High throughput snapshot spectral encoding device for fluorescence spectral microscopy
Systems and methods are provided for multi-spectral or hyper-spectral fluorescence imaging. In one example, a spectral encoding device may be positioned in a detection light path between a detection objective and an imaging sensor of a microscope. In one example, the spectral encoding device includes a first dichroic mirror having a sine transmittance profile and a second dichroic mirror having a cosine transmittance profile. In addition to collecting transmitted light, reflected light from each dichroic mirror is collected and used for total intensity normalization and image analysis.
1. An imaging assembly, comprising:
a first dichroic mirror;
a second dichroic mirror;
wherein a first spectral transmittance curve and a first spectral reflectance curve of the first dichroic mirror have sine wave profiles; and
wherein a second spectral transmittance curve and a second spectral reflectance curve of the second dichroic mirror have cosine wave profiles.
2. The imaging assembly of claim 1 , wherein the first dichroic mirror generates a first spectrally encoded transmitted light portion, and a first spectrally encoded reflected light portion; wherein the second dichroic mirror generates a second spectrally encoded transmitted light portion and a second spectrally encoded reflected light portion; and wherein the first spectrally encoded transmitted light portion, first spectrally encoded reflected light, the second spectrally encoded transmitted light, and the second spectrally encoded reflected light are detected an imaging sensor.
3. The imaging assembly of claim 2 , wherein the imaging sensor is a scientific complementary metal oxide semiconductor sensor (sCMOS sensor).
4. The imaging assembly of claim 2 , further comprising four tube lenses, each positioned to receive one of the four spectrally encoded light portions and focus corresponding spectrally encoded light portions on to the imaging sensor.
5. The imaging assembly of claim 4 , further comprising four adjustment mirrors, each positioned to adjust a corresponding angle of each of the spectrally encoded light portions with respect to the imaging sensor such that each of the spectrally encoded light portions are imaged at different quadrants of the imaging sensor.
6. The imaging assembly of claim 1 , wherein the first dichroic mirror generates a first spectrally encoded transmitted light portion, and a first spectrally encoded reflected light portion; wherein the second dichroic mirror generates a second spectrally encoded transmitted light portion and a second spectrally encoded reflected light portion; and wherein the first spectrally encoded transmitted light portion is detected via a first detector, the first spectrally encoded reflected light is detected via a second detector, the second spectrally encoded transmitted light is detected via a third detector, and the second spectrally encoded reflected light is detected via a fourth detector.
7. The imaging assembly of claim 6 , wherein the first, second, third, and fourth detectors are photomultiplier tubes.
8. The imaging assembly of claim 1 , wherein the first and the second dichroic mirrors each receive fluorescence signals from an imaging objective of a microscope.
9. The imaging assembly of claim 1 , wherein the microscope is any of a light-sheet microscope, a wide field fluorescence microscope, or a confocal microscope.
10. The imaging assembly of claim 1 , further comprising at least one first routing mirror positioned to receive a first spectrally encoded transmitted light or a first spectrally encoded reflected light from the first dichroic mirror; and at least one second routing mirror positioned to receive a second spectrally encoded transmitted light or a second spectrally encoded reflected light portion from the second dichroic mirror; and wherein the first and the second dichroic mirrors, and the at least one first and second routing mirrors generate four spectrally encoded light portions, the four spectrally encoded light portions including the first spectrally encoded transmitted light, the first spectrally encoded reflected light, the second spectrally encoded transmitted light, and the second spectrally encoded reflected light.
11. The imaging assembly of claim 1 , further comprising a beam splitter positioned to receive a fluorescence light signal from an imaging objective of a microscope, the beam splitter configured to split the fluorescence signal equally into a first fluorescence signal and a second fluorescence signal; wherein the first fluorescence signal is directed to the first dichroic mirror, and wherein the second fluorescence signal is directed to the second dichroic mirror, or further comprising one or more pre-filtering optics, the one or more pre-filtering optics configured to filter out signals outside spectral ranges of the first and the second dichroic mirrors.
12. The imaging assembly of claim 11 , further comprising one or more additional routing mirrors to direct one or more of the first and the second fluorescence signals on to one or more of the first and second dichroic mirrors respectively, or further comprising one or more relay lenses positioned between the imaging objective and the beam-splitter.
