UV-excited Autofluorescence Research Articles

Pressure treatment has a differential effect on cyanide- and ethanol-induced autofluorescence response in yeast.

Cellular processes associated with biological membranes and multimeric associations exhibit pressure sensitivity. In cellular respiration for example, pressure-regulated respiratory oxidases and cytochromes are found in piezophilic microbes, and pressure reduces oxygen consumption rates in eukaryotes. The presence of piezotolerant obligate aerobic yeasts in deep sea environments further justifies investigating pressure effects on cellular respiratory mechanisms. Ambient pressure analysis of UV-excited autofluorescence signals used for monitoring respiratory metabolism have identified multiple cellular NADH and NADPH conformations, significant because the distribution of conformational forms depends on metabolic conditions. Here, we use UV-excited autofluorescence spectroscopy with the spectral response quantified using phasor analysis to monitor cellular NADH and NADPH conformation in Saccharomyces cerevisiae (baker's yeast) suspensions after 30-minute pressure treatment at up to 11 kpsi (75.8 MPa). Specifically, we investigate the effects of pressure treatment on the autofluorescence response to the additions of cyanide and ethanol (both respiratory inhibitors at ambient pressure). The autofluorescence response to cyanide measured 30 min after pressure release shows a diminished response, while the response to ethanol shows no detectable difference when compared with the response prior to pressure treatment. Conditions for this study are similar to those previously reported to activate transcription factors in yeast due to pressure-induced stress tolerance, suggesting autofluorescence may be useful for assessing mitochondrial functional under induced stress-tolerance conditions.

Sensing cellular metabolism in turbid media using spectral phasor analysis on UV-excited autofluorescence

Reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) are metabolic cofactors playing significant, distinct roles in cellular metabolism primarily involving cellular respiration and maintaining antioxidant defenses, respectively. Signals from NADH and NADPH are a significant component of cellular autofluorescence and are useful for metabolic sensing. For in vivo sensing of tissues, challenges include the presence of non-specific background fluorescence as well as perturbations of intrinsic NADH/NADPH fluorescence due to scattering and absorption.

Cellular NADH and NADPH Conformation as a Real-Time Fluorescence-Based Metabolic Indicator under Pressurized Conditions.

Cellular conformation of reduced pyridine nucleotides NADH and NADPH sensed using autofluorescence spectroscopy is presented as a real-time metabolic indicator under pressurized conditions. The approach provides information on the role of pressure in energy metabolism and antioxidant defense with applications in agriculture and food technologies. Here, we use spectral phasor analysis on UV-excited autofluorescence from Saccharomyces cerevisiae (baker’s yeast) to assess the involvement of one or multiple NADH- or NADPH-linked pathways based on the presence of two-component spectral behavior during a metabolic response. To demonstrate metabolic monitoring under pressure, we first present the autofluorescence response to cyanide (a respiratory inhibitor) at 32 MPa. Although ambient and high-pressure responses remain similar, pressure itself also induces a response that is consistent with a change in cellular redox state and ROS production. Next, as an example of an autofluorescence response altered by pressurization, we investigate the response to ethanol at ambient, 12 MPa, and 30 MPa pressure. Ethanol (another respiratory inhibitor) and cyanide induce similar responses at ambient pressure. The onset of non-two-component spectral behavior upon pressurization suggests a change in the mechanism of ethanol action. Overall, results point to new avenues of investigation in piezophysiology by providing a way of visualizing metabolism and mitochondrial function under pressurized conditions.

Monitoring Cellular NAD(P)H Conformation under High Pressure during Chemically Induced UV-Excited Autofluorescence Response

Monitoring Cellular NAD(P)H Conformation under High Pressure during Chemically Induced UV-Excited Autofluorescence Response

Distinguishing chemically induced NADPH- and NADH-related metabolic responses using phasor analysis of UV-excited autofluorescence.

NADPH and NADH are well known for their role in antioxidant defense and energy metabolism, respectively, however distinguishing their cellular autofluorescence signals is a challenge due to their nearly identical optical properties. Recent studies applying spectral phasor analysis to autofluorescence emission during chemically induced metabolic responses showed that two-component spectral behavior, i.e., spectral change acting as a superposition of two spectra, depended on whether one or multiple metabolic pathways were affected. Here, we use this property of spectral behavior to show that metabolic responses primarily involving NADPH or NADH can be distinguished. We start by observing that the cyanide-induced response at micro- and millimolar concentrations does not follow mutual two-component spectral behavior, suggesting their response mechanisms differ. While respiratory inhibition at millimolar cyanide concentration is well known and associated with the NADH pool, we find the autofluorescence response at micromolar cyanide concentration exhibits two-component spectral behavior with NADPH-linked EGCG- and peroxide-induced responses, suggesting an association with the NADPH pool. What emerges is a spectral phasor map useful for distinguishing cellular autofluorescence responses related to oxidative stress versus cellular respiration.

