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Multiplexed bioluminescence microscopy via phasor analysis

Abstract

Bioluminescence imaging with luciferase–luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase–luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time and over multiple length scales.

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Fig. 1: Bioluminescent phasor imaging.
Fig. 2: Facile multicomponent imaging with bioluminescent phasor.
Fig. 3: Continuous, excitation-free imaging of tumor spheroids.

Data availability

Source images with unprocessed data are available on Figshare: https://doi.org/10.6084/m9.figshare.19607862.

Code availability

The algorithm that supports the findings of this study is available on GitHub: https://github.com/LorenzoScipioni/Phasor–Bioluminescence.

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Acknowledgements

This research was supported by an Allen Distinguished Investigator Award, a Paul G. Allen Frontiers Group advised grant of the Paul G. Allen Family Foundation (to J.A.P. and M.A.D.), the US National Institutes of Health (grant no. R01 GM107630 to J.A.P., grant no. P41-GM103540 to L.S., E.G., and M.A.D., grant no. R01 DE030123 to A.L.A.) and the UNC University Cancer Research Fund (to A.L.A.). Z.Y. was supported by the National Science Foundation via the BEST IGERT (grant no. DGE-1144901) program and a Graduate Research Fellowship (grant no. DGE-1321846). C.K.B. was supported by the UCI Physical Sciences Machine Learning NEXUS program. We thank members of the Laboratory of Fluorescence Dynamics (LFD, UCI) for discussion. Some experiments were performed at the Laser Spectroscopy Laboratories (LSL) at UCI.

Author information

Authors and Affiliations

Authors

Contributions

Z.Y., C.K.B., L.S., M.A.D. and J.A.P. conceived the project idea. Z.Y., C.K.B., L.S. and H.C. performed the experiments. L.S. and H.C. designed and built the imaging setup. Z.Y., C.K.B. and G.T. prepared the biological samples. L.S. and E.G. wrote the codes and analyzed the raw imaging data. K.K.N., C.K.B., K.P.-S. and A.L.A. generated the reporter constructs. All authors analyzed data and contributed to the writing of the manuscript. All authors have given approval to the final version of the manuscript.

Corresponding authors

Correspondence to Michelle A. Digman or Jennifer A. Prescher.

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The authors declare no competing interests.

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Nature Methods thanks Huiwang Ai, Francesco Cutrale and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Popular bioluminescence reporters produce distinct phasor signatures.

Solutions of recombinant (a) Fluc (500 nM), (b) Akaluc (10 μM), (c) yellow enhanced Nano-lantern (YeNL, 10 nM), and (d) yellow Nano-lantern (YNL, 100 nM) were mixed with their respective luciferin substrates (CycLuc129 or AkaLumine28 = 250 μM with 1 mM ATP; FRZ19 or CTZ17 = 50 μM). Images were acquired using the microscope setup pictured in Supplementary Fig. 1 using a 10 s/frame integration time. A total of 20 frames were collected for each sample and the phasor locations were computed. The emission maximum for each luciferase-luciferin pair is shown for reference.

Extended Data Fig. 2 Phasor signatures from BRET reporters.

HeLa cells were transiently transfected with plasmids encoding (a) Nluc, (b) CeNL, (c) GeNL, or (d) YeNL. The cells were then treated with FRZ (25–50 μM) ~36 h post transfection. Images were acquired using the microscope setup pictured in Supplementary Fig. 1 with a 20X air objective and 10 s/frame integration time. A total of 20 frames were collected for each sample and the phasor locations were computed. Each pixel in the resulting images was false colored according to the phasor signature. The expected phasor for each construct (based on its emission spectrum and BRET efficiency) is also shown for comparison. BRET efficiencies were determined by comparing the acceptor output to the total photon output. The images are color-coded according to the specific reporter, assigned after clustering. These experiments were repeated three times with similar results. Scale bars = 25 μm.

Extended Data Fig. 3 BRET reporters with similar emission maxima can be distinguished via phasor analysis.

