) and Ι_{Αώ), the difference signal AS will be

AS≡ S_t -S_ o ₊(Αω) -

(Αω) + c _Βε _B(Aco) + c_cs_c(Aa>)lflAa)

It is mathematically possible that a design may be positive wherein Ι₊(Αω) and Ι_(Αω) function such that their difference functions satisfy the following orthogonal relations with the Raman spectra of all the interferent species:

|[/₊(Δω)-/_(Δω)]&_Λ(Δω)ί/ΔίΒ = 0 and

|[/₊(ΔΑ -/_(Δω)]ε_ε(Δί»)ί/Δ<» = 0

Note that the Raman shift-dependent Ι₊(Αω) -Ι₊(Αω) has both positive and negative values. As a result, the following quantity may be simplified: [l ₊(Αω) - I _(Αω) [?_ΑΕ _Α(Αω) + c _Βε _Β{Αω) + c_{c r} ( ω)^Αω =

(Αω) - 1 _ (Αω)]=_Αε_Α (Αω)άΑω

The difference signal therefore, is only proportional to the concentration of molecule of interest:

AS≡ S₊ -S_ cc c_A [ΐ₊(Αω)-Ι_(Αω)]ε_Α(Αω)άΑω Because changing different components of excitation spectrum by pulse- shaping does not require the laser to change, fast switching speeds are possible allowing for spectra-temporal shaping of the excitation beams to encode the signal from different chemical species in time (or modulation phase and frequency) instead of optical frequency such that it can be detected with a single detector. This allows implementation of: 1 ) real-time detection of pure signal from target species free from the background signal of interfering species by the subtraction scheme of the two- masks as described above, and 2) simultaneous multicolor imaging with a single detector.

It is desirable to comprise a concrete implementation for fast switching between multiple excitation masks, because laser noise occurs primarily at frequencies < IMHz and different excitation masks need to be probed faster than the laser can change. The sample may also change in between frames (e.g., move), making a frame-by-frame acquisition of different mask impossible.

Technically there are many multiple technologies to achieve such fast spectral modulations, such as: 1 ) spectral modulation with a single device such as an electro- optic or acousto-optic modulator that allows the independent modulation of individual spectral component of the broadband excitation beam, and 2) a combination of a polarization pulse shaper, polarization modulator and polarization analyzer.

Stimulated Raman scattering allows the detection of the vibrational signal with higher signal levels than spontaneous Raman scattering due to stimulated excitation of molecular vibrations and without exciting the non-resonant background signal of CARS microscopy. Spontaneous Raman spectra are thus preserved and the signal strength scales linearly with the concentration allowing for straight forward quantification. Forward- and reverse (epi)-detection is also possible.

In a narrowband SRS flow cytometry, pump and Stokes-beam are used to excite the sample, just as in CARs microscopy. Instead of detecting the newly emitted light at the anti-Stokes frequency, intensity gain (stimulated Raman gain) at the pump frequency or intensity loss (stimulated Raman loss) at the Stokes frequency are detected. As the gain and loss are relatively small a high-frequency detection scheme is often required, in which the SRS signal is modulated at a known frequency that is higher than the laser noise and is extracted with an electronic detector such as a lock-in amplifier to provide the intensity of a pixel. The modulation may be frequency modulation, phase modulation or amplitude modulation.

In this narrowband approach to coherent Raman imaging only a single molecular vibration can be imaged at a time. Thus only single color images may be produced (compared to imaging multiple species simultaneously) and detection is limited to chemical species that have an isolated vibration that does spectrally not interfere with other compounds in the sample. The present invention provides (in certain embodiments) methods and systems to allow coherent Raman imaging based on multiple Raman lines simultaneously.

In particular, a pump beam and a Stokes beam in a sample volume enhance a spontaneous Raman radiation signal. The center frequency of the Stokes beam and the center frequency of the pump beam are separated by an input spectra. SRS leads to an intensity increase in the Stokes beam (stimulated Raman gain or SRG) and an intensity decrease in the pump beam (stimulated Raman loss or SRL).

In accordance with other embodiments, the pump beam is provided as an input pulse train, and the Stokes beam is provided as an input pulse train that is modulated at high frequency / (MHz). The output pulse train includes a resulting amplitude modulation at the high frequency (MHz) due to stimulated Raman loss (SRL) that can only occur if both beams are present. This modulation of the originally non- modulated beam at the same frequency of the modulation / may then be detected by detection electronics and separate it from the laser noise that occurs at other frequencies. Stimulated Raman gain (SRG) of the Stokes-beam can be probed by modulating the pump beam and detecting the Stokes beam.