UV resonance Raman spectroscopy of the supramolecular ligand guanidiniocarbonyl indole (GCI) with 244 nm laser excitation.

Ultraviolet resonance Raman (UVRR) spectroscopy is a powerful vibrational spectroscopic technique for the label-free monitoring of molecular recognition of peptides or proteins with supramolecular ligands such as guanidiniocarbonyl pyrroles (GCPs). The use of UV laser excitation enables Raman binding studies of this class of supramolecular ligands at submillimolar concentrations in aqueous solution and provides a selective signal enhancement of the carboxylate binding site (CBS). A current limitation for the extension of this promising UVRR approach from peptides to proteins as binding partners for GCPs is the UV-excited autofluorescence from aromatic amino acids observed for laser excitation wavelengths >260 nm. These excitation wavelengths are in the electronic resonance with the GCP for achieving both a signal enhancement and the selectivity for monitoring the CBS, but the resulting UVRR spectrum overlaps with the UV-excited autofluorescence from the aromatic binding partners. This necessitates the use of a laser excitation <260 nm for spectrally separating the UVRR spectrum of the supramolecular ligand from the UV-excited autofluorescence of the peptide or protein. Here, we demonstrate the use of UVRR spectroscopy with 244 nm laser excitation for the characterization of GCP as well as guanidiniocarbonyl indole (GCI), a next generation supramolecular ligand for the recognition of carboxylates. For demonstrating the feasibility of the UVRR binding studies without an interference from the disturbing UV-excited autofluorescence, benzoic acid (BA) was chosen as an aromatic binding partner for GCI. We also present the UVRR results from the binding of GCI to the ubiquitous RGD sequence (arginylglycylaspartic acid) as a biologically relevant peptide. In the case of RGD, the more pronounced differences between the UVRR spectra of the free and complexed GCI (1:1 mixture) clearly indicate a stronger binding of GCI to RGD compared with BA. A tentative assignment of the experimentally observed changes upon molecular recognition is based on the results from density functional theory (DFT) calculations.

Using Spectral Phasor Analysis of UV-Excited Autofluorescence to Reveal Small Differences between Chemically-Induced Mitochondrial Responses

Using Spectral Phasor Analysis of UV-Excited Autofluorescence to Reveal Small Differences between Chemically-Induced Mitochondrial Responses

Sensing NADH conformation using phasor analysis on fluorescence spectra

Phasor analysis on fluorescence signals is a sensitive approach for analyzing multicomponent systems. Initially developed for time-resolved measurements, a spectral version has been used for the rapid identification of regions during the spectral imaging of biological systems. Here we show that quantitative information regarding conformation can be obtained from phasor analysis of fluorescence spectrum shape. Methanol denaturation of NADH and NADH binding to various dehydrogenase proteins are used as model reactions. Thermodynamic constants are calculated and compared with previous studies based on more direct measures of conformation. Next, the quantitative monitoring of UV-excited autofluorescence spectrum shape during chemically-induced metabolic transitions is presented and discussed in terms of NADH-utilizing pathways. Results show how phasor analysis is useful in assessing two-state behavior, and in interpreting autofluorescence as emission from an ensemble of cellular NADH forms.

Using Real-Time Quantification of Autofluorescence Spectrum Shape to Distinguish between Metabolic Responses Involving Nadh-Utilizing Pathways

In recent studies, autofluorescence measurement of excited-state lifetime or anisotropy decay have revealed multiple forms (e.g., conformations) of intracellular NADH. Because proportions of these forms respond differently to metabolic conditions, the often-used “free” and “protein bound” assignments for NADH may not be adequately descriptive, possibly leading to ambiguities when identifying metabolic state. Here, we describe an accessible method for sensing changes in cellular NADH forms, based on the real-time tracking of UV-excited autofluorescence spectrum shape quantified using spectral phasor analysis. The analytic power of a phasor approach is utilized, testing for whether changes in spectrum shape are consistent with two-state behavior. We show that spectral phasor quantification of spectrum shape can distinguish between metabolic responses induced by the additions of cyanide, glucose, and various alcohols to Saccharomyces cerevisiae suspensions. Controls are presented, evidencing the physiological origins of these spectral responses. The potential for measuring dynamical aspects of metabolic responses, e.g., in relation to Crabtree-positive overflow metabolism and to cellular respiratory activity, is discussed.

Autofluorescence from NADH Conformations Associated with Different Metabolic Pathways Monitored Using Nanosecond-Gated Spectroscopy and Spectral Phasor Analysis

Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitors—cyanide and ethanol—are similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.

