title: Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection.
abstract: We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization.
authors.affiliation: Division of Scientific Instrumentation, Korea Basic Science Institute, 169-148 Gwahak-ro Yuseong-gu, Daejeon 34133, South Korea.
authors.forename: Kwan Seob
authors.initials: KS
authors.lastname: Park
authors.affiliation: Division of Scientific Instrumentation, Korea Basic Science Institute, 169-148 Gwahak-ro Yuseong-gu, Daejeon 34133, South Korea.
authors.forename: Dong Uk
authors.initials: DU
authors.lastname: Kim
authors.affiliation: Division of Scientific Instrumentation, Korea Basic Science Institute, 169-148 Gwahak-ro Yuseong-gu, Daejeon 34133, South Korea.
authors.forename: Jooran
authors.initials: J
authors.lastname: Lee
authors.affiliation: Division of Scientific Instrumentation, Korea Basic Science Institute, 169-148 Gwahak-ro Yuseong-gu, Daejeon 34133, South Korea.
authors.forename: Geon Hee
authors.initials: GH
authors.lastname: Kim
authors.affiliation: Division of Scientific Instrumentation, Korea Basic Science Institute, 169-148 Gwahak-ro Yuseong-gu, Daejeon 34133, South Korea.
authors.forename: Ki Soo
authors.initials: KS
authors.lastname: Chang
keywords: (100.2000) Digital image processing; (100.5070) Phase retrieval; (180.2520) Fluorescence microscopy