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CHAPTER 20 ANALYTICAL METHODS FOR LANDERS D. G. REA, Editor INTRODUCTION The scientific motivation and goals of exobiology have already been discussed at some length. To achieve these goals, biological laboratories must be landed on the surface and used to provide answers to the initial questions. Although the detailing of such an instrumental package is still in the future, it is of value to consider the various techniques that could be used in tackling the analytical problems. This section is intended to fulfill this function within the following framework. The outstanding subjects for exobiology can be grouped into three general categories: (1) The detection of the presence of life, and its characterization if present. (2) The characterization of any existing organic matter, with particular reference to those molecules and functional groups of importance to terrestrial biology. (3) The characterization of the inorganic and physical environment, including both the surface and atmosphere. The most important question, of course, concerns the existence of life on the planet. The investigator approaching this must consider the possi- bility of different levels of evolution of the Martian biota, and of varying 347

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348 MARTIAN LANDINGS: UNMANNED degrees of divergence from the terrestrial system. Because of its lack of definition, this question may be the most difficult to answer, an unfortu- nate circumstance in view of its importance. A more tractable problem is the characterization of any organic matter present on the surface or in the atmosphere. Any data will be of interest, since, even if life has never existed on Mars, their analysis will be of immense value in studying the chemical development of a sterile planet, and this relates in turn to the origin of life itself. However a complete analysis would be beyond the scope of the biological landers that can be foreseen for the next decade or two. Accordingly, in view of the basic goal of the search for life, the methods discussed here will of necessity be oriented towards compounds and functional groups of known bio- logical interest. Falling in this category are, for example, the amino acids, proteins, purines, pyrimidines, carbohydrates, nucleic acids, liquid water, the secondary amide and the phosphate linkages. It is doubtful whether the presence of such molecules and functional groups would ever be construed as an unequivocal sign of life. But they would be very sug- gestive and of the most direct interest if they were found in objects that had already been characterized as living by other means. Finally there is the problem of determining the nature of the physical and inorganic environment. Experiments designed to elucidate this are the most likely to produce results and such results will be important in interpretations of direct observations of any biological activity. Despite the lower priority accorded these "non-biological" measurements some space in the landers must be devoted to answering some particular ques- tions relevant to the overall problem. These cover such topics as the flux of ultraviolet radiation and of charged particles at the surface, the partial pressures of water vapor and oxygen, the principal mineral components of the top few centimeters of the surface and any minor elements which may play a biological role. In approaching this project the study enumerated the analytical tech- niques that appeared to be the most fruitful for the task. Requests were then made of a number of outstanding scientists, asking them to write a brief note on the applicability of the technique in which they were par- ticularly competent. They were asked to stress the methodology and not to concern themselves unduly with the engineering aspects. Points to be emphasized were the interpretability or ambiguity of the method's indi- cations and quantitative statements on the limits of sensitivity, constraints on type and size of samples and requirements for sample preparation and treatment. The resulting contributions were edited by a subcommittee of the study and details on some existing flight instruments added. They were subse-

