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Fluorescence emission is common in plants. While fluorescence microscopy has been widely used to study living plants, its application in quantifying the fluorescence of fossil plants has been limited. Fossil plant fluorescence, from original fluorophores or formed during fossilization, can offer valuable insights into fluorescence in ancient plants and fossilization processes. In this work, we utilize two-photon fluorescence microspectroscopy to spatially and spectrally resolve the fluorescence emitted by amber-embedded plants, leaf compressions, and silicified wood. The advanced micro-spectroscope utilized, with its pixel-level spectral resolution and line-scan excitation capabilities, allows us to collect comprehensive excitation and emission spectra with high sensitivity and minimal laser damage to the specimens. By applying linear spectral unmixing to the spectrally resolved fluorescence images, we can differentiate between (a) the matrix and (b) the materials that comprise the fossil. Our analysis suggests that the latter correspond to durable tissues such as lignin and cellulose. Additionally, we observe potential signals from chlorophyll derivatives/tannins, although minerals may have contributed to this. This research opens doors to exploring ancient ecosystems and understanding the ecological roles of fluorescence in plants throughout time. Furthermore, the protocols developed herein can also be applied to analyze non-plant fossils and biological specimens.

 

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M 1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M 1 and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M 1 -mEGFP was expressed at levels equal to the M 1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M 1 -mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M 1 -mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M 1 -mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels. 

Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomersbut that the oligomers also may modulate the receptor function provides a promising avenue in the area ofdrug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacywhile reducing side effects. In this regard, determining the various oligomeric configurations and geometricsub-states of a membrane receptor is of utmost importance. Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring thequaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescenceintensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiencyare analyzed using theoretical models of various quaternary structures to determine the geometry andstoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensitiesare analyzed to reveal quantitative information on the size and stability of protein oligomers. Results: We demonstrate the application of these techniques to a number of different fluorescence-based studiesof cells expressing fluorescently labeled membrane receptors, both in the presence and absence of variousligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regardingthe quaternary structure receptors form, as well as FIF and FRET for determining the relative abundanceof different-sized oligomers when an equilibrium forms between such structures. Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers,which are of great benefit to structure‐based drug design. 

Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS‐based method, termed Two‐Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of live cells expressing protein constructs

Basic Protocol 2: Image acquisition and noise correction

Basic Protocol 3: Drawing and segmenting regions of interest

Basic Protocol 4: Calculating the molecular brightness and concentration of individual image segments

Basic Protocol 5: Combining data subsets using a manual procedure (Optional)

Alternate Protocol 1: Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5)

Basic Protocol 6: Performing meta‐analysis of brightness spectrograms

Alternate Protocol 2: Performing meta‐analysis of brightness spectrograms (alternative to Basic Protocol 6)

Basic Protocol 7: Spot extraction and analysis using a manual procedure or by writing a program (Optional)

Alternate Protocol 3: Automated spot extraction and analysis (Optional; alternative to Protocol 7)

Support Protocol: Monomeric brightness determination