Abstract: 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.

Abstract: Various embodiments can provide a method of amplifying a fluorescence signal through the cyclic staining of complementary antibodies conjugated with fluorophores. According to various embodiments, the method of amplifying a fluorescence signal may be configured to prepare a primary antibody and antibody pairs with respect to each target protein, bind the primary antibody to the target protein, and bind the antibody pairs to the primary antibody. The multicolor fluorescence signals may be amplified and fabricated by forming fluorescence signals in a way to prepare different antibodies for which cross reactions with different types of target proteins, respectively, have been removed using an agarose gel and to bind different antibody pairs according to combinations of different antibodies to different primary antibodies bound to target proteins, respectively.

Abstract: 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 obatined from the obtained images while minimizing the mutual information shared by images of each of pairs each consisting of two of the obtained images.

Abstract: 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.