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Analytical Methods for Landers 349 quently examined by the entire group and final amendments made. In view of the editing process the consulting scientists who contributed the working papers should not be held responsible for the final versions that appear below. This is not intended to minimize their contributions, which were of the utmost value in lending authority to the discussions. Their willingness to expend valuable time, often on short notice, is acknowl- edged with the most sincere appreciation. The topics that were treated in this fashion are listed below, together with the names of the contributors; a brief note on morphological criteria is also appended. 1. Separation Methods and Sample Preparation, R. Bock, S. R.Lipsky, F. J. Stevenson and M. J. Johnson. 2. Atomic Spectroscopy, J. Conway. 3. Neutron Activation Analysis, H. Mark, J. Waggoner and C. D. Schrader. 4. Electron and X-Ray Fluorescence, C. C. Delwiche, K. Fredricksson and A. Metzger. 5. X-Ray Diffraction, H. H. Hess, R. C. Speed, D. B. Nash, N. J. Nickle, R. Pepinsky, I. Barshad and C. C. Delwiche. 6. Sensitivity of Fibers to the Physical and Chemical Environment, H. P. Lundgren. 7. Gas Chromatography, J. Oro, S. R. Lipsky, V. I. Oyama, G. R. Shoemake and A. Zlatkis. 8. Mass Spectrometry, K. Biemann. 9. Gas Chromatography—Mass Spectrometry, J. Or6, K. Biemann, R. S. Gohlke, S. R. Lipsky, J. E. Lovelock, F. W. McLafferty, W. G. Meinschein and R. Ryhage. 10. Infrared Spectroscopy, R. C. Lord, N. K. Freeman and C. Sagan. 11. Ultraviolet and Visible Spectroscopy, G. A. Crosby. 12. Fluorimetry, L. Stryer. 13. Optical Shifts in Dye Complexes, D. F. Bradley. 14. Nuclear Magnetic Resonance and Electron Paramagnetic Reso- nance, J. Shoolery and J. D. Roberts. 15. Colorimetry, A. D. McLaren and A. Novick. 16. Optical Microscopy, P. S. Conger. 17. Electron Microscopy and Electron-Optical Techniques, H. Fernan- dez-Moran. 18. A Note on Morphological Criteria for Recognizing Life, D. Schwartz. The list of methods presented, although rather lengthy, is not to be construed as definitive. Some techniques, such as optical activity and the

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350 MARTIAN LANDINGS: UNMANNED use of isotopes, are discussed elsewhere in this report. Of the other omis- sions, wet chemistry stands out as having real possibilities but since any treatment of it would almost necessarily be encyclopedic, and since its applications are widely appreciated, it was not included. Our intention in compiling this section was to be provocative and stimulating, not ex- haustive. Accordingly, the possibilities discussed range from those readily achiev- able even now, to those that will require very extensive development before they are ready for incorporation in lander payloads. Equipment ready for flight or of the "breadboard" type has already been constructed to carry out several kinds of measurements, for example, neutron activation analy- sis, x-ray diffraction, gas chromatography, mass spectrometry, infrared spectroscopy, electronic spectroscopy in the visible and ultraviolet and optical microscopy. Some of these have been designed to analyze the lunar surface, while others have been intended for studying the terrestrial atmosphere or for remote observations. All are at such a stage that adaptation or development into equipment capable of performing on the Martian surface is straightforward. In other cases, however, such as nuclear magnetic resonance and electron microscopy, major technological developments are required before they can be considered seriously. Both methods, under the limits of weight that will probably apply for the first several missions, are dependent on the development of techniques for operating superconducting magnets on the Martian surface after a 6- months' flight time. Such developments will consume many years of in- tensive work, but this should not stand in the way of examining their potentialities. An important constraint that has been generally omitted from the vari- ous papers is that of the data transmission capacity. Early missions may be able to send data back at a rate of only 1 bit per second. To send a picture taken by a camera, optical microscope, or electron microscope, and containing, say 107 bits of information, would require about 120 days. This is inordinately long and would pre-empt the majority of the telemetering capacity to the detriment of other experiments. It is to be hoped that more extensive knowledge of Martian surface conditions will permit the use of more efficient antennas and, with improved power sup- plies, allow a rapid increase in data transmission rates. The sending of pictures of high quality might then become a reality. A final word must be said about what is probably the most critical prob- lem facing us at this time—the acquisition and preparation of a sample. Some of the techniques do not require a sample, e.g., inelastic neutron scattering, but the majority do. Moreover, to make the output data meaningful a certain amount of sample processing is demanded. These

