Light sheet imaging flow cytometer
View Patent ↗The present disclosure relates to systems and methods for cellular imaging and identification through the use of a light sheet flow cytometer. In one implementation, a light sheet flow cytometer may include a light source configured to emit light having one or more wavelengths, at least one optical element configured to form a light sheet from the emitted light, a microfluidic channel configured to hold a sample, and an imaging device. The imaging device may be adapted to forming 3-D images of the sample such that identification tags attached to the sample are visible.
1. A system for an imaging flow cytometer, comprising:
a light source configured to emit light having one or more wavelengths;
at least one optical element adapted to form a light sheet from the emitted light;
an illumination objective;
a microfluidic channel within a focus of the light sheet and configured to hold a sample;
a mirror adapted to reflect the light sheet focused on the sample and form a standing wave;
a detection objective; and
an imaging device, wherein
the imaging device is adapted to capture light from the sample to image a two-dimensional plane of the sample while the sample is illuminated with the light sheet,
the imaging device is adapted to form a three-dimensional image of the sample from successive two-dimensional images,
and the imaging device is adapted to image identification tags contained within the sample.
2. The system of claim 1 , further comprising:
a plurality of dichroic filters, wherein
the light sheet is a multi-color light sheet formed, at least in part, by the at least one optical element,
the imaging device includes a plurality of cameras, and
each of the plurality of dichroic filters is adapted to direct one of a plurality of spectra in the light from the sample toward a corresponding one of the plurality of cameras.
3. The system of claim 1 , further comprising:
a filtering device, wherein
the light sheet is a multi-color light sheet formed, at least in part, by the at least one optical element, and
the filtering device is adapted to select at least one of a plurality of emission spectral bands emitted from the sample while the sample is illuminated with the light sheet.
4. The system of claim 1 , further comprising:
a plurality of illumination objectives configured to focus respective light sheets, including the light sheet, with different illumination spectrums; and
a plurality of detection objectives, each corresponding to a given one of the plurality of illumination objectives, wherein
the imaging device includes a plurality of cameras, each corresponding to a given one of the plurality of detection objectives.
5. The system of claim 1 , further comprising:
a second imaging device adapted to image the sample before the sample is imaged by the first imaging device, wherein the imaging device is the first imaging device.
6. The system of claim 1 , wherein the detection objective is also adapted to function as a second illumination objective for a second light sheet, and the illumination objective is also adapted to function as a second detection objective for the second light sheet.
7. The system of claim 1 , wherein the at least one optical element is adapted to form a plurality light sheets, including the light sheet, from excitation light, and the imaging device is adapted to simultaneously capture the emitted light from each of the plurality of light sheets to form the three-dimensional image.
8. The system of claim 1 , further comprising:
a translation stage affixed to the detection objective, wherein
the at least one optical element is adapted to form a plurality of light sheets from the emitted light, and
the translation stage is adapted to move the detection objective to focus successive emissions from the light source for each of the plurality of light sheets.
9. The system of claim 1 , further comprising:
a pump, adapted to control a flow rate in the microfluidic channel.
10. The system of claim 1 , further comprising:
a DC electric field generator, adapted to pull the sample through the microfluidic chamber.
11. The system of claim 1 , wherein the identification tags include a plurality of beads that are spatially arranged in a well-defined fashion.
12. The system of claim 1 , wherein the identification tags include one or more beads coated with one or more photobleachable dyes.
13. The system of claim 1 , further comprising:
a sequencing assay, wherein
the identification tags are attached to the sample during sequencing.
14. A method of imaging a sample, comprising:
attaching identification tags to a sample;
providing a light source that emits light having one or more wavelengths;
forming a light sheet from the emitted light;
directing the light sheet toward a microfluidic channel within a focus of the light sheet and configured to hold the sample containing identification tags;
forming a standing wave with the light sheet focused on the sample to induce emission light from the sample;
imaging the emission light collected from the sample, wherein each image from the imaging portrays a two-dimensional plane of the sample; and
rendering a three-dimensional image of the sample from successive two-dimensional images from the imaging, wherein the identification tags are visible in the three-dimensional image.
15. The method of claim 14 , further comprising:
sequencing the sample; and
associating the sequence of the sample with the identification tags of the sample.
16. The method of claim 14 , further comprising:
post-processing the successive two-dimensional images.
17. The method of claim 14 , further comprising:
post-processing the rendered three-dimensional image.
18. The method of claim 14 , wherein a plurality of light sources emit a plurality of lights, each having one or more wavelengths.
19. The method of claim 18 , wherein a plurality of light sheets are formed from the plurality of emitted lights.