High-throughput fluorescence lifetime imaging flow cytometry

• Published in: Nature communications Vol. 15; no. 1; pp. 7376 - 15
• Main Authors: Kanno, Hiroshi, Hiramatsu, Kotaro, Mikami, Hideharu, Nakayashiki, Atsushi, Yamashita, Shota, Nagai, Arata, Okabe, Kohki, Li, Fan, Yin, Fei, Tominaga, Keita, Bicer, Omer Faruk, Noma, Ryohei, Kiani, Bahareh, Efa, Olga, Büscher, Martin, Wazawa, Tetsuichi, Sonoshita, Masahiro, Shintaku, Hirofumi, Nagai, Takeharu, Braun, Sigurd, Houston, Jessica P., Rashad, Sherif, Niizuma, Kuniyasu, Goda, Keisuke
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 04.09.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/31; 14; 14/56; 14/63; 38/39; 38/47; 38/62; 631/1647/1407/1492; 631/1647/2163; 631/1647/277; Animals; Antineoplastic drugs; Cell Line, Tumor; Cell Nucleus - metabolism; Continuous radiation; Flow cytometry; Flow Cytometry - methods; Fluctuations; Fluorescence; Fluorescent Dyes - chemistry; Glioma - diagnostic imaging; Glioma - metabolism; Glioma - pathology; Glioma cells; High-Throughput Screening Assays - methods; Humanities and Social Sciences; Humans; Image acquisition; Male; Medical research; Microscopy, Fluorescence - methods; multidisciplinary; Optical Imaging - methods; Rats; Science; Science (multidisciplinary); Subpopulations
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-51125-y

Flow cytometry is a vital tool in biomedical research and laboratory medicine. However, its accuracy is often compromised by undesired fluctuations in fluorescence intensity. While fluorescence lifetime imaging microscopy (FLIM) bypasses this challenge as fluorescence lifetime remains unaffected by such fluctuations, the full integration of FLIM into flow cytometry has yet to be demonstrated due to speed limitations. Here we overcome the speed limitations in FLIM, thereby enabling high-throughput FLIM flow cytometry at a high rate of over 10,000 cells per second. This is made possible by using dual intensity-modulated continuous-wave beam arrays with complementary modulation frequency pairs for fluorophore excitation and acquiring fluorescence lifetime images of rapidly flowing cells. Moreover, our FLIM system distinguishes subpopulations in male rat glioma and captures dynamic changes in the cell nucleus induced by an anti-cancer drug. FLIM flow cytometry significantly enhances cellular analysis capabilities, providing detailed insights into cellular functions, interactions, and environments. Flow cytometry is often hindered by undesired fluctuations in fluorescence intensity. Here, the authors propose high-throughput fluorescence lifetime imaging flow cytometry, which enables imaging at a rate of over 10,000 cells per second and, therefore, enhances the capabilities of cellular analysis.