Controller for an imaging device and method
A controller for an imaging device is configured to control an optical detection unit to receive fluorescence light emitted by first and second fluorophore species excited by first and second excitation light, respectively, to separate the fluorescence light into at least two spectral detection channels corresponding to two wavelength bands, respectively. A first wavelength band includes an emission spectrum of the first fluorophore species. A second wavelength band includes an emission spectrum of the second fluorophore species. The controller is further configured to control the optical detection unit to detect photon arrival times of the fluorescence light relative to light pulses of the second excitation light, to determine a temporal correlation between the light pulses and the photon arrival times, and to determine a first number and a second number of photons received in the first spectral detection channel and/or the second spectral detection channel based on the temporal correlation.
1. A controller for an imaging device, the controller configured to:
control a continuous light source of the imaging device to emit first excitation light in order to excite first fluorophore species, the first excitation light having a first wavelength range;
control a pulsed light source of the imaging device to emit second excitation light in order to excite second fluorophore species, the second excitation light having a second wavelength range;
control an optical detection unit of the imaging device to receive fluorescence light emitted by the excited first fluorophore species and the excited second fluorophore species;
control the optical detection unit of the imaging device to separate the received fluorescence light into at least a first spectral detection channel and a second spectral detection channel, the first spectral detection channel corresponding to a first wavelength band comprising at least a part of an emission spectrum of the first fluorophore species, and the second spectral detection channel corresponding to a second wavelength band comprising at least a part of an emission spectrum of the second fluorophore species;
control the optical detection unit of the imaging device to detect photon arrival times of the received fluorescence light relative to light pulses of the second excitation light;
determine a temporal correlation between the light pulses of the second excitation light and the photon arrival times; and
determine a first number of photons and a second number of photons received in the first spectral detection channel and/or the second spectral detection channel based on the temporal correlation, wherein the first number counts photons emitted by the first fluorophore species, and the second number counts photons emitted by the second fluorophore species.
2. The controller according to claim 1 , further configured to:
control at least one additional continuous light source of the imaging device to emit third excitation light in order to excite third fluorophore species, the third excitation light having a third wavelength range;
control the optical detection unit of the imaging device to separate the received fluorescence light into a third spectral detection channel corresponding to a third wavelength band comprising at least a part of an emission spectrum of the third fluorophore species and a part of the emission spectrum of the second fluorophore species; and
determine a third number of photons received in the third spectral detection channel based on the temporal correlation, wherein the third number counts photons emitted by the third fluorophore species.
3. The controller according to claim 2 , further configured to:
control at least one additional pulsed light source of the imaging device to emit fourth excitation light in order to excite fourth fluorophore species, the fourth excitation light having a fourth wavelength range;
control the optical detection unit of the imaging device to separate the received fluorescence light into a fourth spectral detection channel corresponding to a fourth wavelength band comprising at least a part of the emission spectrum of the fourth fluorophore species and a part of the emission spectrum of the first fluorophore species, the second fluorophore species, and/or the third fluorophore species;
control the optical detection unit of the imaging device to detect photon arrival times of the received fluorescence light relative to light pulses of the fourth excitation light;
determine a second temporal correlation between the light pulses of the fourth excitation light and the photon arrival times of the received fluorescence light relative to pulses of the fourth excitation light; and
determine a fourth number of photons received in the fourth spectral detection channel based on the second temporal correlation, wherein the fourth number counts photons emitted by the fourth fluorophore species.
4. The controller according to claim 3 , wherein the controller is configured to determine the first number and the second number, and/or the third number, and/or the fourth number using machine learning or deep learning.
5. An imaging device, comprising:
a controller according to claim 1 ;
at least one continuous light source configured to emit first excitation light, the first excitation light having a first wavelength range;
at least one pulsed light source configured to emit second excitation light, the second excitation light having a second wavelength range; and
an optical detection unit configured to receive fluorescence light emitted by the excited first fluorophore species and the second fluorophore species, to separate the received fluorescence light into at least the first spectral detection channel and the second spectral detection channel, and to detect photon arrival times of the received fluorescence light relative to light pulses of the second excitation light.
6. The imaging device according to claim 5 , wherein the optical detection unit comprises at least one detector element capable of assigning an arrival time to a detected photon.
7. The imaging device according to claim 5 , wherein the optical detection unit comprises at least one detector element capable of photon counting.
8. The imaging device according to claim 5 , wherein the optical detection unit comprises at least one objective for receiving the fluorescence light emitted by the first fluorophore species and the second fluorophore species.
9. The imaging device according to claim 5 , wherein the continuous light source and/or the pulsed light source comprise at least one laser light source.
10. The imaging device according to claim 9 , wherein the pulsed light source comprises a supercontinuum laser light source configured to emit supercontinuum laser light or a white light laser source.
11. The imaging device according to claim 5 , wherein the optical detection unit comprises at least a first detector element and a second detector element, and a beam splitting element that is configured to direct the received fluorescence light having a wavelength range in the first wavelength band onto the first detector element that is capable of assigning an arrival time to a detected photon, and to direct the received fluorescence light having a wavelength range in the second wavelength band onto the second detector element that is capable of assigning an arrival time to a detected photon.
12. The imaging device according to claim 5 , wherein the optical detection unit comprises a multispectral camera or a hyper spectral camera that is configured to generate the at least first spectral detection channel and the second spectral detection channel, and is capable of assigning an arrival time to a detected photon.
13. The imaging device according to claim 5 , wherein the imaging device comprises a microscope.
14. The imaging device according to claim 13 , wherein the imaging device comprises a confocal microscope.
15. A method for spectral unmixing using an imaging device, the method comprising:
exciting first fluorophore species with first excitation light emitted by a continuous light source, the first excitation light having a first wavelength range;
exciting second fluorophore species with second excitation light emitted by a pulsed light source, the second excitation light having a second wavelength range;
receiving fluorescence light emitted by the excited first fluorophore species and the second fluorophore species;
separating the received fluorescence light into at least a first spectral detection channel and a second spectral detection channel, the first spectral detection channel corresponding to a first wavelength band comprising at least a part of an emission spectrum of the first fluorophore species, and the second spectral detection channel corresponding to a second wavelength band comprising at least a part of an emission spectrum of the second fluorophore species;
detecting photon arrival times of the received fluorescence light relative to light pulses of the second excitation light;
determining a temporal correlation between the light pulses of the second excitation light and the photon arrival times; and
determining a first number of photons and a second number of photons received in the first spectral detection channel and/or the second spectral detection channel based on the temporal correlation, wherein the first number counts photons emitted by the first fluorophore species, and the second number counts photons emitted by the second fluorophore species.