An SRL flow cytometry system may be provided witli either or both forward and epi (reverse) detection. The Stokes beam may be modulated by an electro-optic (or acoustic-optic) modulator and then combined with the pump beam by a beam splitter / combiner. The transmitted or reflected pump beam is filtered by a filter, and detected by a photodiode (PD). For epi detection, the back-scattered beams are collected by the excitation objective lens (OL) and separated from the excitation beams by a combination of a quarter wave plate (λ/4) and polarizing beam splitter (PBS). For forward detection, the forward-scattered beams are collected by a condenser. The SRL is measured by a lock-in amplifier to provide a pixel of the image.

In accordance with various embodiments, the system may be provided using a variety of sources and a variety of modulation techniques. For example, a flow cytometry system 100 in accordance with an embodiment of the invention may include a dual frequency laser source and an optical parametric oscillator. The dual frequency laser source may provide a broadband train of laser pulses at a center frequency (e.g., including a Stokes frequency coy of, for example, 1064 nm), and a train of laser pulses at a more narrow band of frequencies having a center frequency (e.g., 532 nm) to the optical parametric oscillator. The optical parametric oscillator may be, for example, as disclosed in U.S. Patent No. 7,616,304, the disclosure of which is hereby incorporated by reference in its entirety. The output of the optical parametric oscillator provides a train of laser pulses at a center frequency α½ (e.g., a pump frequency) that is selected such that a difference between ω_/ and co_? (e.g., ω_ρ - cos) is resonant with a vibrational frequency of a sample in a flow cytometry interrogation focal volume.

Each pulse of the train of laser pulses is then spectrally shaped by the shaping assembly 3 1 that includes the dispersive element 32, the lens 34 and the spatial light modulator 36. The dispersive element 32 spectrally disperses each broadband pulse, and the spatial light modulator 36 then modulates different frequency components of the specially disperse broadband pulse to provide a train of shaped pulses.

The train of shaped laser pulses is then modulated by the modulator 22, and is then phase adjusted at a translation stage to ensure that the resulting train of modulated shaped laser pulses and the train of laser pulses at the center pump frequency are temporally overlapped. The two trains of laser pulses are combined at a combiner 64 such that they are collinear and spatially overlapped as well.

The illumination from the flow cytometry interrogation volume is directed by the condenser 50 onto the optical detector 28, and the modulated shaped beam (e.g., the Stokes beam) is blocked by the optical filter 26, such that the optical detector 28 measures the intensity of the other beam ω; (e.g., the pump beam) only.

The train of shaped laser pulses is modulated at modulation frequency f (e.g., at least about 100 kHz), by a modulation system that includes, for example, the modulator 22, the controller 54 and a modulation source within the controller. The integrated intensity of substantially all frequency components of the first pulse train from the optical detector is provided to the signal processor, and the intensity modulation due to the non-linear interaction of the train of laser pulses with the train of laser pulses in the interrogation volume is detected at the modulation frequency /to provide information regarding the flow volume. In accordance with an embodiment, the modulation system may provide amplitude modulation of the shaped pulses to provide the modulated shaped pulse train such that only alternating pulses of the shaped pulse train are coincident with the pulses of the ωι pulse train. Such amplitude modulation of the shaped beam may be achieved using a Pockel cell and polarization analyzer as the modulator, and a Pockel cell driver as the controller. In accordance with another embodiment, the modulation rate is half the repetition rate of the laser such that every other pulse of the original o? pulse train is reduced in amplitude to provide that stimulated Raman scattering does not substantially occur in the focal volume with the pulses having the reduced amplitude. If the modulation rate is of the same order of the repetition rate of the laser, countdown electronics can be utilized to guarantee the synchronization (phase) between the modulation and the pulse train. Lower modulation rates are also possible, as long as the modulation frequency is faster that the laser noise. In further embodiments, the contrast pulses may have an amplitude that is substantially zero by switching off the pulses at the modulation frequency, for example using an electro- optic modulator or an acousto-optic modulator.

