In vivo flow cytometry based on cellular autofluorescence
The present invention generally provides methods and systems for performing in vivo flow cytometry by using blood vessels as flow chambers through which flowing cells can be monitored in a live subject in vivo without the need for withdrawing a blood sample. In some embodiments, one or more blood vessels are illuminated with radiation so as to cause a multi-photon excitation of an exogenous fluorophore that was previously introduced into the subject to label one or more cell types of interest. In some other embodiments, rather than utilizing an exogenous fluorophore, endogenous (intrinsic) cellular fluorescence can be employed for in vivo flow cytometry. The emission of fluorescence radiation from such fluorophores in response to the excitation can be detected and analyzed to obtain information regarding a cell type of interest.
1. An apparatus for performing flow cytometry in a subject, comprising
a source of radiation that generates radiation configured to cause multi-photon excitation of one or more naturally-occurring fluorophores in the subject's circulatory system,
an optical system that directs the generated radiation onto a portion of the subject's circulatory system so as to cause multi-photon excitation of said one or more naturally-occurring fluorophores in one or more cells circulating in the subject, and
a detector that detects fluorescence radiation emitted by said naturally-occurring cellular fluorophores in the subject in response to said multi-photon excitation.
2. The apparatus of claim 1 , wherein said source generates the radiation by generating pulses of electromagnetic radiation with temporal durations less than about 1 nanosecond.
3. The apparatus of claim 1 , wherein the generated radiation includes at least one of visible light and infrared light.
4. The apparatus of claim 1 , wherein the one or more naturally-occurring cellular fluorophores are excited by the multi-photon generated radiation without being excited by single-photon generated radiation.
5. The apparatus of claim 1 , further comprising:
a second source of radiation that generates radiation configured to cause single-photon excitation, and
a second detector, wherein
the optical system directs the radiation generated by the second source onto the portion of the subject's circulatory system so as to cause single-photon excitation of the one or more naturally-occurring cellular fluorophores in the one or more cells circulating in the subject, and
the second detector detects fluorescence radiation emitted by the naturally-occurring cellular fluorophores in response to said single-photon excitation.
6. The apparatus of claim 5 , wherein the radiation generated by the second source and directed by the optical system also causes single-photon excitation of one or more exogenous fluorophores bound to one or more cells circulating in the subject, and the second detector detects fluorescence radiation emitted by said exogenous fluorophores in response to said single-photon excitation.
7. The apparatus of claim 1 , wherein the generated radiation directed by the optical system also causes multi-photon excitation of one or more exogenous fluorophores bound to one or more cells circulating in the subject, and the detector detects fluorescence radiation emitted by said exogenous fluorophores in response to said multi-photon excitation.