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Analytical Methods /or Landers 351 problems are treated briefly in the section on Separation Methods and Sample Preparation. They are of such importance, however, and are in such an elementary stage of solution, that a major effort must be ex- pended in the immediate future to solve them. The scientific goals of the mission will never be satisfied by the most sophisticated and ingenious collection of experiments if the sampling device is ineffective or if the necessary sample treatments are not properly applied. After reading the Analytical Techniques section it should be clear that the problems are many and that they range from the trivial to the pro- found. At the same time it is hoped that the feasibility of resolving them has been demonstrated and that scientific observations of the greatest significance can be carried out on the Martian surface and its components, whether organic or inorganic, biogenic or abiogenic. 1. SEPARATION METHODS AND SAMPLE PREPARATION Physical and chemical processes for separation of Martian solids, gases and solutes into simple, convenient or better defined sub-classes will find utility as necessary sample preparation before examination by sophisti- cated analytical methods. If the results of simple separation processes are also observed, useful data can be accumulated about the Martian en- vironment. Examples of suggested questions and methodology are given in this section. Many of the elegant analytical techniques discussed in other sections of this report are less definitive or inapplicable if the sample provided them is an extremely complex mixture or if the class of com- ponents to be detected is a minor constituent of the sample. Many other methods require that the sample be subdivided physically into thin sec- tions, small uniform granules, volatile components or soluble components. These sampling problems are closely related to those of separation and will be discussed in the last half of this section. Specific Questions Answerable By Separation Methods 1. What is the distribution of particle size and particle density found in Martian soils? 2. What is the electrostatic charge of solutes extracted from Martian soil or putative biological objects? Can they exist as polyelectrolytes and as ampholytes with charge dependent upon the acidity of the solvent? 3. What are the fractional solubilities or volatilities of Martian soils or other objects of interest?

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352 MARTIAN LANDINGS: UNMANNED It is evident that answers to these questions will aid in design of future chemical experiments needed for detailed studies of Martian chemistry and biology. These data are essential for any description of the Martian environment. The first question can be probed by simple dry sieving, membrane filtration or gel filtration of soil extracts, and by flotation in solvents of differing density. Each of these methods permits simple sample acquisi- tion, miniature apparatus, and quantitation of the materials separated. The results can be relayed economically in terms of numbers of bits to be transmitted. None of these methods will give so complete a description of the sample as does the automatic particle sedimentation analyzer [Zieg- ler et al., 1964]. In this device, a pressure transducer in a settling tube transmits digitized values of accumulated sediment at predetermined times of settling. If two of these devices contained solvents of differing density the data could be analyzed for particle size distribution and density distri- bution. Direct microscopic observation and Coulter counter size distribu- tion analysis should be evaluated as competing methods for obtaining these data. The density of selected macroscopic objects can be determined in a gas pycnometer. The density range of Earth objects of biological origin is distinctive and in contrast to objects of geological origin. If the limitation on number of data bits transmitted does not rule out video observations at the macro- and microscopic level, the video descrip- tion of particle sizes would be preferred. The second question can be probed by ion exchange adsorption, dialysis through ion exchange membranes or electrophoresis. In each of these approaches the electrolyte or polyelectrolyte behavior of the Martian solutes would be examined in polar solvents (water, formamide and di- methylsulfoxide) in the presence and absence of added acids and bases. Each of these methods can be adapted to simple, durable and miniature devices but the ion exchange adsorption process appears to be both simplest in application and capable of extension to yield the greatest selectivity. The ways of applying this technique are so varied that considerable study and testing on known soils should be conducted. The equilibrium distri- bution of soil solutes among a set of differing ion exchange papers im- mersed in the same medium can be a very simple but informative experi- ment. The quantitative uptake of solutes by a single exchange material will vary as the pH and ionic strength of the solvent is altered. This phe- nomenon can be observed in an array of batch adsorptions or in a single adsorption followed by an array of batch elutions. The latter can be de- signed to give data of higher information content because it is a higher resolution separation process. The third question can be probed by sealing the Martian samples in a