Amplitude modulation of the pump or Stokes pulse trains may therefore be achieved, and the increase of the Stokes pulse train or decrease of the pump pulse train may be measured. By modulating the pump train of pulses and then detecting the Stokes train of pulses from the focal volume, stimulated Raman gain (SRG) may be determined by the processing system. In further embodiments, the Stokes beam may be modulated, the pump beam may be detected from the focal volume, and stimulated Raman loss (SRL) may be determined by the processing system. In still further embodiments, the phase of one of both the shaped beam and the non-shaped beam may be phase modulated or frequency modulated as long as the modulation is done at the modulation frequency such that the detection system is able to extract the signal of interest. In still further embodiments, both the pump and Stokes beams may be modulation by a modulation system.

Systems of various embodiments of the invention, therefore, provide that stimulated Raman scattering flow cytometiy may be achieved using a modulation of one of the pump or Stokes beams as a contrast mechanism. The process may be viewed as a two photon process for excitation of a vibrational transition. The joint action of one photon annihilated from the pump beam and one photon created to the Stokes beam promotes the creation of the molecular vibrational phonon. The energy of the pump photon is precisely converted to the sum of the energy of the Stokes photon and the molecular vibrational phonon. As in any two photon optical process, the transition rate is proportional to the product of the pump beam intensity and the Stokes beam intensity. It is obvious that a molecular vibrational level is necessary for this process to happen, as required by the energy conservation. There is, therefore, no contribution from non-resonant background would be present. This represents a significant advantage over CARS microscopy which is severely limited by non- resonant background which not only distorts the spectrum but also carries unwanted laser noise.

For stimulated Raman gain for Stokes beam, a different third-order nonlinear induced polarization radiates at the Stokes beam frequency. The intensity dependence of this nonlinear radiation scales quadratically with pump beam and linearly with Stokes beam. Its final phase is the same as that of the input Stokes beam at the far field detector. Therefore, the interference between this nonlinear radiation and input Stokes beam results in an increase of the Stokes beam itself. The intensity dependence of the interference term again scales linearly with both the pump beam and Stokes beam.

Although the use of amplitude modulation has the highest modulation depth, this approach may introduce a linear background due to a modulation of the temperature or refractive index of the sample due to the intensity modulation on the sample. In accordance with another embodiment, the modulation system may provide polarization modulation, and may include a polarization device as the modulator, and a polarization controller as the controller. Every other pulse of the ω₂ pulse train has a polarization that is different than that of the other preceding pulse. Each of the ω₂ pulses of the pulse train is coincident with a coi pulse of the a>j pulse train. Different modulation rates other than half of the repetition rate of the laser (in which every other pulse is different) can also be applied.

Polarization modulation also provides that stimulated Raman scattering does not substantially occur in the interrogation volume with the pulses having the altered unparallel polarization. In certain embodiments, the modulator includes a polarization filter to remove one of the sets of pulses as a further contrast mechanism. The polarization of the pulses may therefore, be modulated with respect to each other. In other embodiments, the detector itself may distinguish between the modulated pulses. In particular, when pump and Stokes pulse trains are perpendicular to each other, a different tensor element of the nonlinear susceptibility is probed compared to the case where pump and Stokes field are parallel. Different tensor elements have significantly different magnitudes. This converts the polarization modulation of the excitation beams into amplitude modulations of the gain/loss signal which can then be detected with the lock-in amplifier. Polarization modulation can be implemented with a Pockel cell. This approach has the advantage that it does not introduce a temperature modulation of the sample.

In accordance with other embodiments, one of the pulse trains may be modulated by time-shifting (or phase). For example, one pulse train may include alternating pulses that coincide with a toi pulse, while the remaining pulses are time shifted such that they do not coincide with a ωι pulse. Modulation of one or both of the pump and Stokes beams may also be achieved by frequency modulation as disclosed for CARS microscopy, for example, in U.S. Patent No. 7,352, 458, the disclosure of which is hereby incorporated by reference in its entirety. In a frequency modulation system, the frequency of one or both of the pump and Stokes beams is alternately modulated at a modulation frequency such that a difference frequency between the pump and Stokes beams (e.g., ω_ρ - ω_$> is tuned in and out of a vibrational frequency of the sample. The detector then detects the gain/loss that is generated through non-linear interaction of ω_ρ and α¾ and the sample responsive to the modulation frequency. An output signal may be passed through a lock-in amplifier such that only changes at the time scale of the modulation period are provided in the final output. In accordance with further embodiments, other modulation schemes may be employed such as time-delay modulation, spatial beam mode modulation, etc., which will each introduce a modulation of a generated signal.

