Contains research objectives and reports on seven research projects.Whitaker Health Sciences FundFrancis L. Friedman ChairNational Institutes of Health (Grant AM-31546)National Institutes of Health (Grant AM-25535)International Business Machines, Inc

Essig, Alvin

French, Anthony P.

Jarrell, Joseph A.

King, John G.

Rosenthal, Stanley J.

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Molecule Microscopy19. Molecule MicroscopyAcademic and Research StaffProf. J.G. King, Prof. A.P. French, Dr. A. Essig, Dr. J.A. Jarrell, Dr. S.J. RosenthalWhitaker Health Sciences FundJohn G. King, Joseph A. Jarrell19.1 Research ObjectiveOur principal research objective for the last decade has been to develop a new microscopy thatcan show the location and binding energy of molecules or atoms on or near the surface of asample. We believe that molecule microscopy (MM) will be of unique importance in explainingdiverse phenomena that take place on surfaces of samples of interest in biological and materialsscience without requiring samples that are orderly or flat as are required with diffraction methodsor the newly developed scanning tunnelling microscope (STM). The surface-specific chemicalcontrast mechanism and potentially high spatial resolution of the MM will make it a valuableextension of the various forms of electron microscopy and microanalysis now in use.19.2 Applications of Molecule MicroscopyWhat samples should we examine and what information should we seek initially? We chose toexamine the distribution of water on biological materials, arguing that the presence or absence ofwater would give information about the surface beneath, in particular the electric charge anddipole distributions on the surface which must play important roles in the specific interaction oftissue with ions, macromolecules, antibodies, viruses, etc.In previous studies we have shown that water binds differently to representative frozen lipid,carbohydrate, and protein samples, so that logical initial samples would be frozen artificial lipidmono- and multi!ayers with and without protein. Later we will examine natural material includingcells, cell ghosts, and freeze-fracture samples. In each case we have identified localcollaborators to supply samples and help with sample handling and interpretation of observations.Some freeze-fracture observations of cells, in which water was found to crystallize preferentiallyor "decorate" certain sites forming ice crystals large enough to be seen by electron microscopy,suggest that our approach is valid.We have also considered in the past various applications of the MM to materials science. Theseare now being explored in detail, and we expect to propose studies to be carried out, withappropriate collaboration, of hydrogen transport in metals, and of the interaction of foreign atomsRLE P.R. No. 127115Molecule Microscopywith metal surfaces. The results of such studies would be useful in furthering understanding ofthe mechanisms of hydrogen embrittlement, corrosion and stress-corrosion failure.19.3 Kinds of Molecule MicroscopeMM works by detecting the spatial variations of the emission of neutral molecules from thesample. Various methods of detection are possible, spatial resolution can be achieved in differentways and molecules (of many species, parts of the sample, coming through the sample, or placedon it as a stain) may be emitted from the sample as a result of various stimuli. The earliest MMused a field ionizing point and electron multiplier as a detector, a pinhole to achieve spatialresolution, and a sample which emitted molecules continuously ("self-luminous"). The MM underdevelopment for the biological and metallurgical work mentioned above (called the NanometerResolution Scanning Desorption Molecule Microscope) will use a relatively large (1 fpm) fieldionizing tip which oscillates from above the sample, where it collects neutral molecules emitted bythe sample, to the entrance of a time-of-flight mass spectrometer where the collected moleculesare field desorbed and field ionized. A pulsed beam of focused electrons from an appropriateelectron gun causes the desorption of neutral molecules from the sample surface.This system has the following advantages. The cross-sections for ESD of neutral moleculesfrom samples of interest are large. The ESD is produced by a focused beam of electrons whichcan be of a size comparable to that achieved in EM thus affording high spatial resolution. Therelatively large field ionizing tip close to the sample gives a large solid angle for collecting neutralmolecules. The sticking coefficient of neutral molecules on the tip is near unity. The fielddesorption and field ionization processes make the detection of the neutral molecules highlyefficient.In the MM described above the sample must be in ultra high vacuum, which is permissible forfrozen biological material at 77 K, or metallurgical samples. For live tissue, or some corrosionstudies, the sample must be bathed in an appropriate fluid. We