Commentary on the Pleasures of Solving Impossible Problems of Experimental Physiology

Abstract

This commentary presents a series of examples of "impossible experimental problems" that we have encountered over the years in addressing various challenging questions in physiology. We aim to show how stimulating the challenges of physiology can be and demonstrate how our naive invocation of methods from disparate fields of science and engineering has led to delightful resolutions of physiological challenges that were utterly new to this intrepid interdisciplinary researcher. SEDUCTION BY SOME IMPOSSIBLE PROBLEMS OF PHYSIOLOGY Chemical Kinetics in Physiology by Fluorescence Correlation SpectroscopyThis innocent materials scientist was dragged into molecular physiology in the late 1960s, when I was presented with the challenge of understanding how the genomes of the recently defined DNA double helix could be separated into individual DNA strands for transcription. Physical biochemist Elliot Elson addressed this question when he joined the Cornell faculty in 1968, before the enabling transcription enzymes were characterized and understood. My relevant perspective then was derived from the excitement of measuring (for the first time) the fascinating phase-phase interface fluctuations near continuous phase transitions of molecular mixtures as well as intraphase fluctuations in 3 He-4 He isotopic mixtures at temperatures down to −272.5• C and in quantum superconductors to test the forthcoming theories of their fluctuations and statistical thermodynamics of their phase transitions (cf. 1-6). By comparison, the biophysical challenges of cellular physiology looked feasible even at sparse biomolecular concentrations (thus displaying my ignorance of the complexity of biochemistry).That naive innocence led us to develop Fluorescence Correlation Spectroscopy (FCS) (7,8) But our invention was temporarily confounded by its instrumentation difficulties and so, after approximately 10 publications, we abandoned FCS for a decade. The faithful had to wait nearly 20 years before adequate infrastructure in computer capability, laser stability, and optical components was developed. It is ironic that our use of other indicators for correlation spectroscopies continued unabated. Now the use of FCS is almost a pleasure, yielding hundreds of research papers per year, which we finally recognized when the annual citations of our own FCS papers exceeded 360 during 2004. We now find FCS to be functional in cells as well as on cell membranes and broadly applicable in a variety of geometries (cf. 9-17).FCS did lead us to recognize early on that the sensitivity of fluorescent markers, which can reach single-molecule sensitivity, provided a universal key to molecular signals in molecular cell physiology. We turned to measurement of molecular mobility on the lipid plus protein membranes of living cell surfaces, a challenge not then understood in the light of Singer and Nicolson's (18) fluid mosaic model and still controversial in the age of the mysterious lipid rafts, but improving, as we shall see. Recently our research has led us back...

Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice

“…1996). In fact, the shapes of the recovery curves according to these models are essentially indistinguishable unless data are available over more than five decades in time (Webb, 2006). Other techniques, such as SPT of individual molecules, may be needed in addition to investigate the nature of the immobile fraction as detected by FRAP.…”

Nanofluidic structures for single biomolecule fluorescent detection

“…Membrane-bound receptor binding kinetics, lipid domain dynamics, and membrane-bound ion channel behavior, for exam-ple, may all be studied using fluorescent detection. 7,24,25 In addition to offering roughly an order of magnitude shallower illumination depth than does TIRF microscopy, ZMWs provide high spatial resolution in the transverse plane. Apertures as small as 50 nm can be used to investigate lipid diffusion and binding kinetics for a receptor on a liquid-disordered phase membrane.…”

“…Total internal reflection fluorescent microscopy (TIRF) 3 and near field scanning optical microscopy 4,5 rely on decaying evanescent waves to create a subdiffraction limited focal volume. Twophoton excited fluorescence is also available as a method for shrinking illumination volume and increasing signal-tonoise, 6,7 and can be used noninvasively on living organisms. 8 Nanofabricated, fluid-filled structures have been used to decrease illumination volume, both increasing signal-to-noise and the working concentration at which single molecule experiments may be run.…”

Quantification of receptor targeting aptamer binding characteristics using single‐molecule spectroscopy

“…FCS utilizes nanomolar concentrations of fluorescent probes to ensure that primarily one molecule's trajectory is monitored at a time as it flows through the sub femtoliter confocal volume providing single molecule resolution (Elston and Webb, 1975; Koppel et al, 1976; Magde et al, 1972). With the development of faster data acquisition systems, more stable lasers, and detectors, including avalanche photodiodes (APDs) and photomultiplier tubes (PMTs), the role of FCS in the study of chemical kinetics is becoming more prominent (Webb, 2006). FCS has also been applied in studying singlet and triplet dynamics and in the study of cell membranes including localized regions within the membrane (Pramanik and Rigler, 2001; Vukojevic' et al, 2005; Widengren et al, 1995; Wohland et al, 2001).…”