For example, in accordance with further embodiments, systems of the present invention may employ a dual frequency laser source, a first optical parametric oscillator, as well as an additional optical parametric oscillator that splits the power of the dual frequency laser source. The dual frequency laser source provides a first train of laser broadband pulses (including a pump frequency ω{) and a second train of laser pulses at a center frequency to the optical parametric oscillator and to the optical parametric oscillator. The first train of laser pulses are shaped as discussed above. The output of the optical parametric oscillator provides a third train of laser pulses at a center Stokes frequency £¾ that is selected such that a difference between a>i and <¾ (e.g., op - <ϊ¾) is resonant with a vibrational frequency of a sample (not shown) in a focal volume. The output of the optical parametric oscillator provides a fourth train of laser pulses at a center frequency C02 ' that is selected such that a difference betweeno j and CO2 ' (e.g., ω_ρ- <¾¾*') is not resonant with a vibrational frequency of the sample in the focal volume.

The a>2 pulses are passed through a half wave plate and combined with the ω₂ pulses, which are passed through a different half wave plate. The half wave plates ensure that the pulse trains have different polarization such that one is transmitted by the beam splitter and the other is reflected. At this point, the combined pulse train includes both the a>2 and the ω{ pulses, but with mutually orthogonal polarization. The combined eo^ and the CU2' pulses are passed through a modulator that, responsive to a modulation signal that provides a modulation frequency from a modulation source. Based on the different polarization the modulator together with a polarization analyzer selects a different polarization at the modulation rate i.e., it selects a>2 or ω₂ pulses. The result is that a pulse train of alternating ω₂ and pulses is provided. The first shaped train of laser pulses and the alternating train of laser pulses and are combined at a combiner such that they are collinear and spatially overlapped as well, and the combined pulse trains are directed toward a sample as discussed above.

In accordance with further embodiments, the system may include an electronically locked laser such as an electronically locked mode-locked titanium sapphire laser in place of the optical parametric oscillator. In still further embodiments, the system may include a single optical parametric oscillator for providing both the cu and the cof pulses, and the single optical parametric oscillator may provide the alternating train of laser pulses responsive to a modulation signal that is coupled to the signal processor. In accordance with further embodiments, the system may provide different spectral masks at different modulation frequencies, as well as multiple lock-in detectors tuned to the different modulation frequencies such that a plurality of species may be probed at the same time.

As shown in Figure 2, a system in accordance with a further embodiment of the invention includes the picoseconds (narrowband) optical parametric oscillator 16, the synchronization unit 18, the femtosecond (broadband) Ti:SA laser 20, and the amplitude modulator 22 as discussed above. The pump-beam is modulated at frequency f (>l MHz) with the amplitude modulator 22, and the pump and Stokes beams are provided to the flow cytometry system via combiner 64 such that they are collinear and spatially overlapped. The pump and Stokes beams that are transmitted or reflected through the flow cytometry focal volume are filtered by an optical filter 126 to block the modulated pump beam and the filtered Stokes beam is detected by a photodetector such as a photodiode 128. Again, in various embodiments, the first train of pulses may be femtosecond pulses and the second train of pulses may be picosecod pulses or femtosecond pulses.

The modulation of the detected intensity of the Stokes beam due to the nonlinear interaction with the sample is extracted with an electronic processing unit such as a lock-in amplifier. Excitation spectroscopy is performed by shaping the broadband pulse by the amplitude or polarization pulse shaper 31 that consists of the dispersive element 32 that disperses the individual frequency components of the broadband beam onto the different elements of a multiplex amplitude or polarization shaper such as the spatial light modulator 36. The lens 34 is positioned in a way to refocus the reflected beam such that an un-chirped, spectrally homogenous beam is provided to the spatial light modulator 36. In various embodiments, the settings on the pulse shaper 31 may also be changed or modulated during imaging to provide either a modulation of the pulse train or to provide different sets of pulse shapes for probing multiple species within a sample.

As further shown in Figure 2 therefore, the flow cytometry system in accordance with a further embodiment of the invention includes an in-situ flow cytometry system. In particular, the system includes an objective lens 148 (such as disclosed in Patent Cooperation Treaty Patent Application PCT US2010/54925 filed November 1, 2010, disclosure of which is hereby incorporated by reference in its entirety), a beam splitter 160, the filter 126, the detector 128, a lock-in amplifier 152, a processor 154, and a control computer 156.

