If we break up a living organism by isolating its different parts it is only for the sake of ease in analysis and by no means in order to conceive of them separately. Indeed when we wish to ascribe to a physiological quantity its value and true significance we must always refer to this whole and draw our final conclusions only in relation to its effects in the whole.

Claude Bernard (1865)

Introduction to the Study of Experimental Medicine

1.1 Background

The accurate description of many biological phenomena requires that they be observed in vivo, that is, occurring within the bodies of living organisms. The practice of instrumenting and preparing living organisms so that meaningful measurements can be obtained has evolved significantly, and today a number of sophisticated tools and techniques are available to the experimental biologist. This thesis will investigate two in particular: optical spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. These techniques are extremely attractive because both can be used to non-invasively monitor physiological processes and generate quantitative data. The non-invasive nature of these techniques means that the physiology of the preparation will not be significantly affected by the measurement apparatus and that repeated measures on a subject may be made without deleterious effects.

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This dissertation follows the style and format of the IEEE Transactions on Biomedical Engineering.

Since both NMR and optical spectroscopy derive information about their targets via interaction of non-ionizing electromagnetic energy with the sample, they are well suited to in vivo investigations. In NMR spectroscopy, it is the interaction of radio frequency (RF) energy with the nuclei of atoms contained in chemicals of the sample which yields important information. The characteristics of these interactions are dependent on the local environment of the nuclei and can thus give information about the structure and chemical environment of the compound containing these nuclei. Nuclei sensitive to NMR investigation include hydrogen (¹ H), phosphorus (³¹P), fluorine ( ¹⁹F), carbon (¹³ C), sodium (²³Na), and calcium ( ⁴³Ca) among others. Of particular interest is ³¹P NMR spectroscopy since ³¹ P occurs in 100% natural abundance and many chemicals of fundamental metabolic importance such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and phosphocreatine (PCr), contain phosphorus [1, 2, 3]. The first in vivo ³¹P NMR spectra were published in 1974 by Hoult et al. [4], and since that time the technique has become quite popular. ¹H NMR spectroscopy can be used to analyze physiological processes as well. The ¹H nucleus is the most sensitive and by far the most abundant ([¹H] » 111 molar in water) in the animal. However, the magnitude of ¹H chemical shifts is typically small, and obtaining sufficient spectral resolution is difficult due to the overwhelming presence of water in the body and the subsequent need for specialized water signal suppression techniques [1, 3]. The use of ¹³C NMR spectroscopy is popular for tracking metabolites and or drugs, but, ¹² C (which is not NMR sensitive) and not ¹³ C is the dominant form of carbon and so sensitivity is often low, or specially synthesized chemicals with ¹³C enrichment are required [5].

In optical spectroscopy, it is optical radiation which interacts with the sample through the electron clouds of its constituent chemicals. The information obtained tells of the macroscopic environment of the molecule and its characteristic functional groups rather than specific atomic sites [6]. Optical spectroscopy can be used to identify and quantitate molecules in the blood or organs based on their characteristic interaction signatures or spectra. Compounds like hemoglobin which change their signature depending on their conformational state can be tracked as well [7]. With the aid of specially designed compounds, variables like pH, electrical potential, and the concentrations of many ions can be measured indirectly [8]. Many of these compounds fall into the category of fluorophores - compounds which absorb photons and re-emit part of this energy at different wavelengths.

Because NMR and optical spectroscopy rely on the interaction of samples with very different regions of the electromagnetic spectrum, it is not surprising that the information they can provide about a sample is quite different and often complementary. For example, while near infrared (NIR) optical spectroscopy allows for the direct determination of the saturation percentage of hemoglobin [9], conventional NMR spectroscopy can not. Similarly, NMR spectroscopy is useful for differentiating and determining the relative concentrations of phosphocreatine (PCr) and adenosine triphosphate (ATP), while optical spectroscopy (in its simplest form) is not. It is for these reasons that employing both optical and NMR spectroscopy as tools for physiological investigations is potentially useful. By utilizing both tools simultaneously, the investigator can collect interrelated information inaccessible by a single technique, and thus obtain a more complete picture of the physiological changes and relationships taking place during an experiment.

1.2 Motivation

While non-invasive in nature, NMR spectroscopy does impose stringent limitations on sample preparation. The environment in which NMR studies must be carried out usually involves a large (> 1 Tesla) static magnetic field, high-power RF pulses, and requisitely low levels of electronic noise [10]. This environment is generally inhospitable to or incompatible with other modes of instrumentation. Certain forms of optical spectroscopy, however, are uniquely well suited to the NMR experimental environment, and can provide information which is complementary to or important in understanding that obtained from NMR. Despite this unique compatibility, it is extremely rare that NMR and optical spectroscopy experiments are performed simultaneously. This is partially due to the difficulty and expense of constructing a magnet-compatible optical sensor.

