Unlocking Cellular Metabolism with Two-Photon FLIM-Phasor Analysis
The Bruker Ultima two-photon excitation laser scanning microscope is renowned for its precision and flexibility in multiphoton imaging. Now, by integrating a state-of-the-art Fluorescence Lifetime Imaging Microscopy (FLIM) upgrade (Figure 1), its capabilities have expanded into the dynamic field of metabolic imaging – offering researchers unparalleled insights into cellular energy metabolism.
Figure 1. FLIM Upgrade Schematic for the Bruker Ultima Microscope. The Hamamatsu PMT connects to an active splitter, amplifier, and CFD, sending digitized signals to the FLIM acquisition card (top). The splitter also routes a copy of the signal to the Bruker control unit for parallel imaging. Synchronization signals from the microscope and Mai Tai DeepSee laser are digitized and fed into the FLIM system for TCSPC measurements (center and bottom). FLIM LABS software runs on the same PC as the Bruker system, with data exportable in Python-compatible formats.
FLIM LABS’ advanced FLIM-phasor upgrade transforms the Bruker Ultima into a powerful platform for label-free, high-resolution metabolic imaging based on endogenous NADH fluorescence. Using time-correlated single-photon counting (TCSPC) and the intuitive phasor analysis approach, this system allows real-time mapping of metabolic states in live cells and tissues without the need for complex multi-exponential fitting [1, 2].
The FLIM upgrade for the Bruker Ultima features:
- Two-photon excitation via a Spectra-Physics Mai Tai DeepSee laser, optimized at 780 nm for NADH imaging.
- Sensitive detection using a Hamamatsu H117706-40 gated PMT and dedicated signal processing modules (active splitter, low-noise amplifier, constant fraction discriminators).
- Seamless operation through a compact, USB-powered TCSPC acquisition card, integrated directly into the existing Bruker system without disrupting native functionalities.
- Real-time FLIM analysis using FLIM LABS’ custom FLIM Imager software, offering Python and MATLAB export options for flexible post-processing.
This plug-and-play FLIM extension ensures rapid setup, high photon detection efficiency, and full compatibility with existing Bruker control software.
By converting fluorescence lifetime decays into phasor space, the Bruker Ultima with FLIM LABS’ upgrade delivers an immediate, model-free visualization of metabolic states [3] (Figure 2):
- Glycolysis (short lifetime, ~0.4 ns) and oxidative phosphorylation (longer lifetime, ~3.4 ns) can be readily distinguished.
- Phasor plots allow metabolic mapping without the need for complex fitting algorithms, enabling fast and reproducible data interpretation [4].
Figure 2. FLIM Metabolic Phasor Plot Enables Intuitive Mapping of Cellular Metabolism. Each pixel from the fluorescence image is represented in phasor space, where proximity to reference lifetimes for glycolysis (~0.4 ns) or oxidative phosphorylation (~3.4 ns) determines its metabolic state. This model-free approach eliminates the need for complex fitting, enabling rapid, reproducible metabolic mapping using intuitive color coding (light blue for glycolysis, violet for oxphos).
This is particularly powerful in cancer research, where metabolic reprogramming is a hallmark: healthy cells and tumor cells can be rapidly differentiated based on their distinct lifetime signatures.
Using FLIM-phasor analysis, researchers successfully visualized metabolic differences between healthy and cancerous cells [3] (Figure 3):
- Healthy tissue showed a dominant oxidative phosphorylation signature.
- Cancer cells displayed a strong glycolytic shift, aligning with their known preference for rapid energy production via glycolysis.
Figure 3. The different fluorescence lifetimes of protein-bound and free NADH enable FLIM to recognize cancer cells.
These findings demonstrate the potential of the Bruker Ultima-FLIM platform for uncovering critical biological processes in oncology, neuroscience, and metabolic disease research.
The Bruker Ultima microscope, upgraded with FLIM LABS’ FLIM-phasor technology, offers a turnkey solution for researchers aiming to explore the metabolism of cells and tissues with precision and ease. Its combination of multiphoton resolution and metabolic sensitivity makes it an indispensable tool for the next generation of biological discovery.
Bibliography
[1] Lakner, P. H., Monaghan, M. G., Möller, Y., Olayioye, M. A., & Schenke-Layland, K. (2017). Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models. Scientific Reports, 7(1), 42730.
https://www.nature.com/articles/srep42730
[2] Ranjit, S., Malacrida, L., Stakic, M., & Gratton, E. (2019). Determination of the metabolic index using the fluorescence lifetime of free and bound nicotinamide adenine dinucleotide using the phasor approach. Journal of biophotonics, 12(11), e201900156.
https://onlinelibrary.wiley.com/doi/abs/10.1002/jbio.201900156
[3] Stringari, C., Cinquin, A., Cinquin, O., Digman, M. A., Donovan, P. J., & Gratton, E. (2011). Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue. Proceedings of the national academy of sciences, 108(33), 13582-13587.
https://www.pnas.org/doi/abs/10.1073/pnas.1108161108
[4] Torrado, B., Malacrida, L., & Ranjit, S. (2022). Linear combination properties of the phasor space in fluorescence imaging. Sensors, 22(3), 999.
https://www.mdpi.com/1424-8220/22/3/999