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Analytical Methods for Landers 353 set of containers and then breaking a vial of solvent previously enclosed in that container. The group of solvents chosen should be of low melting point, known to be good solvents and should include representatives of low, medium, and high dielectric constant. After the sample and solvent have been held at a known temperature for a considerable period of time, the connection between an evacuated vial and a porous Teflon filter in the bottom of the solution chamber could be established by breaking a seal. After the vial has filled, it should be separated from the filter, the solvent allowed to evaporate and the mass of the previously weighed vial deter- mined. Alternative methods for detecting the amount and identity of material dissolved should be considered. Devices of this type may be needed for sample processing for such diverse operations as gas chromatog- raphy, optical rotation or infrared spectroscopy. Fundamental Principles and Limitations of Selected Separation Methods The separation methods likely to be utilized in the Martian lander experiments are widely used in chemistry and biology and their power and limitations have been explored. Sedimentation, extraction, electrophoresis, chromatography and other wet separation methods share the limitation that no single solvent is applicable to a wide range of materials. Experi- ments that depend on dissolving the test substance may have to be designed with multiple sets of solvents or as multiple packages each with a different solvent. Vapor phase experiments usually have a wider range of applica- bility in that a broad scan of temperature can successively vaporize or pyrolyze—or both—most chemical species. As will be discussed under sample preparation, this thermal volatilization process is a broad range method of preprocessing for many experiments that requite a sample in the vapor phase. Theoretical studies have indicated that liquid phase chromatographic systems have potentially higher resolution than gas chromatographic pro- cedures. This resolution has not been obtained in practice and the proper- ties of sensitive detection, applicability to a wide range of substances with a single solute (carrier gas)—adsorbant combination and convenience of sample introduction and regeneration of supplies (carrier gas) make gas chromatography the method of choice for separation of complex mixtures. Procedures for Sample Acquisition and Preparation Any sample acquired for analysis should be taken from a definable region of Mars, a known depth of penetration into the soil or height above the soil and should be acquired at a prescribed time of day and season.

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354 MARTIAN LANDINGS: UNMANNED The range of temperatures and pressures during sample acquisition and the intensity of chemical operations during any processing should be known so that judgment may be made on the probability of melting, destruction of morphology, or racemization of optical activity. Video observation of the region from which samples are taken will be a powerful aid to any subse- quent interpretation of results. While many Earth sampling techniques collect large samples, comminute them and mix to randomize before taking a small portion for analysis, the variability thus obscured is an important datum for exploration of Mars and any improvement in statistical weight should be obtained by repeated sampling rather than mixing. Selection of a single object after video or tactile perception can be a powerful means of obtaining a sample of great interest, even though it is apparent that the object is not to be considered representative of the properties of the land- scape in general. Certain processes are of general applicability in that they permit acquisi- tion of a sample of limited size range, density range, of minimally dis- turbed morphology or in an appropriate vapor or solution phase. Drilling a simple core sample followed by extrusion, possibly with simultaneous sectioning, can lead to a good definition of place of acquisition. If the soil is friable, it may be necessary to fix the soil with plastic or low melting waxes which are infused into the core and permitted to harden before sectioning. Particle size selection can be accomplished by using a Venturi tube for sample pickup so that large aggregates are not acquired. The sample size can be narrowed further by passing the pickup gas stream through a cyclone cone dust collector or through a small gas centrifuge with impeller vanes to accelerate dust particles so that they leave the gas stream. Dry sieving of bulk samples or of objects that have been crushed may be useful. Micro-coring and surface sectioning or grinding may be found to be more precise and controllable operations than crushing and sieving. Section by flotation and sedimentation is dependent on density alone if equilibrium methods are used, but on density and shape if rate of sedi- mentation is allowed to influence the fractionation. A simple, equilibrium flotation separation can be effected by dispersing the sample in a low density solvent, allowing ample time for sedimentation and drawing off the liquid including all objects floating in it. A new solvent of significantly higher density is now added and the process repeated until the density range of interest has been covered. Components from a crude sample or from one simplified by any of the above processes may be made volatile by simple slow heating. The vapors produced can be introduced directly into a mass spectrometer, infrared