have developed a MM in which thesample is scanned or probed with a micropipette connected to an ionizer and mass spectrometerfor identifying and detecting molecules emitted by the sample and picked up by the micropipette.Unique information can thus be obtained concerning fluxes of small volatile molecules in tissue.This instrument is called the Scanning Micropipette MM (SMMM).RLE P.R. No. 127 116Molecule Microscopy19.4 Nanometer Resolution Scanning Desorption MoleculeMicroscopyFrancis L. Friedman ChairJohn G. King, Joseph A. JarrellProgressWe have undertaken the detailed design of this instrument. We have also continued thedevelopment of various subsystems, principally the high voltage pulse generators and oscillatingneedle driver systems. We have also made a direct measurement of the cross-section for ESD ofneutral water molecules confirming the indirect results of previous work.19.5 Scanning Micropipette Molecule Microscopy (SMMM)National Institutes of Health (Grant AM-31546)Joseph A. Jarrell, John G. KingThe major goal of this research is the study of water transport at the cellular level acrosshormonally responsive tissues that model the distal tubules of the mammalian kidney, specificallyamphibian urinary bladder. The elucidation of these processes is basic to an understanding ofthe cellular and molecular mechanisms by which the kidney maintains salt and water balance inman.During the past year, we have developed a new flow chamber for the SMMM (boundary layers inthis chamber can be as thin as 10 tim) and a technique for casting composite cellulose acetatemembranes to provide support for our experimental samples. These membranes are 25-30 [mthick except for a central region, 1 mm diameter, which may be as thin as 3 jPm.We have also done preliminary experiments to examine the effects of quin2 (a fluorescentcalcium chelator that has been introduced into some biological cells nondestructively) on thehydro-osmotic vasopressin response in amphibian urinary bladder. If, as has been hypothesizedby others, a change in intracellular calcium activity is an important link in the chain of biochemicalevents that are initiated by vasopressin binding, then the presence of quin2 would delay or bluntthis response. Our results so far are inconclusive. We plan to repeat these experiments withradioactively labelled quin2. This will enable us to make well-defined measurements of thecellular uptake of the chelator.PublicationsJarrell, J.A. and C.A. Rabito, "Development of Gap Junctions Analyzed by Metabolic Cooperationbetween Ouabain-Resistant and Wild-Type LLC-PK1 Cells," submitted for the 1985FASEB meetings.RLIE P.R. No. 127117Molecule Microscopy19.6 Scanning Micropipette Molecule Microscopy - BostonUniversity School of Medicine CollaborationNational Institutes of Health (Grant AM-25535)Stanley J. Rosenthal, John G. King, Alvin EssigA SMMM has recently been made operational and applied to tissue transport studies with thefollowing initial results. sampling tracer concentrations above a transporting Necturusgallbladder. Experiments were conducted with a probe of drawn quartz with tip dimensions 4.5[Lm I.D., 11 pm O.D., plugged with DMS in a thickness less than 0.5 pm and protruding beyond thetip. This probe provided an initial sensitivity of 7 x 10 -9 amps/mole/cc, which degraded to 2 x10- 9 amps/mole/cc after one week of use. Necturus gallbladder was dissected, rinsed, andquickly mounted with moderate stretching in a chamber exposing a tissue area of 0.22 cm2 . Themucosal bath contained a standard Ringer solution with 2.5 mM HCO 3 and either 100 mM sucroseor 10- 3 M EGTA. The small volume of serosal Ringer required was made using 50% H20 18The epithelium and probe were easily observed simultaneously under a Leitz microscope. Cellborders were discernible at 400X magnification. The positioning of the probe at a border or cellcenter was reliable as was the ability to lower the probe, touch the cell (as indicated by a cellshape change), and raise the probe 2 Ipm above the surface.The existence of unstirred surface layers was made tangible as the tracer signal dramaticallyincreased when a probe was within 300 [pm of the surface. Quantitative studies of theconcentration profile in this region are in progress.Four tissue were mounted and explored for localized sites of tracer water flux. The firstmounting had too much tissue movement for reliable probe positioning due to inadequatestretching of the bladder. In one tissue the serosal compartment was closed and sucrose addedto the mucosal bath to encourage a directional water flux, but hydrostatic pressure was poorlycontrolled so that net flux was uncertain. Under these conditions the concentration differencebetween junctional and cell-centered sites was insignificant, 0.03 ± 0.09 mM/cc (N = 15 that is,15 observations). We added EGTA to remove Ca +' , which has been reported to loosenjunctions. An opening in the cell continuum appeared and was scanned with the probe.A 0.5 mM/cc concentration increase