Current capabilities and future perspectives of FCS: super-resolution microscopy, machine learning, and in vivo applications

Sankaran, Jagadish; Wohland, Thorsten

doi:10.1038/s42003-023-05069-6

Cited by 15 publications

(4 citation statements)

References 138 publications

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“…Analyzing the intensity fluctuations caused by molecules diffusing in and out of the focus yields information on kinetic (diffusion coefficient) and thermodynamic (concentration, brightness/oligomeric state) properties of the system. These parameters are obtained by fitting the autocorrelation curves to an appropriate model 83 , 87 , 88 and can then be statistically compared for different conditions.…”

Section: Tpe Fcsmentioning

confidence: 99%

More than double the fun with two-photon excitation microscopy

Luu,

Fraser,

Schneider

2024

Commun Biol

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For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy users interested in adopting TPE for intravital imaging. We focus on applications in neurobiology, present current trends towards faster, wider and deeper imaging, discuss the combination with photon counting technologies for metabolic imaging and spectroscopy, as well as highlight outstanding issues and drawbacks in development and application of these methodologies.

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Section: Tpe Fcsmentioning

confidence: 99%

More than double the fun with two-photon excitation microscopy

Luu,

Fraser,

Schneider

2024

Commun Biol

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“…By choosing various fluorescent probes, FCS can be broadly used to investigate the dynamic behavior and interactions of amphiphilic molecules like surfactants. In summary, FCS’s advantages in high sensitivity, real-time dynamic analysis, non-destructive measurements, small sample volumes, high spatial resolutions, and versatility make it a powerful tool for researching molecular dynamics and interactions, particularly in low-concentration and complex environments [ 47 ].…”

Section: Microemulsion Characterization Techniquesmentioning

confidence: 99%

Advancements in Characterization Techniques for Microemulsions: From Molecular Insights to Macroscopic Phenomena

Li,

Qu,

Liu

et al. 2024

Molecules

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Microemulsions are thermodynamically stable, optically isotropic, transparent, or semi-transparent mixed solutions composed of two immiscible solvents stabilized by amphiphilic solutes. This comprehensive review explores state-of-the-art techniques for characterizing microemulsions, which are versatile solutions essential across various industries, such as pharmaceuticals, food, and petroleum. This article delves into spectroscopic methods, nuclear magnetic resonance, small-angle scattering, dynamic light scattering, conductometry, zeta potential analysis, cryo-electron microscopy, refractive index measurement, and differential scanning calorimetry, examining each technique’s strengths, limitations, and potential applications. Emphasizing the necessity of a multi-technique approach for a thorough understanding, it underscores the importance of integrating diverse analytical methods to unravel microemulsion structures from molecular to macroscopic scales. This synthesis provides a roadmap for researchers and practitioners, fostering advancements in microemulsion science and its wide-ranging industrial applications.

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“…High resolution structure approaches provide atomic level details, but quaternary interactions between membrane proteins need to be resolved in situ because of the chemical complexity of the plasma membrane. Several fluorescence-based methods have been developed to quantify protein interactions in live cells(Martin-Fernandez 2023; Sankaran and Wohland 2023; Stoneman and Raicu 2023). Fluorescence fluctuation spectroscopy (FFS) methods have become valuable for analyzing membrane protein interactions(Bacia et al 2006; Christie et al 2020).…”

Section: Introductionmentioning

confidence: 99%

HER4 is a high affinity dimerization partner for all EGFR/HER/ErbB-family proteins

Singh,

Kim,

Smith

2024

Preprint

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Human epidermal growth factor receptors (HER), also known as EGFR or ErbB receptors, are a subfamily of receptor tyrosine kinases (RTKs) that play crucial roles in cell growth, division, and differentiation. HER4 (ErbB4) is the least studied member of this family, partly because its expression is lower in later stages of development. Recent work has suggested that HER4 can play a role in metastasis through cell migration and invasiveness; however, unlike EGFR and HER2, the precise role that HER4 plays in tumorigenesis is still unresolved. Early work on HER family proteins suggested that there are direct interactions between the four members, but to date, there has been no single study of all four receptors in the same cell line studied with the same biophysical method. Here, we quantitatively measure the degree of association between HER4 and the other HER-family proteins in live cells with a time-resolved fluorescence technique called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is sensitive to the oligomerization state of membrane proteins in live cells, while simultaneously measuring protein expression levels and diffusion coefficients. Our PIE-FCCS results demonstrate that HER4 interacts directly with all HER family members in the cell plasma membrane. The interaction between HER4 and other HER family members intensified in the presence of a HER4-specific ligand. Our work suggests that HER4 is a preferred dimerization partner for all HER family proteins, even in the absence of ligands.

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Current capabilities and future perspectives of FCS: super-resolution microscopy, machine learning, and in vivo applications

Cited by 15 publications

References 138 publications

More than double the fun with two-photon excitation microscopy

More than double the fun with two-photon excitation microscopy

Advancements in Characterization Techniques for Microemulsions: From Molecular Insights to Macroscopic Phenomena

HER4 is a high affinity dimerization partner for all EGFR/HER/ErbB-family proteins

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