HeLa cells were transiently transfected with plasmids encoding (a) OeNL (b) ReNL, or (c) LumiScarlet. The cells were then treated with FRZ (25–50 μM) ~36 h post transfection. Images were acquired using the microscope setup pictured in Supplementary Fig. 1 with a 20X air objective and 10 s/frame integration time. A total of 20 frames were collected for each sample and the phasor locations were computed. Each pixel in the resulting images was false colored according to the phasor signature. The expected phasor for each construct (based on its emission spectrum and BRET efficiency) is also shown for comparison. The BRET efficiency was determined by comparing the acceptor output to the total photon output. The images are color-coded according to the specific reporter, assigned after clustering. These experiments were repeated three times with similar results. Scale bars = 25 μm.

Extended Data Fig. 4 Bioluminescent phasor reveals heterogeneous BRET efficiencies with single cell resolution.

(a) Pixels from images acquired with OeNL-expressing HeLa cells (Extended Data Fig. 3a) were re-assigned based on the computed BRET efficiency. The donor was assigned based on the phasor signature from Nluc-expressing cells (Extended Data Fig. 2a), and the acceptor location was assigned based on the fluorescent spectrum of mKOκ. (b) BRET heterogeneity observed with OeNL-expressing cells was validated on a confocal microscope (Zeiss LSM880) equipped with a 32-channel spectral detector. Multiple cells were analyzed per field of view, with similar results. (c) Pixels from images acquired with ReNL-expressing HeLa cells (Extended Data Fig. 3b) were re-assigned based on the computed BRET efficiency. The donor location was assigned based on the phasor signature from Nluc-expressing cells (Extended Data Fig. 2a), and the acceptor location was assigned based on the fluorescent spectrum of tdTomato. (d) BRET heterogeneity was validated on a confocal microscope (Zeiss LSM880) equipped with a 32-channel spectral detector. Multiple cells were analyzed per field of view, with similar results. Scale bars = 25 μm.

Extended Data Fig. 5 Multicomponent imaging with BRET reporters.

HeLa cells were transfected with either Nluc or a BRET reporter (CeNL, GeNL, YeNL, ReNL, OeNL, or LumiScarlet). The transfected cells were then randomly mixed and plated onto a glass slide 16 h post-transfection. The cells were treated with FRZ (25–50 μM). Images were acquired using the microscope setup pictured in Supplementary Fig. 1 with a 20X air objective and 10 s/frame integration time. A total of 20 frames were collected for each sample and the phasor locations were computed. Each pixel in the resulting image was false colored according to the phasor signature. Selected examples of (a, b) two-component and (c, d) four-component mixtures are shown. Each component in the mixture was assigned by referencing to the single populations shown in Extended Data Figs. 2 and 3. Images are representative of n > 3 independent experiments. Scale bars = 25 μm.

Extended Data Fig. 6 Phasor analysis is required to differentiate probes with similar acceptor fluorophores.

HeLa cells were transfected with plasmids encoding either ReNL or LumiScarlet. The transfected cells were then mixed and plated onto a glass slide 16 h post-transfection. The ‘TRITC filter’ image was obtained by summing the channels corresponding to the interval 578-633 nm, mimicking a 605/55 emission filter (left). The ‘fluorescence color’ image (center left) was obtained by transforming all channels of the spectral detector in RGB and combining them. The ‘phasor’ image (center right) was false-colored after phasor clustering (right). Multiple cells were analyzed per field of view, with similar results. Scale bars = 25 μm.

Extended Data Fig. 7 Spectral unmixing cannot resolve probes that can be separated via bioluminescent phasor.

HeLa cells were transfected with plasmids encoding either ReNL or LumiScarlet. (a) The transfected cells were plated across 96-well plates either as single populations (ReNL: 2.5×104 cells/well, LumiScarlet: 5×104 cells/well) or a mixed population (1.25×104 ReNL expressing cells and 2.5×104 LumiScarlet-expressing cells). (b) The samples were incubated with FRZ (50 µM) and photon outputs were measured on an IVIS imaging station with either no filter (open) or the conventional filters shown (GFP, DsRed, Cy5.5, ICG). Data are presented as mean values ± SEM of n = 3 biologically independent samples. (c) Images collected during the imaging session. (d) The cell mixtures were inseparable with a conventional bioluminescence spectral unmixing algorithm25. This experiment was repeated two times with similar results.