13. The imaging assembly of claim 12 , further comprising one or more pre-filtering optics, the one or more pre-filtering optics configured to filter out signals outside spectral ranges of the first and the second dichroic mirrors, and/or one or more filters configured to filter out wavelengths corresponding to one or more excitation light sources illuminating a sample imaged via the imaging objective.
14. The imaging assembly of claim 1 , wherein each of the first and the second dichroic mirrors have a spectral range from 400 nm to 700 nm.
15. A spectral encoding assembly for integration with a microscope, the spectral encoding assembly comprising:
an encoding portion configured to encode emission light in to a plurality of encoded light channels, the emission light received from an imaging objective of the microscope;
wherein the encoding portion includes at least two dichroic mirrors; and
wherein each of the at least two dichroic mirrors generate encoded light having a periodic waveform.
16. The assembly of claim 15 , wherein the spectral encoding assembly is positioned within infinity space of the microscope and between the imaging objective of the microscope and an imaging sensor, or wherein the encoding portion further includes at least one beam splitter for directing emission light equally to the at least two dichroic mirrors, or wherein the microscope is any of a light-sheet microscope, a wide field fluorescence microscope, or a confocal microscope.
17. The assembly of claim 16 , further comprising one tube lens corresponding to each encoded light channel, the one tube lens focusing light from each channel on to a separate portion of the imaging sensor.
18. The assembly of claim 17 , wherein the encoding portion includes one or more adjustment mirrors for directing each of the plurality of encoded light channels from each of the tube lens on to the separate portions of the imaging sensor.
19. The assembly of claim 17 , wherein the imaging sensor is a cMOS sensor.
20. The assembly of claim 17 , further comprising a pair of relay lenses and a ring actuated iris diaphragm at an intermediate image plane of the pair of relay lenses; wherein an opening amount of the ring actuated iris diaphragm is based on an imaging area on the imaging sensor such that respective images from each of the plurality of channels do not overlap.
21. The assembly of claim 16 , wherein the imaging sensor is communicatively coupled to an imaging processor.
22. The assembly of claim 15 , further comprising one or more pre-filtering optics between the imaging objective and the encoding portion, the one or more pre-filtering optics configured to filter out emission signals outside spectral ranges of the at least two dichroic mirrors, and/or one or more filters configured to filter out wavelengths corresponding to one or more excitation light sources illuminating a sample imaged via the imaging objective, or further comprising relay optics between the imaging objective and the encoding portion.
23. The assembly of claim 15 , wherein each of the at least two dichroic mirrors have a spectral range including wavelengths in visible spectrum.
24. An imaging system, comprising:
an imaging objective acquiring fluorescence signal from a sample;
an imaging sensor; and
a spectral encoding device positioned between the imaging objective and the imaging sensor;
wherein the spectral encoding device includes a first dichroic mirror and a second dichroic mirror; and
wherein the spectral encoding device generates four encoded light channels via the first and the second dichroic mirrors, wherein each of the four encoded light channels have a periodic waveform.
25. The imaging system of claim 24 , wherein the four encoded light channels include a first transmitted light channel and a first reflected light channel from the first dichroic mirror, and a second transmitted light channel and a second reflected light channel from the second dichroic mirror.
26. The imaging system of claim 25 , further comprising a controller including executable instructions stored in non-transitory memory that when executed cause the controller to:
determine a normalization intensity by calculating an integral of intensity values of the four encoded light channels;
obtain corresponding channel images, via the imaging sensor, for each of the four encoded light channels; and
normalize each corresponding channel image according to the normalization intensity.
27. The imaging system of claim 26 , wherein the controller includes further executable instructions stored in non-transitory memory that when executed cause the controller to:
generate a hyper-spectral or multi-spectral image according to each normalized channel image.
28. A method for spectral fluorescence imaging, comprising:
receiving, at a spectral encoding device, fluorescence signal from a biological sample;
generating, via the spectral encoding device, at least two transmitted light channels and at least two reflected light channels; and
imaging, at an imaging sensor, the at least two transmitted light channels and the at least two reflected light channels;
wherein the spectral encoding device includes at least two dichroic mirrors, each of the two dichroic mirrors having a transmittance and reflectance profile resembling a periodic waveform.
29. The method of claim 28 , further comprising:
registering the images formed by each of the at least two transmitted light channels and each of the at least two reflected light channels.
30. The method of claim 29 , further comprising:
performing phasor analysis on the registered images to generate a phasor plot; and
generating an unmixed image according to the phasor plot.