During use, the objective lens 148 directs the interrogating pump and Stokes fields into an interrogation volume within a subject, and in particular for example, within a portion of a vein 160 of a subject 158. As blood flows within the vein 160, the system may monitor images of the interrogation volume using the stimulated Raman scattering processes described above.

During the non-linear interaction of the modulated Stokes train of pulses and the pump train of laser pulses when focused through optics 148 toward a interrogation volume, both the pump and Stokes pulses are directed in a forward direction. Some pump and Stokes pulses are however, initially forward directed but are then reflected by non-uniformities within the sample back toward the optics 148. The detector 128 may therefore, be positioned in the reverse direction with respect to the incoming pump and Stokes pulse trains that are directed into the focal volume. In such as reverse direction detection system, the detector will detect reflected pump pulses.

As the signal and excitation beams have the same optical frequency, the system may provide that the beam splitter 160 is a 50/50 splitter that reflects 50% of an incident beam and ti ansmits 50% of the incident beam through the beam splitter onto a heat absorber (not shown). This would ideally provide that 25% of the Stokes beam would be transmitted back into the detector 128. In further embodiments, the beam splitter 160 may be a 20/80 splitter that reflects 20% of an incident beam and tiansmits 80% of the incident beam through the beam splitter, resulting in 4% signal on the detector 128.

As with the embodiments discussed above, the system may provide modulation at a modulation frequency f, such as amplitude modulation, polarization modulation, phase modulation or frequency modulation, and the processor 154 (using a phase locked loop) detects a modulation (amplitude and/or phase) of the integrated intensity of substantially all of the optical frequency components of the Stokes pulse train due to the non-linear interaction of the Stokes pulse train with the pump pulse train within the common focal volume.

The specificity of the SRS signal for a certain target species of the presented single-band approach with narrowband lasers is, however, limited, as different chemical bonds may have the same vibrational frequencies. The full specificity for Raman spectroscopy may be exploited only if the full vibrational spectrum of all bonds of a compound are probed rather than simply a single frequency.

In accordance with various embodiments of the invention therefore, spectral masks may be used to provide improved imaging. For example, none of the individual Raman peaks may be isolated from those of other molecules, but the molecule's overall vibrational fingerprint, however, may be unique. Complex molecules have several Raman active peaks, which combined result in a characteristic vibrational signature of the molecules. Vibrational spectra can thus be used as a label-free contrast mechanism for biomedical imaging.

If only a single Raman peak is used as a marker-band, crosstalk between different compounds is possible. This can limit the specificity of the methods in many cases. It is possible to probe the a bigger portion Raman spectrum with SRS, by using at least one broadband laser source as pump- and Stokes beam.

As discussed above, suppression of spectral cross-talk may be achieved by subtracting the signal from mask 2 (mainly containing the spectral components resonant with the interfering molecules) from the signal from mask 1 (mainly containing the spectral components resonant with the target molecules). As laser noise scales with the absolute signal, i.e., with the signal from the target component and the interfering species, the signal from the target molecules can easily be buried in the laser noise of the interfering species, when its concentration is much lower or the Raman scattering cross-section is much weaker. For this reason the subtraction from mask 1 and mask 2 has to be accomplished at a MHz rate since laser noise occurs mainly at lower frequencies. As such multivariate optical computation applied to excitation spectroscopy in SRS microscopy is equivalent to a complex frequency modulation scheme between two arbitrarily shaped excitation spectra.

Although the above discussion is directed to an application involving SRS microscopy, the ideas are valid for any type of contrast in microscopy that is based on excitation spectroscopy such as CARS, one- and two-photon absorption and emission, stimulated emission, photo-thermal scattering and photo-acoustic scattering. It is also possible to use a femtosecond - femtosecond configuration (i.e., both pump and Stokes beam are broadband), for which one or even both beams are shaped. The excitation masks are not as obvious in this situation, as all frequency combinations between the two pulses need to be considered, but they can be determined as the spectral resolution is solely determined by the spectral resolution of the pulse-shaper and not the bandwidth of the lasers.

