Compact and cost-effective handheld multispectral fluorescence lifetime imaging (FLIM) system for oral mucosa in vivo imaging based on a frequency-domain implementation (Conference Presentation)

2018 ◽  
Author(s):  
Shuna Cheng ◽  
Michael Serafino ◽  
Rodrigo Cuenca ◽  
Beena Ahmed ◽  
Brian Applegate ◽  
...  
Keyword(s):  
Frequency Domain ◽  
Oral Mucosa ◽  
Fluorescence Lifetime ◽  
In Vivo Imaging ◽  
Cost Effective ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

scholarly journals Linear Behavior of the Phase Lifetime in Frequency-Domain Fluorescence Lifetime Imaging of FRET Constructs

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniel Sumetsky ◽  
James Y. Jiang ◽  
Marina A. Ayad ◽  
Timothy Mahon ◽  
Audrey Menaesse ◽  
...  
Keyword(s):  
Frequency Domain ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cost Effective ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Linear Behavior ◽  
Fret Efficiency ◽  
Linear Fit

We utilize a cost-effective frequency-domain fluorescence lifetime imaging microscope to measure the phase lifetime of mTFP1 in mTFP1-mVenus fluorescence resonance energy transfer (FRET) constructs relevant to the VinTS molecular tension probe. Our data were collected at 15 modulation frequencies ω/2π selected between 14 and 70 MHz. The lifetime of mTFP1 was τD = 3.11 ± 0.02 ns in the absence of acceptor. For modulation frequencies, ω, such that (ω · τD) < 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), τϕDA, was directly related to the amplitude-weighted lifetime τaveDA inferred from the known FRET efficiency (EFRETtrue) of the constructs. A linear fit to a plot of (ω·τϕDA) vs. (ω·τaveDA) yielded a slope of 0.79 ± 0.05 and intercept of 0.095 ± 0.029 (R2 = 0.952). Thus, our results suggest that a linear relationship exists between the apparent EFRETapp based on the measured phase lifetime and EFRETtrue for frequencies such that (ω · τD) < 1.1. We had previously reported a similar relationship between EFRETapp and EFRETtrue at 42 MHz. Our current results provide additional evidence in support of this observation, but further investigation is still required to fully characterize these results. A direct relationship between τϕDAand τaveDA has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs.

scholarly journals High-speed, long-term, 4D in vivo lifetime imaging in intact and injured zebrafish and mouse brains by instant FLIM

2020 ◽  
Author(s):  
Yide Zhang ◽  
Ian H. Guldner ◽  
Evan L. Nichols ◽  
David Benirschke ◽  
Cody J. Smith ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Fluorescence Lifetime ◽  
High Speed ◽  
Super Resolution ◽  
Cost Effective ◽  
Fluorescence Lifetime Imaging ◽  
Imaging Data ◽  
Lifetime Imaging ◽  
Effective Components

AbstractTraditional fluorescence microscopy is blind to molecular microenvironment information that is present in fluorescence lifetime, which can be measured by fluorescence lifetime imaging microscopy (FLIM). However, existing FLIM techniques are typically slow to acquire and process lifetime images, difficult to implement, and expensive. Here, we present instant FLIM, an analog signal processing method that allows real-time streaming of fluorescence intensity, lifetime, and phasor imaging data through simultaneous image acquisition and instantaneous data processing. Instant FLIM can be easily implemented by upgrading an existing two-photon microscope using cost-effective components and our open-source software. We further improve the functionality, penetration depth, and resolution of instant FLIM using phasor segmentation, adaptive optics, and super-resolution techniques. We demonstrate through-skull intravital 3D FLIM of mouse brains to depths of 300 μm and present the first in vivo 4D FLIM of microglial dynamics in intact and injured zebrafish and mouse brains up to 12 hours.

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

2009 ◽  
Vol 72 (5) ◽  
pp. 371-379 ◽  
Author(s):  
Aymeric Leray ◽  
Franck B. Riquet ◽  
Elodie Richard ◽  
Corentin Spriet ◽  
Dave Trinel ◽  
...  
Keyword(s):  
Frequency Domain ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Optimized Protocol

scholarly journals In Vivo Fluorescence Lifetime Imaging for Monitoring the Efficacy of the Cancer Treatment

2014 ◽  
Vol 20 (13) ◽  
pp. 3531-3539 ◽  
Author(s):  
Yasaman Ardeshirpour ◽  
Victor Chernomordik ◽  
Moinuddin Hassan ◽  
Rafal Zielinski ◽  
Jacek Capala ◽  
...  
Keyword(s):  
Cancer Treatment ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
In Vivo Fluorescence

scholarly journals Coarse-grained modeling of mitochondrial metabolism enables subcellular flux inference from fluorescence lifetime imaging microscopy

2020 ◽  
Author(s):  
Xingbo Yang ◽  
Daniel J. Needleman
Keyword(s):  
Fluorescence Lifetime ◽  
Metabolic Flux ◽  
Living Cells ◽  
Coarse Grained ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Metabolic Fluxes ◽  
Subcellular Resolution

AbstractMitochondria are central to metabolism and their dysfunctions are associated with many diseases1–9. Metabolic flux, the rate of turnover of molecules through a metabolic pathway, is one of the most important quantities in metabolism, but it remains a challenge to measure spatiotemporal variations in mitochondrial metabolic fluxes in living cells. Fluorescence lifetime imaging microscopy (FLIM) of NADH is a label-free technique that is widely used to characterize the metabolic state of mitochondria in vivo10–18. However, the utility of this technique has been limited by the inability to relate FLIM measurement to the underlying metabolic activities in mitochondria. Here we show that, if properly interpreted, FLIM of NADH can be used to quantitatively measure the flux through a major mitochondrial metabolic pathway, the electron transport chain (ETC), in vivo with subcellular resolution. This result is based on the use of a coarse-grained NADH redox model, which we test in mouse oocytes subject to a wide variety of perturbations by comparing predicted fluxes to direct biochemical measurements and by self-consistency criterion. Using this method, we discovered a subcellular spatial gradient of mitochondrial metabolic flux in mouse oocytes. We showed that this subcellular variation in mitochondrial flux correlates with a corresponding subcellular variation in mitochondrial membrane potential. The developed model, and the resulting procedure for analyzing FLIM of NADH, are valid under nearly all circumstances of biological interest. Thus, this approach is a general procedure to measure metabolic fluxes dynamically in living cells, with subcellular resolution.

Towards in-vivo assessment of fluorescence lifetime: imaging using time-gated intensified CCD camera

2018 ◽  
Vol 38 (4) ◽  
pp. 966-974 ◽  
Author(s):  
Piotr Sawosz ◽  
Stanislaw Wojtkiewicz ◽  
Michal Kacprzak ◽  
Elzbieta Zieminska ◽  
Magdalena Morawiec ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Ccd Camera ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
In Vivo Assessment
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