scholarly journals Phasor-based single-molecule fluorescence lifetime imaging using a wide-field photon-counting detector

2009 ◽  
Author(s):  
R. Colyer ◽  
O. Siegmund ◽  
A. Tremsin ◽  
J. Vallerga ◽  
S. Weiss ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Photon Counting ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Single Molecule Fluorescence ◽  
Photon Counting Detector ◽  
Lifetime Imaging

scholarly journals Development of new photon-counting detectors for single-molecule fluorescence microscopy

2013 ◽  
Vol 368 (1611) ◽  
pp. 20120035 ◽  
Author(s):  
X. Michalet ◽  
R. A. Colyer ◽  
G. Scalia ◽  
A. Ingargiola ◽  
R. Lin ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Single Photon ◽  
Photon Counting ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Large Area ◽  
Single Molecule Fluorescence ◽  
Single Photon Counting ◽  
Single Molecule Fluorescence Microscopy

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

scholarly journals In situ real-time monitoring the mechanism of self-assembly of short peptide supramolecular polymers

2021 ◽  
Author(s):  
Mari C. Mañas-Torres ◽  
Cristina Gila-Vilchez ◽  
Juan Antonio Gonzalez Vera ◽  
Francisco Conejero-Lara ◽  
Victor Blanco ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Self Assembly ◽  
Correlation Spectroscopy ◽  
Supramolecular Polymers ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Single Molecule Fluorescence ◽  
Lifetime Imaging

Making use of the combination of multiparametric Fluorescence Lifetime Imaging Microscopy (FLIM) and single-molecule Fluorescence Lifetime Correlation Spectroscopy (FLCS), we have been able to study early stages of Fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF)...

scholarly journals Optical Estimation of Absolute Membrane Potential Using One- and Two-Photon Fluorescence Lifetime Imaging Microscopy

2021 ◽  
Author(s):  
Julia R. Lazzari-Dean ◽  
Evan W. Miller
Keyword(s):  
Membrane Potential ◽  
Fluorescence Lifetime ◽  
Single Photon ◽  
Photon Counting ◽  
Fluorescence Lifetime Imaging ◽  
Single Photon Counting ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Wide Range ◽  
Relative Measurements

AbstractBackgroundMembrane potential (Vmem) exerts physiological influence across a wide range of time and space scales. To study Vmem in these diverse contexts, it is essential to accurately record absolute values of Vmem, rather than solely relative measurements.Materials & MethodsWe use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane potential.ResultsWe test the consistency of VF2.1.Cl lifetime measurements performed on different single photon counting instruments and find that they are in striking agreement (differences of <0.5 ps/mV in the slope and <50 ps in the y-intercept). We also demonstrate that VF2.1.Cl lifetime reports absolute Vmem under two-photon (2P) illumination with better than 20 mV of Vmem resolution, a nearly 10-fold improvement over other lifetime-based methods.ConclusionsWe demonstrate that VF-FLIM is a robust and portable metric for Vmem across imaging platforms and under both one-photon and two-photon illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.

scholarly journals Macroscopic fluorescence-lifetime imaging of NADH and protoporphyrin IX improves the detection and grading of 5-aminolevulinic acid-stained brain tumors

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mikael T. Erkkilä ◽  
David Reichert ◽  
Johanna Gesperger ◽  
Barbara Kiesel ◽  
Thomas Roetzer ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Brain Tumors ◽  
Fluorescence Lifetime ◽  
Tumor Tissue ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Low Grade ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

AbstractMaximal safe tumor resection remains the key prognostic factor for improved prognosis in brain tumor patients. Despite 5-aminolevulinic acid-based fluorescence guidance the neurosurgeon is, however, not able to visualize most low-grade gliomas (LGG) and infiltration zone of high-grade gliomas (HGG). To overcome the need for a more sensitive visualization, we investigated the potential of macroscopic, wide-field fluorescence lifetime imaging of nicotinamide adenine dinucleotide (NADH) and protoporphyrin IX (PPIX) in selected human brain tumors. For future intraoperative use, the imaging system offered a square field of view of 11 mm at 250 mm free working distance. We performed imaging of tumor tissue ex vivo, including LGG and HGG as well as brain metastases obtained from 21 patients undergoing fluorescence-guided surgery. Half of all samples showed visible fluorescence during surgery, which was associated with significant increase in PPIX fluorescence lifetime. While the PPIX lifetime was significantly different between specific tumor tissue types, the NADH lifetimes did not differ significantly among them. However, mainly necrotic areas exhibited significantly lower NADH lifetimes compared to compact tumor in HGG. Our pilot study indicates that combined fluorescence lifetime imaging of NADH/PPIX represents a sensitive tool to visualize brain tumor tissue not detectable with conventional 5-ALA fluorescence.

scholarly journals Optically sectioned wide-field fluorescence lifetime imaging microscopy enabled by structured illumination

2017 ◽  
Vol 8 (3) ◽  
pp. 1455 ◽  
Author(s):  
Taylor Hinsdale ◽  
Cory Olsovsky ◽  
Jose J. Rico-Jimenez ◽  
Kristen C. Maitland ◽  
Javier A. Jo ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Structured Illumination ◽  
Lifetime Imaging
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