Extended Data Fig. 8 Single cell bioluminescence imaging.

(a) HeLa cells stably expressing CeNL were treated with FRZ (50 μM). Images were acquired using the microscope setup pictured in Supplementary Fig. 1 with a 20X air objective and 10 s/frame integration time. A total of 20 frames were collected and the phasor location was computed. Analysis of a single CeNL-expressing HeLa cell was sufficient for a robust phasor output. (b) CeNL-expressing HeLa cells were serially diluted and seeded in a 96-well plate (1×104 – 1×100 cells/well). Samples were incubated with FRZ and light emission was recorded using an IVIS detector. Data are presented as mean values ± SEM of n = 3 biologically independent samples. (c) Photon output of individual CeNL-expressing cells. Cells were seeded a in 96 well plate (5 cells/well) and allowed to adhere. The next day, the cells were washed with PBS (100 µL) and fresh media (100 µL) was added. Samples were incubated with FRZ and light emission was recorded. Signal was normalized to the number of cells observed in the well. Data are presented as mean values ± SEM of n = 3 biologically independent samples.

Extended Data Fig. 9 Subcellular bioluminescent phasor imaging.

HeLa cells were transiently transfected with plasmids encoding YeNL localized to (from left to right): the membrane (CD8LS-YeNL-CD4), actin (YeNL-actin), mitochondria (Cox8×2-YeNL), ER (Carl-YeNL-KDEL) or nucleus (YeNL-H2B). The cells were treated with FRZ (25–50 μM) ~36 h post transfection. Images were acquired using the microscope setup pictured in Supplementary Fig. 1 with a 60x oil objective and 10 s/frame integration time. A total of 20 frames were collected for each sample and the raw intensity images (a) were subjected to image processing to remove out-of-focus signal. The resulting structural-enhanced images are shown in (b) and phasors computed for each construct are shown in (c). The phasor location of cytosolic YeNL is shown in black for comparison. These experiments were repeated three times with similar results. Scale bars = 25 μm.

Extended Data Fig. 10 Simultaneous tracking of two subcellular features.

HeLa cells were transiently transfected with plasmids encoding YeNL localized to the nucleus (YeNL-H2B) and GeNL localized to the membrane (CD8LS-GeNL-CD4). The cells were treated with FRZ (25 µM) ~36 h post transfection. Images were acquired using the microscope setup pictured in Supplementary Fig. 1 with a 60x oil objective and 10 s/frame integration time. A total of 360 frames were collected and the raw intensity images from (a) were subjected to image processing to remove out-of-focus signal. The resulting structural-enhanced images are shown in (b). Images are representative of n = 3 independent experiments. Scale bars = 25 µm.

Supplementary information

Supplementary Information

Supplementary Table 1 and Figs. 1–6.

Reporting Summary

Supplementary Video 1

Longitudinal tracking of tumor spheroid via bioluminescent phasor. MDA-MB231 spheroids were formed with three cell lines, stably expressing CeNL, YeNL or LumiScarlet and embedded in collagen. The spheroids were treated with FRZ (50 µM) and images were acquired using the microscope setup pictured in Supplementary Fig. 1 with a ×10 objective and 10-s frame integration time. The sample was continuously imaged for 2.5 h (900 frames total). Selected snapshots are shown in Fig. 2. Scale bars, 100 µm.

Supplementary Video 2

Longitudinal multi-organelle tracking via bioluminescent phasor. HeLa cells were transiently transfected with constructs comprising nuclear-localized YeNL and membraned-localized GeNL. The cells were treated with FRZ (25–50 μM) ~36 h post transfection. Images were acquired using the microscope setup pictured in Supplementary Fig. 1 with a ×60 oil objective and integration time of 10 s per frame. The sample was continuously imaged for 1 h (360 frames total). Selected snapshots at 0, 15, 30 and 45 min are shown in Fig. 2.

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Yao, Z., Brennan, C.K., Scipioni, L. et al. Multiplexed bioluminescence microscopy via phasor analysis. Nat Methods 19, 893–898 (2022). https://doi.org/10.1038/s41592-022-01529-9

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