A novel imaging flow cytometer for preparative fluorescence-activated live cell sorting

Session

Using FLIM and FCS to Determine Interactions and Dynamics

Authors

Ani Augustine Jose (1), Hanning Mai (2), Conor Tracey (1, 2), Jacub Nebdal (1), Thomas Kavanagh (1), Robert Henderson (2), Simon Ameer-Beg (1), Simon Poland (1)

Affiliations

1. Kings Collage
2. University of Edinburgh

Keywords

Fluorescence Lifetime Imaging (FLIM), Flow Cytometry, Cell Sorting, SPAD Array

Abstract text

We propose a novel microfluidics-based multi-beam FLIM (Fluorescence Lifetime Imaging) flow cytometer, which employs a distinctive imaging method to capture high-resolution volumetric images of cells flowing through the microfluidic chip. 

FLIM is a powerful imaging technique that provides information on various cellular parameters like pH, temperature, viscosity, refractive index, and ion concentrations, and extracts metabolic information from tissue autofluorescence. When combined with Förster resonance energy transfer (FRET), FLIM can be used to quantify protein-protein interactions and conformational changes to understand complex biological systems. Developing FLIM-based technologies for high-throughput applications, such as preparative cell sorting of heterogeneous cell populations, is challenging due to its complexity, large data sizes, and analysis procedures [1].

Current cell sorting and analysis pipelines have a significant bottleneck in separating wanted from unwanted cells after sampling. Sterile preparative fluorescence-activated live cell sorting (FACS) [2] has been the primary solution, but the technique has limitations such as the lack of spatial information and high pressures during sorting that compromise cell integrity [3]. To overcome these limitations, we propose integrating real-time fluorescence lifetime (FL) quantification into FACS with microfluidic cell sorting. This approach enables gentle cell sorting based on cellular redox status and multiplexing based on fluorescence lifetime differences. Our solution provides an innovative approach to overcome the limitations of existing imaging and analysis pipelines. Our goal is to develop user-friendly and accessible FLIM-based technologies.

We will develop a novel multibeam FLIM-Flow cytometry imaging modality, utilizing a 32x32 complementary metal-oxide-semiconductor (CMOS) single-photon avalanche diode (SPAD) array with time-correlated single-photon counting (TCSPC)-based timing electronics (Megaframe) [4] and a multifocal fluorescence microscope platform. Based on previous work [5], the platform incorporates a custom diffractive optical element (Holoeye) to generate a beamlet array using picosecond pulsed laser light from a frequency-doubled Ti:Sapphire laser source, with high optical efficiency (~70%). Fluorescence associated with the flow is optically relayed onto the detector array. With the beamlet array stationary, the flow of the sample in the microfluidic chip provides the necessary scan. High resolution image reconstruction is facilitated by the orientation of beamlet projection and camera detection array in relation to the one-dimensional sweep provided by the flow.

To enable high-speed volumetric imaging, arbitrary de-focus terms will be applied to adjacent beamlets using the multifocal multiplane imaging approach reported previously [6]. Validating the effectiveness of the system will involve using well-established live-cell models. Once validated with a single wavelength, multispectral FL detection will be incorporated into the platform with accurate spatial and temporal control over the cell's path in a miniaturized setting. We will also discuss on-chip analysis and rapid cell sorting mechanisms currently being developed which will allow for accurate and high-throughput sorting of live cells based on their fluorescence properties, providing crucial information for the study of biological systems.

References

 

 

[1]         Y. Han, Y. Gu, A. C. Zhang, and Y.-H. Lo, “Review: imaging technologies for flow cytometry,” Lab Chip, vol. 16, no. 24, pp. 4639–4647, Nov. 2016, doi: 10.1039/C6LC01063F.

[2]         A. Cossarizza et al., “Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition),” Eur J Immunol, vol. 49, no. 10, pp. 1457–1973, Oct. 2019, doi: 10.1002/eji.201970107.

[3]         K. Yamada, Y. Saijo, H. Nakagami, and Y. Takano, “Regulation of sugar transporter activity for antibacterial defense in Arabidopsis,” Science, vol. 354, no. 6318, pp. 1427–1430, Dec. 2016, doi: 10.1126/science.aah5692.

[4]         J. A. Richardson, L. A. Grant, and R. K. Henderson, “Low Dark Count Single-Photon Avalanche Diode Structure Compatible With Standard Nanometer Scale CMOS Technology,” IEEE Photonics Technology Letters, vol. 21, no. 14, pp. 1020–1022, Jul. 2009, doi: 10.1109/LPT.2009.2022059.

[5]         S. M. AMEER-BEG, S. Poland, J. LEVITT, and J. NEDBAL, “Luminescence imaging apparatus and methods,” US20200132976A1, Apr. 30, 2020 Accessed: Feb. 24, 2023. [Online]. Available: https://patents.google.com/patent/US20200132976A1/en?q=(Simon)&inventor=poland&assignee=King%27s+College+London

[6]         S. P. Poland et al., “Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo,” Opt. Lett., OL, vol. 43, no. 24, pp. 6057–6060, Dec. 2018, doi: 10.1364/OL.43.006057.