Real-Time Quantification of Time-Gated Autofluorescence Spectrum Shape to Track Mitochondrial Metabolism

Recent studies by various groups report the use of NADH as an intrinsic optical biomarker and metabolic indicator. Using measurements of excited-state lifetime or anisotropy decay, different forms of intracellular NADH were identified and monitored, with the response of NADH to metabolic conditions being more complex than a simple concentration change. Recently, we report an approach for the real-time tracking of UV-excited autofluorescence based on the rapid quantification of spectrum shape. Here, we show that nanosecond-gated spectral acquisition - combined with spectrum-shape quantification (such as with spectral phasor analysis) - can be used to track the physiologic response of a cellular system (Saccharomyces cereviseae) to the addition of mitochondrial functional modifiers (e.g., cyanide) and metabolic substrates (e.g., ethanol and glucose). We demonstrate that time-gated detection allows for the rejection of short excited-state-lifetime emission. We observe that the spectrum shape of long excited-state-lifetime autofluorescence exhibits a different response to chemical additions than that of the time-integrated autofluorescence, even allowing for the discrimination between nominally similar responses. Results are consistent with the existence of multiple NADH forms, as changes in autofluorescence spectrum shape cannot be accounted for by a two spectra model. Time-gated spectroscopy combined with rapid spectrum-shape analysis may lead to new and useful approaches for the real-time tracking of cellular metabolic state.

Note: A micro-perfusion system for use during real-time physiological studies under high pressure

We construct a micro-perfusion system using piston screw pump generators for use during real-time, high-pressure physiological studies. Perfusion is achieved using two generators, with one generator being compressed while the other is retracted, thus maintaining pressurization while producing fluid flow. We demonstrate control over perfusion rates in the 10-μl/s range and the ability to change between fluid reservoirs at up to 50 MPa. We validate the screw-pump approach by monitoring the cyanide-induced response of UV-excited autofluorescence from Saccharomyces cerevisiae under pressurization.

In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5–6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.

The Necdin-Wnt Pathway Causes Epigenetic Peroxisome Proliferator-activated Receptor γ Repression in Hepatic Stellate Cells

Hepatic stellate cells (HSCs), vitamin A-storing liver pericytes, undergo myofibroblastic trans-differentiation or "activation" to participate in liver wound healing. This cellular process involves loss of regulation by adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ). Necdin, a melanoma antigen family protein, promotes neuronal and myogenic differentiation while inhibiting adipogenesis. The present study demonstrates that necdin is selectively expressed in HSCs among different liver cell types and induced during their activation in vitro and in vivo. Silencing of necdin with adenovirally expressed shRNA, reverses activated HSCs to quiescent cells in a manner dependent on PPARγ and suppressed canonical Wnt signaling. Promoter analysis, site-directed mutagenesis, and chromatin immunoprecipitation demonstrate that Wnt10b, a canonical Wnt induced in activated HSCs, is a direct target of necdin. Necdin silencing abrogates three epigenetic signatures implicated in repression of PPARγ: increased MeCP2 (methyl CpG binding protein 2) and HP-1α co-repressor recruitments to Pparγ promoter and enhanced H3K27 dimethylation at the exon 5 locus, again in a manner dependent on suppressed canonical Wnt. These epigenetic effects are reproduced by antagonism of canonical Wnt signaling with Dikkopf-1. Our results demonstrate a novel necdin-Wnt pathway, which serves to mediate antiadipogenic HSC trans-differentiation via epigenetic repression of PPARγ.

Biochemical Imaging of Human Atherosclerotic Plaques with Fluorescence Lifetime Angioscopy

A prototype angioscopy system with fluorescence lifetime imaging microscopy (FLIM) capabilities was built and applied for biochemical imaging of human coronary atherosclerotic plaques. The FLIM angioscopy prototype consisted of a thin flexible angioscope suitable for UV-excited autofluorescence imaging, and a FLIM detection system based on a pulse sampling approach. The angioscope was composed of an imaging bundle attached to a gradient index objective lens and surrounded by a ring of illumination fibers (2 mm outer diameter, 50 microm spatial resolution). For FLIM detection based on the pulse sampling approach, a gated-intensified charge-couple device camera (200 ps temporal resolution) was used. Autofluorescence was excited with a pulsed UV laser (337 nm) and FLIM images were acquired at three emission bands (390/40 nm, 450/40 nm, 550/88 nm). The system was characterized on standard fluorophores and then used to image postmortem human coronary arteries. The FLIM angioscope allowed us to distinguish elastin-dominant plaques (peak emission at 450 nm, approximately 1.5 ns lifetimes) from collagen-dominant plaques (peak emission at 390 n, approximately 2-3 ns lifetimes) based on their intrinsic fluorescence spectral and lifetime differences. This study demonstrates the potential of FLIM angioscopy for biochemical imaging of human coronary atherosclerotic plaques.