Several fields of study utilize the combination of optical and NMR spectroscopy. For example, characterization of manufactured chemicals can rely on correlation of separately collected optical spectroscopy and NMR spectroscopy data [11]. In biology, NMR spectroscopy has been used to track intracellular pH while optical fluorescence spectroscopy was used to determine cellular calcium concentrations [12]. Finally, in clinical medicine, optical near infrared (NIR) spectroscopy has been used in conjunction with ³¹P NMR spectroscopy in an attempt to correlate changes in tissue oxygenation state with pathological deficiencies in phosphorus metabolism [13, 14], and some investigators have attempted to use NIR spectroscopy and ¹H magnetic resonance imaging (MRI) in order to make better functional imaging maps of brain activity during certain tasks [15]. In most other cases, though, experiments are usually duplicated to separately collect NMR and optical data. In many cases this experimental replication is unnecessary and can lead to unwanted treatment variation [16]. We are particularly interested in in vivo physiological studies where optical spectroscopy and NMR spectroscopy have been separately used to monitor physiological variables in real time. It is here that the development of a probe to concomitantly collect optical and NMR data would be a boon. Simultaneous access to both optical and NMR measurands would allow for reduction in subject and treatment variability, better time correlation between variable measurements, and an increase in the amount of information which can be derived from a single experiment. Finally, the cost of such a sensor is offset if it is sufficiently versatile in its design so as to be useful for many different types of studies.

1.3 Problem Statement

The purpose of this research is to design, develop, and test a new sensor that combines the collection of both nuclear magnetic resonance spectra and optical fluorescence spectra simultaneously. The combination of these modalities increases the amount of information which can be learned from a single experiment since NMR sensitive and fluorescence sensitive variables may be monitored together. Additionally, techniques including the use of appropriately designed coil arrays will be investigated as a means to maximize the signal-to-noise ratio (SNR) over a particular small volume, e.g. the turtle heart. The application of this research will be to demonstrate the sensor as a tool useful to biologists and physiologists studying cardiac function in vivo by simultaneously monitoring intracellular calcium levels and phosphate metabolism.

1.3.1 Physiological Background

Mammalian cardiac cells are markedly intolerant to hypoxic stress which results in (among other things) a breakdown of intracellular calcium homeostasis [17, 18]. Anoxic stress in mammalian cardiomyocytes leads within minutes to a decrease of high-energy phosphates and a breakdown of normal cellular calcium homeostasis [16]. This breakdown allows [Ca²⁺ ] levels in the intracellular space to rise and for the cell to enter a hypercontractile state. It has been postulated that this rise in intracellular [Ca²⁺] is due to a depleted energy store of high-energy phosphates such as ATP and PCr [19]. However, a definite causal relationship has not yet been established in part because calcium and phosphate levels have not been measured simultaneously. During anoxia, oxidative phosphorylation ceases, and cardiac cells deplete their stores of phosphate energy. This decrease in ATP inhibits active Ca ²⁺-ATPase transport of Ca²⁺ out of the cytosol and into the extracellular space of internal sequestration stores. Concentration gradient driven Na⁺ /Ca²⁺ exchangers may also fail as intracellular [Na⁺] rises with a breakdown of active Na⁺/K⁺-ATPase pumps. In fact, Na⁺/Ca²⁺ exchange may actually reverse as intracellular [Na⁺ ] rises [18].

In contrast, a particular turtle species, the western painted turtle ( Chrysemys picta bellii), exhibits cardiac tolerance to extreme and prolonged hypoxia [18, 20]. If the current model linking metabolic energy state and calcium regulation is correct, these turtles should exhibit normal tissue energy levels and normal [Ca²⁺] levels even during prolonged hypoxic stress. While experiments measuring calcium concentrations in preparations of isolated cells exposed to anoxia have been performed [18] and these data have been compared with ³¹ P-NMR data corresponding to similar treatments of isolated, perfused hearts [21], simultaneous measurement of both calcium and high-energy phosphate stores in vivo has not been attempted. Further, some researchers have suggested that in vivo circulating plasma factors may be at work in hypoxia tolerance [22]. It is for these reasons that simultaneous measurement of both phosphorus energy level and intracellular [Ca ²⁺] levels in vivo is important as it will allow more accurate time correlation of high-energy phosphate depletion and calcium deregulation [16]. This, in turn, may also allow observation of a cause and effect relationship between cell energetics and calcium homeostasis.

1.3.2 Scope of Work

The scope of this project includes 1) development of an optical fluorescence spectrometer compatible with the NMR environment, 2) design, fabrication, and investigation of an array of surface coils or other suitable detection technique for the collection of ³¹P NMR spectra, 3) combination of the two probes into a single sensor for measuring NMR and optical spectra, and 4) simultaneous in vivo monitoring of high energy phosphate and intracellular free [Ca²⁺ ] levels in a living heart. This dissertation describes in detail the construction of a CCD-based fiber-optic fluorescence spectrometer, the design of an NMR surface coil array suitable for optimizing the SNR from a specific portion of the in vivo turtle heart, the design of a double-tuned NMR probe for in vivo phosphorus spectroscopy, specialized preparatory techniques developed during the course of this work, and the combined, simultaneous collection of ³¹P NMR and optical fluorescence spectroscopy data from an in vivo animal preparation. Preliminary experiments verifying the function of each component and technique of the experiment are presented, and finally, data collected using western painted turtles in a 2T, 30 cm horizontal bore superconducting MRI magnet is presented and discussed with respect to known physiological principles.

Finally, although this research is targeted toward development of a probe for a specific dual spectroscopy application, it should be noted that simultaneous optical and NMR spectroscopy are of value in other investigations as well. The design principles and compatibility issues addressed here will be useful in development of probes for other applications such as chemical reaction kinetics or brain metabolism studies.