Apparatus and methods for fluorescence imaging using radiofrequency-multiplexed excitation
Apparatus and methods for fluorescence imaging using radiofrequency multiplexed excitation. One apparatus splits an excitation laser beam into two arms of a Mach-Zehnder interferometer. The light in the first beam is frequency shifted by an acousto-optic deflector, which is driven by a phase-engineered radiofrequency comb designed to minimize peak-to-average power ratio. This RF comb generates multiple deflected optical beams possessing a range of output angles and frequency shifts. The second beam is shifted in frequency using an acousto-optic frequency shifter. After combining at a second beam splitter, the two beams are focused to a line on the sample using a conventional laser scanning microscope lens system. The acousto-optic deflectors frequency-encode the simultaneous excitation of an entire row of pixels, which enables detection and de-multiplexing of fluorescence images using a single photomultiplier tube and digital phase-coherent signal recovery techniques.
1. A method of fluorescence imaging, the method comprising:
(a) creating a first beam of frequency shifted light;
(b) creating a second beam of frequency shifted light;
(c) interrogating a sample with the first and second beams of light; and
(d) detecting a response in the sample from exposure to the beams of frequency shifted light;
(e) applying a Fast Fourier Transform to a reference comb to identify comb-line frequencies;
(f) dividing a received signal into frames;
(g) applying a Fast Fourier Transform to the divided signal;
(h) recording values of transformed signal at comb-line frequencies; and
(i) forming an image from the recorded values.
2. The method as recited in claim 1 , further comprising:
combining the first beam and the second beam to produce a combined beam; and
interrogating the sample with the combined beam.
3. The method as recited in claim 1 , wherein said first beam is created with an acousto-optic deflector driven by an RF comb and an excitation laser beam.
4. The method as recited in claim 1 , wherein said second beam is created with an acousto-optic frequency shifter driven by an RF tone and an excitation laser beam.
5. The method as recited in claim 1 , further comprising:
assigning a distinct radiofrequency to each pixel in a row of pixels.
6. A method of fluorescence imaging, the method comprising:
(a) creating a first beam of frequency shifted light;
(b) creating a second beam of frequency shifted light;
(c) interrogating a sample with the first and second beams of light;
(d) detecting a response in the sample from exposure to the beams of frequency shifted light;
(e) obtaining a signal from the detector;
(f) calculating comb tones;
(g) mixing the signal with each comb tone to produce a mixed signal;
(h) passing the signal through a low pass filter to determine temporal resolution;
(i) undersampling the filtered signal; and
(j) forming an image.
7. The method as recited in claim 6 , further comprising:
combining the first beam and the second beam to produce a combined beam; and
interrogating the sample with the combined beam.
8. The method as recited in claim 6 , wherein said first beam is created with an acousto-optic deflector driven by an RF comb and an excitation laser beam.
9. The method as recited in claim 6 , wherein said second beam is created with an acousto-optic frequency shifter driven by an RF tone and an excitation laser beam.
10. The method as recited in claim 6 , further comprising:
assigning a distinct radiofrequency to each pixel in a row of pixels.
11. A method of fluorescence imaging, the method comprising:
(a) creating a first beam of frequency shifted light;
(b) creating a second beam of frequency shifted light;
(c) interrogating a sample with the first and second beams of light;
(d) detecting a response in the sample from exposure to the beams of frequency shifted light;
(e) obtaining a signal from the detector;
(f) calculating comb tones;
(g) mixing the signal with each comb tone to produce a mixed signal;
(h) passing each comb tone of the signal through a digital low pass filter in parallel;
(i) oversampling the filtered signals; and
(j) forming an image.
12. The method as recited in claim 11 , further comprising:
combining the first beam and the second beam to produce a combined beam; and
interrogating the sample with the combined beam.
13. The method as recited in claim 11 , wherein said first beam is created with an acousto-optic deflector driven by an RF comb and an excitation laser beam.
14. The method as recited in claim 11 , wherein said second beam is created with an acousto-optic frequency shifter driven by an RF tone and an excitation laser beam.
15. The method as recited in claim 11 , further comprising:
assigning a distinct radiofrequency to each pixel in a row of pixels.