Device and method for in vivo flow cytometry using the detection of photoacoustic waves

Abstract

A photoacoustic flow cytometry (PAFC) device for the in vivo detection of cells circulating in blood or lymphatic vessels is described. Ultrasound transducers attached to the skin of an organism detect the photoacoustic ultrasound waves emitted by target objects in response to their illumination by at least one pulse of laser energy delivered using at least one wavelength. The wavelengths of the laser light pulse may be varied to optimize the absorption of the laser energy by the target object. Target objects detected by the device may be unlabelled biological cells or cell products, contrast agents, or biological cells labeled with one or more contrast agents.

1 . A device for detecting the presence of moving target objects within a circulatory vessel of a living organism at up to 15 cm away from the vessel, comprising:
 a. at least one tunable pulsed laser source functioning at one or more wavelengths between about 10 Å and about 1 cm, a pulse width ranging between about 0.1 ps and about 1000 ns, a pulse repeat rate ranging between about 1 Hz and about 500,000 Hz, and a pulse energy fluence ranging between about 0.1 mJ/cm 2  and about 1000 J/cm 2 ;   b. at least one optical element operating to direct a pulse from the tunable laser source through the vessel in a beam with an elliptically-shaped cross-section, wherein the beam cross-section has a maximum dimension ranging between about 1 μm and about 150 μm; and,   c. at least one ultrasound transducer with a sample rate ranging between about 10 kHz and about 100 MHz.   
   
   
       2 . The device of  claim 1 , wherein the device further includes an amplifier with a high frequency boundary between about 10 kHz and about 200 kHz, a low frequency boundary between about 1 MHz and about 100 MHz, and a resonance bandwidth between about 0.3 MHz and about 5 MHz. 
   
   
       3 . The device of  claim 1 , wherein the device further includes a data-recording device. 
   
   
       4 . The device of  claim 1 , wherein the device further comprises a computer with a stored analysis program. 
   
   
       5 . The device of  claim 1 , wherein the pulses from the tunable pulsed laser source are emitted at a single wavelength. 
   
   
       6 . The device of  claim 1 , wherein the tunable pulsed laser source emits a repeating series of at least two pulses, wherein each pulse in the series is emitted at one of at least two different wavelengths. 
   
   
       7 . The device of  claim 6 , wherein each pulse in the repeating series is separated from the preceding pulse in the series by a time delay ranging between about 0.1 μs and about 100 μs. 
   
   
       8 . A device for performing in vivo photoacoustic flow cytometry to detect the presence of moving target objects within a circulatory vessel of a living organism at up to 15 cm away from the vessel, comprising:
 a. at least one tunable pulsed laser source functioning at one or more wavelengths between about 10 Å and about 1 cm, a pulse width ranging between about 0.1 ps and about 1000 ns, a pulse repeat rate ranging between about 1 Hz and about 500,000 Hz, and a pulse energy fluence ranging between about 0.1 mJ/cm 2  and about 1000 J/cm 2 ;   b. at least one optical element operating to direct a pulse from the tunable laser source through the vessel in a beam with an elliptically-shaped cross-section, wherein the beam cross-section has a maximum dimension ranging between about 1 μm and about 150 μm;   c. at least one ultrasound transducer with a sample rate ranging between about 10 kHz and about 100 MHz;   d. an amplifier with a high frequency boundary between about 10 kHz and about 200 kHz, a low frequency boundary between about 1 MHz and about 100 MHz, and a resonance bandwidth between about 0.3 MHz and about 5 MHz;   e. a data-recording device; and,   f. a computer with a stored analysis program.   
   
   
       9 . The device of  claim 8 , wherein the tunable pulsed laser source emits a repeating series of at least two pulses, wherein each pulse in the series is emitted at one of at least two different wavelengths. 
   
   
       10 . The device of  claim 9 , wherein each pulse in the repeating series is separated from the preceding pulse in the series by a time delay ranging between about 0.1 μs and about 100 μs. 
   
   
       11 . A method for detecting at least one type of moving target object within a circulatory vessel of a living organism, the method comprising:
 a. pulsing the target object moving through a vessel with at least one pulse of laser energy;   b. detecting at least one photoacoustic pulse emitted by the target object; and,   c. analyzing at least one characteristic of the detected photoacoustic pulse to determine at least one characteristic of the detected target objects.   
   
   
       12 . The method of  claim 11 , wherein the at least one pulse of laser energy is delivered at a one wavelength. 
   
   
       13 . The method of  claim 11 , wherein the at least one pulse of laser energy comprises a repeating series of at least two pulses, wherein each pulse in the series is emitted at one of at least two different wavelengths. 
   
   
       14 . The method of  claim 13 , wherein each pulse in the repeating series is separated from the preceding pulse in the series by a time delay ranging between about 0.1 μs and about 100 μs. 
   
