Apparatus and method for acquiring fluorescence image
Provided is a fluorescence image acquisition apparatus for acquiring fluorescence images and phase images using optical signals that are modulated at the same frequency and that have different time delays. The fluorescence image acquisition apparatus may include a light source configured to generate, at different time delays, a plurality of optical signals that are modulated at the same frequency, an illuminator configured to control paths of the plurality of modulated optical signals so that the plurality of modulated optical signals are illuminated onto a sample including a plurality of fluorescent materials, a photodetector configured to detect a plurality of fluorescence signals that are emitted from the plurality of fluorescent materials, respectively, and a controller configured to acquire a plurality of fluorescence images and a plurality of phase images from the plurality of detected fluorescence signals.
1. A fluorescence image acquisition apparatus comprising:
a light source configured to generate, at different time delays, a plurality of optical signals that are modulated at the same frequency;
an illuminator configured to control paths of the plurality of modulated optical signals so that the plurality of modulated optical signals are illuminated onto a sample comprising a plurality of fluorescent materials;
a photodetector configured to detect a plurality of fluorescence signals that are emitted from the plurality of fluorescent materials, respectively; and
a controller configured to acquire a plurality of fluorescence images and a plurality of phase images from the plurality of detected fluorescence signals,
wherein an intensity value of each pixel in the plurality of phase images is based on a phase magnitude of the plurality of detected fluorescence signals.
2. The fluorescence image acquisition apparatus of claim 1 , wherein the controller is configured to acquire a phase image corresponding to each of the plurality of fluorescence signals and configured to separate the fluorescence images for each wavelength corresponding to each of the fluorescence signals based on a fluorescence image comprising the plurality of fluorescence signals.
3. The fluorescence image acquisition apparatus of claim 1 , wherein the controller is configured to acquire a fluorescence image comprising the plurality of fluorescence signals and a phase image corresponding to the fluorescence image based on a four-bucket scheme using a frame rate that varies depending on the modulation frequency.
4. The fluorescence image acquisition apparatus of claim 3 , wherein the controller is configured to extract a phase image corresponding to each of a plurality of pieces of phase information included in the plurality of fluorescence signals.
5. The fluorescence image acquisition apparatus of claim 1 , wherein the photodetector is configured to detect the plurality of fluorescence signals based on a frame rate of 4f when the modulation frequency is f.
6. The fluorescence image acquisition apparatus of claim 1 , wherein the illuminator is configured to separate, using a polychroic mirror, the plurality of modulated optical signals generated by the light source and a plurality of fluorescence signals that are emitted from the sample.
7. A method of separating fluorescence images, the method comprising:
determining whether a fluorescence signal is present, based on a fluorescence image comprising a plurality of fluorescence signals;
extracting a binary fluorescence image determined based on whether the fluorescence signal is present;
removing a random phase of a phase image based on the extracted binary fluorescence image;
extracting, from the phase image from which the random phase is removed, each of phase images separated based on phase information corresponding to each of the fluorescence signals; and
acquiring multi-wavelength fluorescence images separated based on phase information by applying a fluorescence image comprising a plurality of fluorescence signals to the extracted phase images.
8. The method of claim 7 , wherein the fluorescence image comprising the plurality of fluorescence signals is acquired by detecting fluorescence signals generated by illuminating, at different time delays, a plurality of optical signals modulated at the same frequency onto a sample comprising a plurality of fluorescent materials.
9. The method of claim 8 , wherein each of the fluorescence signals is modulated after an arbitrary time delay based on the same modulation frequency of a modulated optical signal absorbed by each of the plurality of fluorescent materials.
10. A method of processing fluorescence images, the method comprising:
generating, at different time delays, a plurality of optical signals that are modulated at the same frequency;
controlling paths of the plurality of modulated optical signals so that the plurality of modulated optical signals are illuminated onto a sample comprising a plurality of fluorescent materials;
detecting a plurality of fluorescence signals that are emitted from the plurality of fluorescent materials, respectively; and
acquiring a plurality of fluorescence images and a plurality of phase images from the plurality of detected fluorescence signals,
wherein an intensity value of each pixel in the plurality of phase images is based on a phase magnitude of the plurality of detected fluorescence signals.
11. The method of claim 10 , further comprising:
acquiring a fluorescence image comprising the plurality of fluorescence signals and a phase image corresponding to the fluorescence image based on a four-bucket scheme using a frame rate that varies depending on the modulation frequency.
12. The method of claim 10 , further comprising:
acquiring a phase image corresponding to each of the plurality of fluorescence signals and separating fluorescence images for each wavelength corresponding to each of the fluorescence signals based on a fluorescence image comprising the plurality of fluorescence signals.