Half-frequency spectral signatures
View Patent ↗A technique for determining whether or not a fluorescent material exhibits a directionally dependent property, such as anisotropy or chirality, involves illuminating the particle at its excitation wavelength to stimulate fluorescent emission at both a full-frequency (fundamental) wavelength and a half-frequency wavelength. The ratio of the full-frequency signal strength to the half-frequency signal strength provides an indication of the sample's directionally dependent property. This half-frequency spectral analysis can be used to sort anisotropic particles suspended in fluid flowing through a flow cytometer. For instance, the present technique may be used to separate racemic mixtures of chiral enantiomers of cells, pharmaceutical compounds, and other samples.
1. A method of determining a nonlinear optical property of a fluorescent material, the method comprising:
irradiating the fluorescent material at an excitation frequency;
detecting a fluorescence spectrum of the fluorescent material, wherein the fluorescence spectrum includes a first fluorescence peak at a first frequency and a second fluorescence peak at a second frequency equal to about half the first frequency;
performing a comparison of the first fluorescence peak to the second fluorescence peak;
determining the nonlinear optical property of the fluorescent material based on the comparison, wherein the nonlinear optical property of the fluorescent material includes an anisotropy associated with the fluorescent material; and
sorting the fluorescent material based on the nonlinear optical property of the fluorescent material.
2. The method of claim 1 , wherein the nonlinear optical property of the fluorescent material further includes a chirality associated with the fluorescent material.
3. The method of claim 1 , wherein the fluorescent material includes a cell with a fluorescent tag.
4. The method of claim 1 , wherein the fluorescent material is a molecule with a fluorescent tag.
5. The method of claim 1 , wherein the fluorescent material includes a pharmaceutical compound.
6. The method of claim 1 , wherein irradiating the fluorescent material further comprises selecting radiation at the excitation frequency in a first polarization state; and
wherein detecting the fluorescence spectrum further comprises selecting radiation at the first frequency and the second frequency in a second polarization state.
7. The method of claim 6 , wherein the first polarization state is orthogonal to the second polarization state.
8. The method of claim 6 , further comprising:
changing at least one of the first and second polarization states; and
detecting another fluorescence spectrum.
9. The method of claim 1 , wherein irradiating the fluorescent material further comprises irradiating the fluorescent material at a first angle and detecting the fluorescence spectrum further comprises detecting radiation at a second angle with respect to the fluorescent material.
10. The method of claim 9 , wherein the first angle is different than the second angle.
11. The method of claim 9 , wherein the first angle and the second angle are 90° apart.
12. The method of claim 1 , wherein the excitation frequency is about 750 THz to about 30,000 THz.
13. The method of claim 1 , wherein the excitation frequency is about 375 THz to about 750 THz.
14. The method of claim 1 , wherein the excitation frequency is about 187.5 THz to about 375 THz.
15. The method of claim 1 , wherein detecting the fluorescence spectrum further comprises:
diffracting radiation emitted by the fluorescent material off a grating;
filtering radiation at an excitation wavelength from the radiation emitted by the fluorescent material; and
sensing the radiation emitted by the fluorescent material.
16. The method of claim 15 , wherein sensing the radiation emitted by the fluorescent material includes detecting the radiation at the first frequency and the second frequency in parallel.
17. The method of claim 15 , wherein sensing the radiation emitted by the fluorescent material includes detecting the radiation at the first frequency and the second frequency in series.
18. The method of claim 1 , wherein the first frequency is about 375 THz to about 750 THz.
19. The method of claim 1 , wherein the first frequency is about 187.5 THz to about 375 THz.
20. The method of claim 1 , wherein performing the comparison of the first fluorescence peak to the second fluorescence peak comprises:
estimating a first area under the first fluorescence peak;
estimating a second area under the second fluorescence peak; and
calculating a ratio of the first area to the second area.
21. The method of claim 1 , wherein, if the fluorescent material is chiral, further comprising determining whether the fluorescent material has left-handed chirality or right-handed chirality based on a reference.
22. The method of claim 1 , further comprising:
determining whether the fluorescent material includes a monomer or a dimer based on the nonlinear optical property.
23. The method of claim 1 , wherein the first frequency comprises a full-frequency wavelength and the second frequency comprises a half-frequency wavelength.