3-D holographic imaging continuous flow cytometry
Refractive index of biological specimens is a source of intrinsic contrast that can be explored without any concerns of photobleaching or harmful effects caused by extra contrast agents. This feature also contains rich information that can be related to the metabolism of cells at the cellular and subcellular levels. The present invention relates to systems and methods that can provide, without any moving parts, the 3-D refractive index map of continuously flowing biological samples in a micro-fluidic channel, for example.
1. A method of flow cytometry comprising:
continuously moving a sample in a flowing medium through a channel, the channel having a size to enable flow of the sample during illumination of the sample;
illuminating the moving sample passing through a sample light path with converging light within the channel using a single shot of illuminating light at each of a plurality of locations of the sample within the channel, the converging light forming a line focused beam having a plurality of plane waves;
detecting light from the continuously moving sample in response to the illuminating light and reference light directed along a reference light beam path with a detector at each of the plurality of locations, the reference light beam path and the sample light path being angularly offset at the detector;
processing the detected light from the continuously moving sample and the reference light with a data processor to form a plurality of angular spectra related to the plurality of locations;
mapping phase data and amplitude data of the plurality of angular spectra to a three-dimensional representation;
forming a two-dimensional Fourier transform of the mapped angular spectra; and
mapping the Fourier-transformed angular spectrum to a scattering potential of the sample in a spatial frequency space.
2. The method of claim 1 further comprising generating a plurality of phase images of the sample.
3. The method of claim 1 further comprising illuminating the channel on a line extending across the channel.
4. The method of claim 1 further comprising displaying the three-dimensional representation on a display.
5. The method of claim 1 further comprising determining a refractive index or refractive index distribution of the sample.
6. The method of claim 1 further comprising focusing light onto the channel with a converging cylindrical optical element.
7. The method of claim 1 further comprising adjusting a flow speed of a fluid within the channel.
8. The method of claim 1 further comprising generating an interferogram image of the sample.
9. The method of claim 1 further comprising performing an iterative computation to provide a computed scattering potential.
10. The method of claim 1 further comprising performing an inverse Fourier transform to determine a three dimensional scattering potential.
11. The method of claim 1 further comprising tilting with a mirror the reference light beam path relative to the sample light path to select the angular offset.
12. The method of claim 11 wherein the reference light beam path and the sample light beam path are tilted at an angle greater than zero.
13. The method of claim 1 further comprising moving an illumination beam with an actuated optical element.
14. The method of claim 13 wherein the actuated optical element comprises a movable mirror.
15. The method of claim 13 wherein the actuated optical element comprises a scanner.
16. The method of claim 1 further comprising processing image data with at least one of a smoothness constraint or a positivity constraint.
17. The method of claim 1 further comprising generating data to populate a region of a frequency space.
18. The method of claim 1 further comprising illuminating the sample at a first wavelength and at a second wavelength.
19. The method of claim 1 wherein the detector generates data and further comprising regularizing the data.
20. The method of claim 1 further comprising measuring a rotation of a region of the sample in the channel.
21. The method of claim 1 further comprising detecting fluorescence in the sample.
22. The method of claim 1 further comprising programming a control system with scan parameters to scan light on the sample.
23. The method of claim 1 further comprising delivering illuminating light into a full field of view of the channel.
24. The method of claim 1 further comprising obtaining a confocal image of the sample in the channel.