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.

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.

A metabolic interpretation for the response of cellular autofluorescence to chemical perturbations assessed using spectral phasor analysis.

Analytical approaches for sensing cellular NADH conformation from autofluorescence signals have significance because NADH is a metabolic indicator and endogenous biomarker. Recently, spectral detection of multiple cellular NADH forms during chemically-induced metabolic response was reported, however because NADH is solvatochromic and the spectral change is small, the possibility of a non-metabolic interpretation needs to be considered. Here we investigate the response of UV-excited autofluorescence to a range of well-known chemicals affecting fermentation, respiration, and oxidative-stress pathways in Saccharomyces cerevisiae. The two-component nature of the spectral response is assessed using phasor analysis. By considering a series of physically similar and dissimilar chemicals acting on multiple pathways, we show how the two-component nature of a spectral response is of metabolic origin, indicative of whether a single or several pathways have been affected.

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.

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 inhibitors­cyanide and ethanol­are 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.

The real-time quantification of autofluorescence spectrum shape for the monitoring of mitochondrial metabolism.

The cellular proportion of free and protein-bound NADH complexes is increasingly recognized as a metabolic indicator and biomarker. Because free and bound forms exhibit different fluorescence spectra, we consider whether autofluorescence shape sufficiently correlates with mitochondrial metabolism to be useful for monitoring in cellular suspensions. Several computational approaches for rapidly quantifying spectrum shape are used to detect Saccharomyces cereviseae response to oxygenation, and to the addition of mitochondrial functional modifiers and metabolic substrates. Observed changes appear consistent with previous studies probing free/protein-bound proportions, making this a potentially useful approach for the real-time monitoring of metabolism. (© 2015 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim).

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.

Fluorescent probe dyes for metabolic-ion sensing under high hydrostatic pressures.

Fluorescence spectroscopy and microscopy imaging are widely used at ambient pressure for analytical studies on biological systems. Before using fluorescence-based methods at high pressures, biochemical and metabolic probes need to be characterized under pressure to ensure valid quantitative results. In this review, we describe the principles behind the use of fluorescent probe dyes for ion sensing and provide models for interpreting the dye spectrum under pressure. We then review results from three studies using the excited-state emission from probe dyes sensitive to pH and calcium-ion concentration, demonstrating some ways pressure may affect probe operation.

Using LysoSensor Yellow/Blue DND-160 to sense acidic pH under high hydrostatic pressures.

LysoSensor Yellow/Blue DND-160, a dual-wavelength fluorophore commonly used for sensing pH in acidic organelles, possesses solvatochromic behavior believed to originate from an intramolecular charge transfer (ICT). Given this, we investigated whether DND-160 can be used for acidic pH sensing under hydrostatic pressures up to 510 atm, a range suitable for studying a wide variety of cellular processes. We found that the emission spectrum of the protonated form does not exhibit sensitivity to pressure, whereas the deprotonated form shows a piezochromic shift consistent with increased ICT character. Although pressure effects on the apparent pKa are buffer solvent dependent, DND-160 retains two-state behavior, making it a useful acidic pH probe under pressure.

Calibration approach for fluorescence lifetime determination for applications using time-gated detection and finite pulse width excitation.

Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is noniterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5-12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime, which can be determined with good reproducibility (typically <100 ps) even for data with poor signal-to-noise ratios ( approximately 20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.

A fluid handling system with finger-tightened connectors for biological studies at kiloatmosphere pressures.

We present a high-pressure fluid handling system based around a simple-to-construct seal for applications in the biologically relevant kiloatmosphere range. Connectors are compact and finger tightened, as compared to the wrench tightening required of cone-type seals commonly used. The seal relies on an O-ring compression, and the system has been tested up to 2000 atm. While the system was designed for biological studies, it should be versatile enough for a wide range of applications, thus contributing finger-tightened convenience to the kiloatmosphere range.

Characterization of dual-wavelength seminaphthofluorescein and seminapthorhodafluor dyes for pH sensing under high hydrostatic pressures.

Hydrostatic pressure is an important physical parameter in biology, with pressures in the few-hundred-atm range having significant effects on cellular morphology, metabolism, and viability. To ensure valid results when studying pressure effects using fluorescence spectroscopy and imaging methods, metabolic probes need to be characterized for high-pressure use. Of interest is the sensing of pH at high pressures due to the key role that pH plays in cellular function. Despite the availability of pH-sensitive dyes, only a few have been characterized for high-pressure use. Here we present the effects of pressure on the acid-base equilibria of four dual-wavelength seminaphthorhodafluor and seminaphthofluorescein dyes (pK(a)=6.6-7.8). Using phosphate buffers as high-pressure pH references, we investigate the pressure dependence of pK(a) for these dyes and determine the volume change associated with the acid-dissociation reaction. We find that if pressure-induced pK(a) changes are not accounted for during interpretation of emission spectra, systematic errors of up to 0.02 pH units per 100atm would result, comparable to previously measured pressure-induced pH changes in vivo. Results are validated by correctly sensing pH changes in Tris and acetate solutions. Methods presented here are applicable to other metabolic probes utilizing dual-wavelength ratiometric sensing modes.

Probing substates in sperm whale myoglobin using high-pressure crystallography.

Pressures in the 100 MPa range are known to have an enormous number of effects on the action of proteins, but straightforward means for determining the structural basis of these effects have been lacking. Here, crystallography has been used to probe effects of pressure on sperm whale myoglobin structure. A comparison of pressure effects with those seen at low pH suggests that structural changes under pressure are interpretable as a shift in the populations of conformational substates. Furthermore, a novel high-pressure protein crystal-cooling method has been used to show low-temperature metastability, providing an alternative to room temperature, beryllium pressure cell-based techniques. The change in protein structure due to pressure is not purely compressive and involves conformational changes important to protein activity. Correlation with low-pH structures suggests observed structural changes are associated with global conformational substates. Methods developed here open up a direct avenue for exploration of the effects of pressure on proteins.