Korea Advanced Institute of Science and Technology

Yeonbo Sim

Young-Gyu Yoon

Junyoung Seo

Jae-Byum Chang

Jee Won Kim

The present disclosure provides a method and apparatus for biomolecular multiplexed imaging through the iterative unmixing of fluorophores. According to the first embodiments of the present disclosure, although signals of two fluorophores are detected in the first fluorescent detection spectral range, the signals of the two images obtained from two detection spectral ranges can be unmixed through the iterative minimization of mutual information. Furthermore, the present disclosure provides a multi-color unmixing method and apparatus through the iterative minimization of mutual information. In the second embodiments of the present disclosure, a plurality of images of a plurality of fluorophores marking different biomolecules, respectively, for example, N fluorophores are obtained. Images, each containing the signals of single fluorophore can be obtained from the obtained images while minimizing the mutual information shared by images of each of pairs each consisting of two of the obtained images.

Method and apparatus for multiplexed imaging of biomolecule through iterative unmixing of fluorophore signals

Multiplexed fluorescent imaging which is essential for finding out how various biomolecules are spatially distributed in cells or tissues is disclosed. The present disclosure may obtain 10 or more different biomolecule images with one labeling and imaging by newly designing selection of fluorophores, detection spectral ranges, and signal unmixing algorithm. The present disclosure is a blind unmixing technology for unmixing an image without an emission spectrum of fluorophore, and in this technology, 4 pairs of fluorophores are used, and each pair consists of two fluorophores in which emission spectra are overlapped. Each pair of fluorophores is strongly excited by only one excitation laser. Two images with different detection spectral ranges are obtained for each pair, and two images are unmixed via mutual information minimization without fluorophore emission spectrum information. Two images also may be unmixed via Gram-Schmidt orthogonalization and fluorescence measurement based unmixing. This signal unmixing is repeated for each pair of fluorophores. Furthermore, a total of 10 or more fluorophores may be simultaneously used by adding two large stoke's shift fluorophores emitting light in wavelength ranges that does not overlap with the emission spectra of the above 8 fluorophores.

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Daejeon, South Korea

Yeonbo Sim