   
       15 . The method of  claim 11 , wherein the at least one pulse of the laser energy is delivered at a wavelength ranging between about 10 Å and about 1 cm, a pulse width ranging between about 0.1 ps and about 1000 ns, a pulse repeat rate ranging between about 1 Hz and about 500,000 Hz, a pulse energy fluence ranging between about 0.1 mJ/cm 2  and about 1000 J/cm 2 , and wherein the at least one laser pulse is delivered as a beam with an elliptically-shaped cross-section with a maximum dimension ranging between about 1 μm and about 150 μm. 
   
   
       16 . The method of  claim 11 , wherein the at least one photoacoustic pulse is detected with at least one ultrasound transducer with a sample rate ranging between about 10 kHz and about 100 MHz. 
   
   
       17 . A method of detecting moving target objects in blood or lymphatic vessels of a living organism at up to 15 cm away from the vessels, comprising:
 a. pulsing the target objects with at least one pulse of laser energy at one or more wavelengths ranging between about 400 nm and about 2500 nm, a pulse width ranging between about 0.1 ps and about 1000 ns, a pulse repeat rate ranging between about 1 Hz and about 500,000 Hz, and a pulse energy fluence ranging between about 0.1 mJ/cm 2  and about 1000 J/cm 2 ;   b. detecting the resulting ultrasonic photoacoustic waves emitted by the target objects at a sample rate ranging between about 10 kHz and about 100 MHz; and,   c. analyzing at least one characteristic of the detected photoacoustic pulse to determine at least one characteristic of the detected target objects.   
   
   
       18 . The method of  claim 17 , wherein the at least one pulse of laser energy is delivered at a one wavelength. 
   
   
       19 . The device of  claim 17 , wherein the at least one pulse of laser energy comprises a repeating series of at least two pulses, wherein each pulse in the series is emitted at one of at least two different wavelengths. 
   
   
       20 . The device of  claim 19 , wherein each pulse in the repeating series is separated from the preceding pulse in the series by a time delay ranging between about 0.1 μs and about 100 μs. 
   
   
       21 . The method of  claim 17 , wherein the analysis of the detected photoacoustic pulse comprises determining an onset time and a termination time of a photoacoustic wave using a wave detection algorithm, subtracting the onset time from the termination time to determine the pulse duration, determining the maximum signal amplitude detected during the photoacoustic wave, performing spectral analysis of the photoacoustic pulse to obtain a pulse frequency spectrum, and subtracting onset time from the time at which the laser energy pulse was delivered to determine the time delay between the pulse of laser energy and the emitted photoacoustic pulse, and combinations thereof. 
   
   
       22 . The method of  claim 17 , wherein the characteristic of the detected target object is determined by matching the characteristic of the detected photoacoustic pulse to a previously determined characteristic of the target object. 
   
   
       23 . A device for detecting the presence of moving target objects within a circulatory vessel of a living organism at up to 15 cm away from the vessel, comprising:
 a. at least one tunable pulsed laser source functioning at one or more wavelengths between about 10 Å and about 1 cm, a pulse width ranging between about 0.1 ps and about 1000 ns, a pulse repeat rate ranging between about 1 Hz and about 500,000 Hz, and a pulse energy fluence ranging between about 0.1 mJ/cm 2  and about 1000 J/cm 2 .   
   
   
       24 . The device of  claim 23 , wherein the tunable pulsed laser source emits a repeating series of at least two pulses, wherein each pulse in the series is emitted at one of at least two different wavelengths. 
   
   
       25 . The device of  claim 24 , wherein each pulse in the repeating series is separated from the preceding pulse in the series by a time delay ranging between about 0.1 μs and about 100 μs. 
   
   
       26 . A method for in vivo detection of a circulating, unlabelled metastatic melanoma cell, the method comprising:
 a. pulsing an area of an organism with at least one pulse of NIR laser energy at a wavelength ranging between about 650 nm and about 950 nm and a laser fluence ranging between about 20 mJ/cm 2  and about 100 mJ/cm 2 ;   b. detecting at least one photoacoustic pulse emitted by the melanoma cell; and,   c. analyzing the at least one detected photoacoustic pulse to indicate the presence of the metastatic melanoma cell in circulation.   
   
   
       27 . The method of  claim 26 , wherein metastatic melanoma cells are detected at a resolution of at least 1 metastatic melanoma cell per 10 8  cells in circulation. 
   
   
       28 . A method for selectively destroying target objects circulating in a vessel of a living organism in vivo, comprising:
 a. detecting the target objects circulating in the vessels;   b. triggering a pulse of laser energy delivered at a wavelength and energy level sufficient to cause the destruction of the detected target objects;   c. monitoring frequency of detection of target objects circulating through the vessel; and,   d. terminating when the frequency of detection of target objects falls below a threshold level.   
   
   
       29 . The method of  claim 28 , in which the target objects are detected using an in vivo flow cytometry device using laser-excited photoacoustic waves emitted by the target objects. 
   
   
       30 . The method of  claim 28 , in which the threshold level of the frequency of detection ranges between about 10 −3  target objects/min and about 10 2  target objects/min.