FLIM & PLIM

Applications in FLIM and PLIM

Many biological processes including synaptic plasticity require dynamic interactions between proteins. Unfortunately, classical biochemical approaches or the normal diffraction-limited fluorescence microscopy to study these sometimes transient but intimate interactions do not provide required sensitivity or resolution (1).

Fluorescence Lifetime Imaging Microscopy (FLIM) is the most rigorous method for measuring molecular environment parameters, protein-interactions by Fӧrster resonance energy transfer (FRET) (2), and measurements of the metabolic state of cells and tissue via their autofluorescence (3). FLIM-based FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with metabolic state of the cells or the tissue.

Recent development of two-photon-excitable oxygen-sensing probes made Phosphorescence Lifetime Imaging Microscopy (PLIM) increasingly popular (4). PLIM allows for quantitative in vivo oxygen measurements at high spatial and temporal resolution with high specificity.

Bruker developed a FLIM module based on Becker & Hickl product. The module is fully integrated with all Bruker’s multiphoton systems. FLIM and PLIM data acquisition is enabled in Bruker’s proprietary Prairie View software. Both upright and inverted configuration of the scope are available.

References

1. Datta R, Heaster TM, Sharick JT, Gillette AA, Skala MC. Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications. J Biomed Opt. 2020 May;25(7):1-43. doi: 10.1117/1.JBO.25.7.071203.

2. Algar WR, Hildebrandt N, Vogel SS, Medintz IL. FRET as a biomolecular research tool - understanding its potential while avoiding pitfalls. Nat Methods. 2019 Sep;16(9):815-829. doi: 10.1038/s41592-019-0530-8.

3. Jones JD, Ramser HE, Woessner AE, Veves A, Quinn KP. Quantifying Age-Related Changes in Skin Wound Metabolism Using In Vivo Multiphoton Microscopy. Adv Wound Care (New Rochelle). 2020 Mar 1;9(3):90-102. doi: 10.1089/wound.2019.1030.

4. Esipova TV, Barrett MJP, Erlebach E, Masunov AE, Weber B, Vinogradov SA. Oxyphor 2P: A High-Performance Probe for Deep-Tissue Longitudinal Oxygen Imaging. Cell Metab. 2019 Mar 5;29(3):736-744.e7. doi: 10.1016/j.cmet.2018.12.022.