The objective is to design positive excitation spectral shapes. In a mixture of A, B and C with unknown concentrations c of each one, two positive excitation spectral shapes Ι_÷{Αω) and I_ (Aa>) (i.e., masks) may be designed such that the difference signal AS from these two excitation masks can selectively predict the concentration of molecule A without getting interference from molecules B and C. For a given excitation spectral shape I(Aco) , the obtained absorption signal S may be described as discussed above.

In accordance with certain embodiments, therefore, the spectral shaper (e.g., a spatial light modulator), may be set to provide a first mask having a first polarization at the same time that the spectral shaper is set to provide a second mask having a second polarization. The spectral shaper, therefore, provides two polarization distinct masks at the same time without changing. A polarization modulator may then switch between the two masks very quickly, permitting real-time subtraction of the results obtained using the second mask from the results obtained using the first mask.

It is also possible to use a femtosecond - femtosecond configuration (i.e., both pump and Stokes beam are broadband), for which one or even both beams are shaped, as the spectral resolution is solely determined by the spectral resolution of the pulse- shaper and not the bandwidth of the lasers. Those skilled in the art will appreciate that numerous modifications and variations may be made to the above disclosed embodiments without departing from the spirit and scope of the claims.

What is claimed is:

Claims

1. A flow cytometry system comprising:

a light source system for providing a first train of pulses including a first broadband range of frequency components, and a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second optical frequency is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;

a spectral shaper for spectrally modifying an optical property of at least some frequency components of the broadband range of frequency components such that the broadband range of frequency components is shaped producing a shaped first train of pulses to specifically probe a spectral feature of interest from a sample, and to reduce information from features that are not of interest from the sample;

a modulator system for modulating a property of at least one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses;

an optics system for directing and focusing the shaped first train of pulses and the second train of pulses as modulated toward a common focal volume;

an optical detector for detecting an integrated intensity of substantially all optical frequency components of a train of pulses of interest transmitted or reflected through the common focal volume;

a flow conveyance system for providing flow path of a fluid through the common focal volume; and

a processor for detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the train of pulses of interest due to the non-linear interaction of the shaped first train of pulses with tlie second train of pulses as modulated in tlie common focal volume, and for providing an output signal for the flow cytometry system.

2. The flow cytometry system as claimed in claim 1 , wherein said system further includes a switching device in the flow path and wherein the switching device is responsive to the output signal.

3. The flow cytometry system as claimed in claim 1 , wherein said system receives said modulation of the integrated intensity of substantially all of the optical frequency components of the train of pulses in an epi-direction.

4. The flow cytometry system as claimed in claim 1 , wherein only one of the shaped train of laser pulses or the second train of pulses is modulated at the modulation frequency to provide the modulated train of pulses such that the other of the shaped train of laser pulses and the second train of pulses remains a non- modulated train of pulses;

wherein the optical detector detects the integrated intensity of substantially all optical frequency components of the non-modulated train of pulses transmitted or reflected through the common focal volume by blocking the modulated train of pulses; and

wherein the processor detects a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the non-modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non-modulated train of pulses in the common focal volume.

5. The flow cytometry system as claimed in claim 4, wherein said optical property of either the shaped first train of pulses or the second train of pulses that is modulated is amplitude.

6. The flow cytometry system as claimed in claim 4, wherein said optical property of either the shaped first train of pulses or the second train of pulses that is modulated is polarization and wherein said system further includes a polarization analyzer.

7. The flow cytometry system as claimed in claim 1 , wherein said spectral shaper and said modulator system are included in the same device.

8. The flow cytometry system as claimed in claim 4, wherein said broadband range of frequency components includes a range of frequency components of at least 0.5 nm.

9. The flow cytometry system as claimed in claim 1 , wherein said broadband range of frequency components is discontinuous.

10. The flow cytometry system as claimed in claim 4, wherein said second train of pulses also has a broadband range of frequency components.

1 1. The flow cytometry system as claimed in claim 4, wherein the modulation frequency is at least 100kHz.

12. The flow cytometry system as claimed in claim 4, wherein said system employs stimulated Raman spectroscopy as a contrast mechanism, and wherein the first train of pulses provides a pump beam, and the second train of pulses is modulated by the modulator system at the modulation frequency to provide one of a pump beam and a Stokes beam such that a Raman loss is detected at the signal processor at the modulation frequency.

