Multiplexed Multidimesional Fluorescence Lifetime Imaging Microscopy

Comparing with conventional wide-field imaging microscopes, confocal microscopy hold significant advantages in image contrast enhancement, 3D sectioning capabilities, and compatibility with specialized detectors. For applications such as live cell imaging, slow acquisition speed is a key barrier to adaption of confocal microscopy. While wide-field microscopy is typically faster, multiplexed confocal schemes such as using a spinning foci array can significantly increase the image acquisition rate. The moving foci array in the spinning disc, however, prevents the use of specialized discrete photo detectors arrays.

We have developed a suite of technologies to generate, scan, and measure 1000+ confocal foci simultaneously, while is compatible with stationary discrete detectors. Another key feature of the technique is that it can be retrofitted to a conventional wide-field fluorescence microscope. We are also developing various related technologies for its applications in drug discovery and in vivo imaging.

Publications:

• Morgan Richards, Elizabeth Osterlund, Andrea Buendia, Nikolina Malic, Ray Truant, Qiyin Fang, “Multispectral multiplexed confocal FLIM for live cell imaging,” J. Phys. Photonics 7(1):015003, 2025 (DOI 10.1088/2515-7647/ad936b)
• Qiyin Fang, Morgan Richards, Yiping Wang, “Sampling-Based Two-Dimensional Temporal Imaging,” In: Liang, J. (eds) “Coded Optical Imaging,” Springer, Cham. 2024, doi:10.1007/978-3-031-39062-3_24
• Xu Mei, Qiyin Fang, and P. Ravi Selvaganapathy, “Three-dimensional oxygen concentration monitoring in hydrogels using low-cost phosphorescence lifetime imaging for tissue engineering,” Biomed. Opt. Express 14, 4759-4774 (2023) Open Access
• Elizabeth J. Osterlund, Nehad Hirmiz, James M. Pemberton, Adrien Nougarede, Qian Liu, Brian Leber, Qiyin Fang, and David W. Andrews, “Efficacy and specificity of inhibitors of BCL-2 family protein interactions assessed by affinity measurements in live cells,” Science Advances, 8(16): 2022, doi: 10.1126/sciadv.abm7375 (Open Access)
• Nehad Hirmiz, Anthony Tsikouras, Elizabeth J. Osterlund, Morgan Richards, David W. Andrews, and Qiyin Fang, “Highly Multiplexed Confocal Fluorescence Lifetime Microscope Designed for Screening Applications,” IEEE Selected Topics in Quantum Electronics, 27(5):1-9, 2020, doi.org/10.1109/JSTQE.2020.2997834 (Preprint PDF)
• Nehad Hirmiz, Anthony Tsikouras, Elizabeth J. Osterlund, Morgan Richards, David W. Andrews, and Qiyin Fang, “Multiplexed confocal microscope with a refraction window scanner and a single-photon avalanche photodiode array detector,” Opt. Lett. 45(1), 69-72 (2020), https://doi.org/10.1364/OL.45.000069 (online)
• Nehad Hirmiz, Anthony Tsikouras, Elizabeth J. Osterlund, Morgan Richards, David W. Andrews, and Qiyin Fang, “Cross-talk reduction in a multiplexed synchroscan streak camera with simultaneous calibration,” Opt. Express 27, 22602-22614 (2019), doi.org/10.1364/OE.27.022602 (online)
• Anthony Tsikouras, Pietro Peronio, Ivan Rech, Nehad Hirmiz, M. Jamal Deen, and Qiyin Fang, “Characterization of SPAD Array for Multifocal High-Content Screening Applications,” Photonics 3(4):56, 2016; doi: 10.3390/photonics3040056, (Open Access)
• Bo Xiong, Qiyin Fang, “Luminescence lifetime imaging using a cellphone camera with an electronic rolling shutter”, Optics Letters, 45(1): 81-84, 2020, doi.org/10.1364/OL.45.000081 (https://www.osapublishing.org/ol/abstract.cfm?uri=ol-45-1-81)
• Anthony Tsikouras, Richard Berman, David W. Andrews and Qiyin Fang, “High-speed multifocal array scanning using refractive window tilting,” Biomedical Optics Express 6, 3737-3757, 2015. (Open Access)
• Zhaojun Nie, Ran An, Joseph E. Hayward, Thomas J. Farrell, Qiyin Fang, “Hyperspectral fluorescence lifetime imaging for optical biopsy,” Journal of Biomedical Optics 18 (9):096001, 2013, doi: 10.1117/1.JBO.18.9.096001, (PDF)
• Anthony Tsikouras, Jin Ning, Sandy Ng, Richard Berman, David W. Andrews, and Qiyin Fang, “Streak camera crosstalk reduction using a multiple decay optical fiber bundle,” Optics Letters 37(2): 250-252, 2012. (PDF)