scholarly journals Metabolic Imaging Using Two-Photon Excited NADH Intensity and Fluorescence Lifetime Imaging

2012 ◽  
Vol 18 (4) ◽  
pp. 761-770 ◽  
Author(s):  
Jorge Vergen ◽  
Clifford Hecht ◽  
Lyandysha V. Zholudeva ◽  
Meg M. Marquardt ◽  
Richard Hallworth ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Intensity ◽  
Organ Of Corti ◽  
Metabolic Imaging ◽  
Fluorescence Lifetime Imaging ◽  
Metabolic State ◽  
Intact Mouse ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Reduced Nicotinamide Adenine Dinucleotide

AbstractMetabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

scholarly journals Metabolic state of human blastocysts measured by fluorescence lifetime imaging microscopy

2021 ◽  
Author(s):  
Marta Venturas ◽  
Jaimin S Shah ◽  
Xingbo Yang ◽  
Tim H Sanchez ◽  
William Conway ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Metabolic Imaging ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Embryo Viability ◽  
Metabolic State ◽  
Lifetime Imaging ◽  
Non Invasive

Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in Assisted Reproductive Technologies (ART) to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state. Here, we explore the application of metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) for studying human blastocysts. We use FLIM to measure the autofluorescence of two central coenzymes, NAD(P)H and FAD+, in 215 discarded human blastocysts from 137 patients. We find that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We show that the metabolic state of human blastocysts changes continually over time, and that variations between blastocyst are partially explained by both the time since fertilization and their developmental stage, but not their morphological grade. We also observe significant metabolic heterogeneity within individual blastocysts, including between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida. Taken together, this work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism.

scholarly journals Sperm metabolism is altered during storage by female insects: evidence from two-photon autofluorescence lifetime measurements in bedbugs

2015 ◽  
Vol 12 (110) ◽  
pp. 20150609 ◽  
Author(s):  
Klaus Reinhardt ◽  
Hans Georg Breunig ◽  
Aisada Uchugonova ◽  
Karsten König
Keyword(s):  
Fluorescence Lifetime ◽  
Sperm Cells ◽  
Fluorescence Lifetime Imaging ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Photon Excitation ◽  
The Common ◽  
The Mean ◽  
The Difference ◽  
Male Genotype

We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius , was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, τ m = 1.54–1.84 ns) than in that extracted from the female sperm storage organ (tau m, τ m = 1.26–2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype.

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 High-speed imaging of transient metabolic dynamics using two-photon fluorescence lifetime imaging microscopy

Optica ◽  
2018 ◽  
Vol 5 (10) ◽  
pp. 1290 ◽  
Author(s):  
Andrew J. Bower ◽  
Joanne Li ◽  
Eric J. Chaney ◽  
Marina Marjanovic ◽  
Darold R. Spillman ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
High Speed ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
High Speed Imaging ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence
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