13. The flow cytometry system as claimed in claim 4, wherein said system employs stimulated Raman spectroscopy as a contrast mechanism, and wherein the first train of pulses provides a pump beam, and the second train of pulses is modulated by the modulator system at the modulation frequency to provide one of a pump beam and a Stokes beam such that a Raman gain is detected at the signal processor at the modulation frequency.

14. The flow cytometry system as claimed in claim 1 , wherein said processor detects a modulation of the integrated intensity of substantially all of the optical frequency components of a train of anti-Stokes pulses due to the non-linear interaction of the shaped first train of pulses with the second train of pulses as modulated in the common focal volume.

15. The flow cytometry system as claimed in claim 1 , wherein said system employs two-photon absorption as a contrast mechanism in which one photon from the first train of pulses and a second photon from the second train of pulses are simultaneously absorbed.

16. The flow cytometry system as claimed in claim 4, wherein said spectral shaper includes one of a spatial light modulator, a dazzler system, a multiplex electro-optic modulator, a multiplexed electro-acoustic modulator, or an acousto-optic tunable filter.

17. The flow cytometry system as claimed in claim 4, wherein said spectral shaper differently modifies a polarization of different frequency components of the broadband range of frequency components of the first train of pulses.

18. The flow cytometry system as claimed in claim 4, wherein said spectral shaper differently modifies an amplitude of different frequency components of the broadband range of frequency components of the first train of pulses.

19, The flow cytometry system as claimed in claim 4, wherein said spectral shaper includes a spectral dispersion unit and a polarization spatial light modulator, and wherein said modulator is a polarization modulator, and wherein said system further includes a polarization analyzer that is positioned before or after the modulator.

20. A method of performing flow cytometiy using frequency modulation comprising the steps of:

providing a first train of pulses at including a first broadband range of optical frequency components;

providing a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second optical frequency is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;

spectrally modifying an optical property of at least some frequency components of the first broadband range of frequency components to provide a shaped first train of pulses that is shaped to specifically probe a spectral feature of interest from a sample, and to reduce information from features that are not of interest from the sample; modulating an optical property of one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses and providing the other of the shaped first train of pulses and the second train of pulses as a non-modulated train of pulses;

directing and focusing the modulated train of pulses and the non-modulated train of pulses toward a common focal volume;

providing flow path of a fluid through the common focal volume;

detecting an integrated intensity of substantially all optical frequency components of the other of the modulated train of pulses and the non-modulated train of pulses transmitted or reflected through the common focal volume by blocking the modulated train of pulses;

detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the non-modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non- modulated train of pulses in the common focal volume; and

providing the detected modulation as the signal for flow cytometry.

21. The method as claimed in claim 20, wherein said method further includes the step of controlling a switching device in the flow path responsive to the output signal.

22. The method as claimed in claim 20, wherein said modulation of the integrated intensity of substantially all of the optical frequency components of the train of pulses is received by a detector in an epi-direction.

23. The method as claimed in claim 20, wherein said step of spectrally modifying an optical property of at least some frequency components and the step of modulating an optical property of one of the shaped first train of pulses and the second train of pulses to provide a modulated train of pulses is performed by the same device.

24. The method as claimed in claim 20, wherein said method further includes the steps of:

further spectrally modifying an optical property of at least further frequency components of the broadband range of frequency components of the first train of pulses to provide a further shaped first train of pulses to specifically probe a spectral feature from a sample that interferes with the spectral feature of interest from the sample;

subtracting the detected modulation of the integrated intensity of substantially all of the optical frequency components of the non-modulated train of pulses due to the non-linear interaction of the further shaped first train of pulses and the second train of pulses in the focal volume from the detected modulation of the integrated intensity of substantially all of the optical frequency components of non-modulated train of pulses due to the non-linear interaction of the originally shaped first train of pulses and the second train of pulses in the focal volume; and providing the difference as the signal for flow cytometry.

25. The method as claimed in claim 22, wherein said step of further spectrally modifying an optical property of at least further frequency components of the broadband range of frequency components of the first train of pulses is performed for an entire scan area prior to the step of subtracting the detected modulation of the integrated intensity of substantially all of the optical frequency components obtained thereby from the detected modulation of the integrated intensity of substantially all of the optical frequency components of non-modulated train of pulses.

26. The method as claimed in claim 23, wherein said step of further spectrally modifying an optical property of at least further frequency components of the broadband range of frequency components of the first train of pulses and the step of subtracting the detected modulation of the integrated intensity of substantially all of the optical frequency components obtained thereby from the detected modulation of the integrated intensity of substantially all of the optical frequency components of non-modulated train of pulses are performed.

