Label-Free Fluorescence Detection of Aromatic Compounds in Chip Electrophoresis Applying Two-Photon Excitation and Time-Correlated Single-Photon Counting

2013 ◽  
Vol 85 (17) ◽  
pp. 8150-8157 ◽  
Author(s):  
Reinhild Beyreiss ◽  
David Geißler ◽  
Stefan Ohla ◽  
Stefan Nagl ◽  
Tjorben Nils Posch ◽  
...  
Keyword(s):  
Fluorescence Detection ◽  
Aromatic Compounds ◽  
Single Photon ◽  
Photon Counting ◽  
Label Free ◽  
Single Photon Counting ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

scholarly journals Two-photon fluorescence lifetime for label-free microfluidic droplet sorting

2021 ◽  
Author(s):  
Sadat Hasan ◽  
Maximilian E. Blaha ◽  
Sebastian K. Piendl ◽  
Anish Das ◽  
David Geissler ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Single Photon ◽  
Photon Counting ◽  
Droplet Microfluidics ◽  
Label Free ◽  
Single Photon Counting ◽  
Two Photon ◽  
Deep Uv ◽  
Droplet Sorting ◽  
532 Nm

AbstractMicrofluidic droplet sorting systems facilitate automated selective micromanipulation of compartmentalized micro- and nano-entities in a fluidic stream. Current state-of-the-art droplet sorting systems mainly rely on fluorescence detection in the visible range with the drawback that pre-labeling steps are required. This limits the application range significantly, and there is a high demand for alternative, label-free methods. Therefore, we introduce time-resolved two-photon excitation (TPE) fluorescence detection with excitation at 532 nm as a detection technique in droplet microfluidics. This enables label-free in-droplet detection of small aromatic compounds that only absorb in a deep-UV spectral region. Applying time-correlated single-photon counting, compounds with similar emission spectra can be distinguished due to their fluorescence lifetimes. This information is then used to trigger downstream dielectrophoretic droplet sorting. In this proof-of-concept study, we developed a polydimethylsiloxane-fused silica (FS) hybrid chip that simultaneously provides a very high optical transparency in the deep-UV range and suitable surface properties for droplet microfluidics. The herein developed system incorporating a 532-nm picosecond laser, time-correlated single-photon counting (TCSPC), and a chip-integrated dielectrophoretic pulsed actuator was exemplarily applied to sort droplets containing serotonin or propranolol. Furthermore, yeast cells were screened using the presented platform to show its applicability to study cells based on their protein autofluorescence via TPE fluorescence lifetime at 532 nm. Graphical abstract

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.

Autofluorescence of epithelial tissue: single-photon versus two-photon excitation

2008 ◽  
Vol 13 (5) ◽  
pp. 054010 ◽  
Author(s):  
Wei Zheng ◽  
Yicong Wu ◽  
Dong Li ◽  
Jianan Y. Qu
Keyword(s):  
Single Photon ◽  
Epithelial Tissue ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

A custom confocal and two-photon digital laser scanning microscope

2000 ◽  
Vol 278 (6) ◽  
pp. H2150-H2156 ◽  
Author(s):  
W. Gil Wier ◽  
C. William Balke ◽  
Jeffrey A. Michael ◽  
Joseph R. H. Mauban
Keyword(s):  
Laser Scanning ◽  
Photon Counting ◽  
Objective Lens ◽  
Scanning Microscope ◽  
Fluorescence Detector ◽  
Laser Scanning Microscope ◽  
Ti:Sapphire Laser ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

We describe a custom one-photon (confocal) and two-photon all-digital (photon counting) laser scanning microscope. The confocal component uses two avalanche photodiodes (APDs) as the fluorescence detector to achieve high sensitivity and to overcome the limited photon counting rate of a single APD (∼5 MHz). The confocal component is approximately nine times more efficient than our commercial confocal microscope (fluorophore fluo 4). Switching from one-photon to two-photon excitation mode (Ti:sapphire laser) is accomplished by moving a single mirror beneath the objective lens. The pulse from the Ti:sapphire laser is 109 fs in duration at the specimen plane, and average power is ∼5 mW. Two-photon excited fluorescence is detected by a fast photomultiplier tube. With a ×63 1.4 NA oil-immersion objective, the resolution of the confocal system is 0.25 μm laterally and 0.52 μm axially. For the two-photon system, the corresponding values are 0.28 and 0.82 μm. The system is advantageous when excitation intensity must be limited, when fluorescence is low, or when thick, scattering specimens are being studied (with two-photon excitation).

In vivo label-free two-photon excitation autofluorescence microscopy of microvasculature using a 520 nm femtosecond fiber laser

2020 ◽  
Vol 45 (10) ◽  
pp. 2704
Author(s):  
Ting Wu ◽  
Jiuling Liao ◽  
Jia Yu ◽  
Yufeng Gao ◽  
Hui Li ◽  
...  
Keyword(s):  
Fiber Laser ◽  
Label Free ◽  
Two Photon ◽  
Photon Excitation ◽  
Femtosecond Fiber Laser ◽  
Two Photon Excitation

scholarly journals A novel method for observing proteins in vivo using a small fluorescent label and multiphoton imaging

2005 ◽  
Vol 390 (3) ◽  
pp. 787-790 ◽  
Author(s):  
Stanley W. Botchway ◽  
Ignasi Barba ◽  
Randolf Jordan ◽  
Rebecca Harmston ◽  
Peter M. Haggie ◽  
...  
Keyword(s):  
Fluorescence Detection ◽  
Fluorescence Emission ◽  
Yeast Cells ◽  
Fluorescent Label ◽  
Multiphoton Imaging ◽  
Two Photon ◽  
Photon Excitation ◽  
Novel Method ◽  
Two Photon Excitation

A novel method for the fluorescence detection of proteins in cells is described in the present study. Proteins are labelled by the selective biosynthetic incorporation of 5-hydroxytryptophan and the label is detected via selective two-photon excitation of the hydroxyindole and detection of its fluorescence emission at 340 nm. The method is demonstrated in this paper with images of a labelled protein in yeast cells.

Photochemistry with high power ultraviolet lasers

1983 ◽  
Vol 61 (5) ◽  
pp. 1023-1026 ◽  
Author(s):  
R. J. Donovan ◽  
C. Fotakis ◽  
A. Hopkirk ◽  
C. B. McKendrick ◽  
A. Torre
Keyword(s):  
Energy Distribution ◽  
High Power ◽  
Single Photon ◽  
Laser Induced Fluorescence ◽  
Multiphoton Excitation ◽  
Dissociation Process ◽  
Two Photon ◽  
Photon Excitation ◽  
Ultraviolet Lasers ◽  
Two Photon Excitation

Rotationally resolved photofragment fluorescence from OH(A2Σ+) following the coherent two-photon excitation of H2O with a KrF laser (248 nm), is reported and the dynamics of the dissociation process are discussed. Fluorescence from CN(B2Σ+) following two-photon excitation of ICN is also described. In both cases the energy distribution in the photofragments is shown to differ significantly from that observed with single-photon excitation at closely similar energies.Two further examples of multiphoton excitation, involving CS2 and SO2, are briefly discussed. In these cases absorption of a further photon, by fragments produced in the primary step, gives rise to strong laser-induced fluorescence.

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