Paul Urayama

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.

Capillary-based, high-pressure chamber for fluorescence microscopy imaging

We present a high-pressure chamber suitable for the fluorescence microscopy imaging of cellular systems, consisting of a quartz capillary attached to high-pressure tubing with the capillary walls acting as the optical window and mechanical support. The chamber withstands pressures up to 1000atm, spanning most of the biosphere’s pressure range. We validate the chamber for high-pressure studies by using a pH-sensitive fluorophore to sense the pressure-induced acidification of a phosphate buffer. Next, because the capillary walls result in image aberrations, we demonstrate the ability to use spatial deconvolution to improve image resolution. Capillary wall thicknesses are less than 0.5mm, making the chamber compatible with high-numerical-aperture optics.

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).

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.

Characterizing the dual-wavelength dye indo-1 for calcium-ion sensing under pressure

Indo-1 is a dual-wavelength fluorophore widely used for calcium-ion sensing at ambient pressure. Because understanding the physico-chemical nature of high-pressure effects on probe dyes extends the range over which quantitative sensing is possible and informs the development of more robust probes, we report the high-pressure characterization of indo-1 for potential use in calcium-ion sensing under pressure. We find that indo-1 emission remains consistent with a two-state binding model when pressurized to 510 atm, accompanied by a decrease in calcium-binding affinity and an observation of piezochromic behavior. An estimate of the thermodynamic volume change of the indo-1 calcium dissociation reaction is consistent with values for other metal-ion chelators, and piezochromic shifts appear to be due to a pressure-induced change in solvent polarity versus a change in solvent viscosity. The two-state behavior and the linear response of parameters needed for dye calibration makes indo-1 amenable for use under pressure.

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.

An application of spatial deconvolution to a capillary-based high-pressure chamber for fluorescence microscopy imaging

Capillary‐based high‐pressure chambers for which the wall serves as both the optical window and mechanical support have been reported for fluorescence microscopy imaging. Although capillary chambers are straightforward and economical to construct, the curved capillary wall introduces image aberrations. The significance of these aberrations in imaging sub‐cellular‐dimension objects has yet to be assessed. Using a capillary chamber that is routinely pressurized to between 20 and 30 MPa, a pressure range suitable for studying a wide variety of cellular processes, we demonstrate sub‐cellular‐dimension spatial resolution in the imaging of fluorescent micro‐spheres. Objectives with a range of numerical apertures (0.5–1.3) and working distances (0.1–7.4 mm) are considered. We show that spatial (or point‐spread function, PSF) deconvolution improves image contrast in capillary‐based images by comparing deconvolution results with those obtained from slide‐mounted controls. Furthermore, similar deconvolution results between a measured PSF and a calculated, flat‐geometry PSF indicate that the capillary wall is optically flat on cellular length scales. Results here facilitate the application of contemporary techniques in fluorescence microscopy to high‐pressure imaging fields.

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.

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.

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.