27. Tire method as claimed in claim 20, wherein said step of spectrally modifying an optical property of at least some frequency components of the broadband range of frequency components of the shaped first train of pulses involves amplitude modulation.

28. The method as claimed in claim 20, wherein said step of spectrally modifying an optical property of at least some frequency components of the broadband range of frequency components of the shaped first train of pulses involves polarization modulation.

29. The method as claimed in claim 20, wherein said method includes the steps of providing different spectral masks at different modulation frequencies, as well as the steps of detecting multiple trains of pulses of interest using multiple lock-in detectors tuned to the different modulation frequencies such that a plurality of species may be probed at the same time.

30. A method of performing flow cytometry comprising the steps of:

a) providing a first train of pulses at including a first broadband range of optical frequency components;

b) providing a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second frequency component is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;

c) spectrally modifying an optical property of at least some frequency components of the first broadband range of frequency components such that the first train of pulses is shaped to provide a shaped first train of pulses to specifically probe a spectral feature of interest from a sample; d) modulating a property of one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses and to provide the other of the shaped first train of pulses and the second train of pulses as a non-modulated train of pulses;

e) directing and focusing the modulated train of pulses and the non-modulated train of pulses toward a common focal volume;

f) providing flow path of a fluid through the common focal volume;

g) detecting an integrated intensity of substantially all optical frequency components of the non-modulated train of pulses at a modulation frequency transmitted or reflected through the common focal volume by blocking the modulated train of pulses;

h) detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non-modulated train of pulses in the common focal volume;

i) further spectrally modulating an optical property of at least some frequency components of the first broadband range of frequency components such that the first train of pulses is negatively shaped to provide to provide a negatively shaped first train of pulses to specifically probe a spectral feature from a sample that interferes with the spectral feature of interest from the sample;

j) modulating a property of one of the negatively shaped first train of pulses and the second train of pulses at a modulation frequency to provide a further modulated train of pulses to provide the other of the shaped first train of pulses and the second train of pulses as a non-further modulated train of pulses;

k) directing and focusing the further modulated train of pulses and non-further modulated train of pulses toward a common focal volume; 1) detecting an modulation of an integrated intensity of substantially all optical frequency components of non-further-modiilated train of pulses and the further modulated train of pulses at a modulation frequency transmitted or reflected tlirough the common focal volume by blocking the further modulated train of pulses;

m) subtracting the modulation of the integrated intensity of substantially all of the optical frequency components obtained from the modulation of the integrated intensity of substantially all of the further modulated train of pulses due to the non- linear interaction of the further modulated train of pulses and the non-further modulated train of pulses in the common focal volume to obtain a difference signal; and

n) providing an output signal for flow cytometry.

31 . A flow cytometry system comprising:

a spectral modulator for independently modulating the optical properties of the frequency components of the first train at independent electrical frequencies and phases. an optics system for directing and focusing the shaped first train of pulses and the second train of pulses as modulated toward a common focal volume; an optical detector for detecting an integrated intensity of substantially all optical frequency components of a train of pulses of interest transmitted or reflected through the common focal volume;

a processor for analyzing the output of the optical detector to provide the independent signals of the modulations at the independent electrical frequencies and phases due to the non-linear interaction of the first train of pulses with the second train of pulses in the common focal volume, and for providing an output signal for the flow cytometry system.

32. The flow cytometry system as claimed in 31 , where independent optical frequency components of the first train of pulses are modulated at different electrical frequencies and the processor computed the Fourier transform of the output of the optical detector to provide the amplitude of the modulation at the different electrical frequencies, which correspond to the optical frequencies of the first train of pules.

33. The flow cytometry system as claimed in claim 31 , which further included a high- frequency modulator for modulating an optical property of the first or the second train of pulsed at a high frequency and the processor includes a lock-in amplifier referenced to the frequency of the high-frequency modulator.

34. The flow cytometry system as claimed in claim 31 , in which the optical detector has multiple elements.

35. The flow cytometry system as claimed in claim 31 , in which the optical detector is a smart-CMOS camera.

36. The flow cytometry system as claimed in claim 31 , in which the spectral modulator provides a frequency-modulated pulse train for the detection of FM-SRS signal.