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A single-biomolecule optical toggle-switch is demonstrated based on a mutated green fluorescent protein (GFP). We have exploited molecular biology techniques to tailor GFP molecular structure and photophysical properties and give it…

Here we describe the coupled standardisation of two complementary fluorescence imaging techniques and apply it to liquid-liquid phase separated condensates formed from an EGFP fluorescent reporter of Flowering Control Locus A (FCA), a…

Biomolecules · Quantitative Biology 2023-01-25 Alex L. Payne-Dwyer , Mark C. Leake

We demonstrate sequential two-photon fluorescence microscopy using forbidden state transitions. Nonlinear red excitation leads to green fluorescence in live cells expressing eYFP, maintaining optical sectioning and allowing deep tissue…

In this work, we study the conditions under which fluorescent beads can be used to emulate single fluorescent molecules in the calibration of optical microscopes. Although beads are widely used due to their brightness and easy manipulation,…

Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled…

Biological Physics · Physics 2023-01-25 Jack W Shepherd , Alex L Payne-Dwyer , Ji-Eun Lee , Aisha Syeda , Mark C Leake

Over the last decade, single-molecule optical microscopy has become the gold-standard approach to decipher complex molecular processes in cellular environments. [1-3] Single-molecule fluorescence microscopy has several advantages such as…

Biological Physics · Physics 2017-11-15 Edakkattuparambil Sidharth Shibu , Nadezda Varkentina , Laurent Cognet , Brahim Lounis

Patient-specific therapies require that cells be manufactured in multiple batches of small volumes, making it a challenge for conventional modes of quality control. The added complexity of inherent variability (even within batches)…

Biological Physics · Physics 2018-04-17 Derrick Yong , Ahmad Amirul Abdul Rahim , Jesslyn Ong , May Win Naing

Using the ultraviolet autofluorescence of tryptophan aminoacids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labelling. However, the low autofluorescence signals have so far limited the UV…

We report the cell biological applications of a recently developed multiphoton fluorescence lifetime imaging microscopy system using a streak camera (StreakFLIM). The system was calibrated with standard fluorophore specimens and was shown…

Cell Behavior · Quantitative Biology 2009-11-10 R. V. Krishnan , Eva Biener , Jian-Hua Zhang , Robert Heckel , Brian Herman

The importance of spin-correlated radical pairs in biology is increasingly recognized, with roles in biological effects of weak magnetic fields and emerging quantum spin-based biomedical applications. Fluorescence microscopy offers…

Biological Physics · Physics 2025-10-08 Noboru Ikeya , Jonathan R. Woodward

The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single molecule and single cell level can add…

Quantitative Methods · Quantitative Biology 2012-11-21 Alex Robson , Kevin Burrage , Mark Leake

Fluorescence Correlation Spectroscopy (FCS) is a powerful single-molecule technique which allows for measuring motion (diffusion, flow), concentration, and molecular interaction kinetics of fluorescent molecules from picomolar to micromolar…

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded…

Single-molecule fluorescence techniques have revolutionized our ability to study proteins. However, the presence of a fluorescent label can alter the protein structure and/or modify its reaction with other species. To avoid the need for a…

Biological Physics · Physics 2022-04-07 Aleksandr Barulin , Prithu Roy , Jean-Benoît Claude , Jérôme Wenger

Recent experimental results have shown that active enzymes can diffuse faster when they are in the presence of their substrates. Fluorescence correlation spectroscopy (FCS), which relies on analyzing the fluctuations in fluorescence…

Subcellular Processes · Quantitative Biology 2019-09-25 Mengqi Xu , Lyanne Valdez , Aysuman Sen , Jennifer L. Ross

We experimentally investigate the fluorescence photon emission characteristics for single q-dots by using optical nanofibers. We demonstrate that single q-dots can be deposited along an optical nanofiber systematically and reproducibly with…

Mesoscale and Nanoscale Physics · Physics 2015-06-03 Ramachandrarao Yalla , K. P. Nayak , K. Hakuta

Autofluorescence lifetime images reveal unique characteristics of endogenous fluorescence in biological samples. Comprehensive understanding and clinical diagnosis rely on co-registration with the gold standard, histology images, which is…

Image and Video Processing · Electrical Eng. & Systems 2022-02-17 Qiang Wang , Susan Fernandes , Gareth O. S. Williams , Neil Finlayson , Ahsan R. Akram , Kevin Dhaliwal , James R. Hopgood , Marta Vallejo

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as F\"orster…

The poor photostability and low brightness of protein autofluorescence have been major limitations preventing the detection of label-free proteins at the single molecule level. Overcoming these issues, we report here a strategy to promote…

Chemical Physics · Physics 2020-02-25 Aleksandr Barulin , Jérôme Wenger

Optical detection and spectroscopy of single molecules has become an indispensable tool in biological imaging and sensing. Its success is based on fluorescence of organic dye molecules under carefully engineered laser illumination. In this…

Chemical Physics · Physics 2011-01-18 Ilja Gerhardt , Lijian Mai , Antia Lamas-Linares , Christian Kurtsiefer
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