was observed above that site, demonstrating that the probefunction had not been compromised in the above series of measurements.The need for careful control of hydrostatic pressure became obvious and, a manometer andcalibrated capillary tube were added. The pressure gradient could now be measured to within1/2 millibar and flow rates to within a few microliters per hour. A tissue exposed to a 5 mbar(serosal above musocal) hydrostatic pressure gradient responded with a 130 fl/hr-cm2 flow(s - m). Again junctional sites showed no significant concentration difference to withinRLE P.R. No. 127 118Molecule Microscopy0.3 mM/cc. The appearance of hemispherical blisters 50 pjm to 300 lim in radius was noted.These areas of tightly stretched cells showed no difference in junctional permeability. But theblisters were easily damaged when poked too hard by the probe. A defect thus produced showeda high water flow rate, as indicated by a high concentration (1.7 ± 0.4 mM/cc (N= 4)). As afurther demonstration of feasibility, we increased the pressure gradient to 14 mbar (s > m) andthe flow rate went up to 400 pl/hr-cm2. Sampling above three different junctional sites showed asignificant increase in tracer concentration as compared with that over cell-centered sites,although the variability was high. The mean difference was 2.2 ± 2.1 mM/cc (N = 6).In a final tissue we were able to control blisters (which might distort the unstirred layer) bykeeping the pressure below 1 mbar (s > m) and the flow rate less than 47 ml/hr-cm2; however anoverheating wire in the vacuum caused spurious noise and forced termination of this run. Thesetests show the feasibility of the approach and that quantitative results can be obtained fromcareful data accumulation.19.7 Desorption StudiesWhitaker Health Sciences FundJohn G. King, Joseph A. JarrellWe are continuing the experiments begun by D. Lysy and B. Silver on the thermal and electronstimulated desorption of water and other small molecules from biological materials for fourreasons. (1) To understand the contrast mechanism in molecule microscopy; (2) to measure thetotal water content of various samples; (3) to measure the binding energy of this water; and (4) todetermine the order of the kinetics of the desorption process. The importance of theseexperiments comes from the central role of water in biological processes at the molecular level.ProgressMost of 1984 was spent in apparatus improvement and training new students in an effort toestablish a routine to permit 100 desorptions per day instead of 10 or 1.19.8 Electrical Neutrality of MoleculesInternational Business Machines, Inc.Anthony P. French, John G. KingThe present experimental upper limit on any departure from neutrality of molecules isapproximately 10-21 of one elementary charge. This can be interpreted as a limit on theproton-electron charge difference, or on the charge on the neutron, or on some combination ofthese. The occurrence of innumerable transformations involving both leptons and baryons, andRLE P.R. No. 127119Molecule Microscopyour long-standing belief in the conservation of electric charge as a fundamental law of nature,certainly support the assumption of exact equality of charges of either sign on all elementaryparticles. Nevertheless, the possibility that there might be some minute difference betweenelectron and proton charges is something that cannot be ruled out on the basis of any existingexperimental evidence or theoretical scheme, and its existence would have profoundimplications.The fundamental interest of this question has led to a number of experiments by variousinvestigators. These experiments have involved three different approaches: (1) direct tests forany net charge in a volume of gas released from a container; (2) tests for a charge on an isolatedbody (analogous to the Millikan oil-drop experiment); (3) molecular-beam deflection experiments.In 1973 King and Dylla 1 pioneered a new technique based on the fact that an electric field,periodic in time, applied to a homogeneous medium will excite acoustic waves at the samefrequency if the molecules of the medium are not completely neutral. (This is separable from anyinduced polarization effects, which will occur at doubled frequency).ProgressThe apparatus described in RLE Progress Report No. 126 has been assembled and tested. Thefirst runs in air show that there is a fundamental frequency signal present that is ca.10 3 larger thanthe minimum detectable signal. We attribute this to forces arising from the interaction of theelectric field and frozen-in charges in the mylar rings used to separate the copper equipotentialrings. Other arrangements to avoid this effect are being studied. We have begun development ofhigh voltage audio-frequency sources with exceptionally low harmonic content as needed toexploit the intrinsic sensitivity of this technique.References1. H. Dylla and J. King, Phys. Rev. A 7, 1224 (1973).RLE P.R. No. 127 120

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