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Analytical Methods for Landers 355 spectrometer [Bartz and Ruhl, 1964] or a specific detector designed to search for certain vapors (water, COL., NH3), or may be condensed in a form appropriate for convenient application to other analytical techniques. If the vapors are condensed on the surface of a thermoelectrically cooled film or plate, the condensate can periodically be transferred to an infrared spectrometer or a polarimeter for observation. If the sample vapors are passed through an indium capillary chilled over a small region, the capillary may be crushed (swaged) periodically to entrap the condensate in a con- tainer convenient for transfer to a gas chromatogram or mass spectrometer. The indium capillary is placed in a heatable receptacle and melts at 153°C to release a concentrated sample for analysis [Wilkins Instrument Co., 1964]. If the vapors are condensed on a chilled silica rod, the rod may be transferred to an attenuated total reflectance device for ultrasensitive qualitative detection of its infrared spectrum [Harrick, 1963]. The same sample trapping devices may be employed on the effluent stream of a gas chromatogram. The thermal volatilization and pyrolysis experiment gains value if it can be coupled with measurement of the change of mass of the sample as the temperature increases (thermogravimetry) and of the heat input needed to cause the temperature rise and volatilization [Watson et al., 1964]. A detailed discussion of these techniques is given in another section of this chapter. Samples that are later to be examined for optical rotation need addi- tional care in that the temperature, acid and base strength of solvents and surfaces of contact should be chosen to minimize the degree of racemization of any optically active compounds present. This is not difficult to manage but is a precaution which must be borne in mind during design of separa- tion processes. The problem of introduction of samples into an evacuated chamber is shared by several analytical procedures. Design of simple, efficient ports for entry into vacuum chambers will be essential for the Mars mission. Be- cause of its simplicity, the condensation of vapors into a swagable indium tube should be considered for atmospheric sampling and subsequent infra- red or mass spectral analysis. The finely divided samples, thin sections of fixed soil cores or of objects and microobjects discussed above can be examined by x-ray fluoroscopic, x-ray diffractometric, electron microbeam [Buhler, 1964] and electron microscopic methods. Of these methods, only electron microscopy con- ventionally uses samples which have been stained or shadowed. These operations can now be avoided if the Westinghouse or Argonne versions of scanning electron microscopes [Westinghouse Electric Corp., 1964] are

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356 MARTIAN LANDINGS: UNMANNED employed. The simplification of sample processing should be considered as a reasonable exchange for the slightly more complex instrumentation in the newer electron microscopes. Apparatus for Spacecraft A vacuum aerosolizer with dust collection impeller action has been de- signed and constructed in a form near that for flight testing. It is now a component of the Multivator project under Lederberg at Stanford. The vacuum cleaner method of sample collection is employed in the bread- board models now constructed for the Wolf Trap biological probe developed by Vishniac at the University of Rochester. A sampling device has been built at flight scale breadboard stage for the Gulliver project under Horo- witz at California Institute of Technology. In this device a chenille string is ejected from the landed probe and upon dragging across soil, entraps small particles and drags them back into the probe when the string is rewound. This principle can also be used for collecting samples to be pyrolized if the chenille is fabricated from stainless steel or glass fibers. Design of an atmospheric gas collector for infrared spectroscopy of Martian atmospheres is under investigation by Pimentel at Berkeley. Core drilling devices have been designed and constructed for the Sur- veyor moonshot. The sampling device is near flight capability for simple coring operations. REFERENCES Bartz, A. and H. Ruhl (1964), Rapid Scan Infrared Spectrometer. Anal. Chem. 36, 1892. Buhler, J. (1964), The New Era of X-ray Analysis and Control. Instruments and Control Systems, 37, 77. Harrick, N. J. (1963), Attenuated Internal Reflectance Spectroscopy. Ann. N. Y. Acad.Sci. 101,928. Watson, E., M. O'Neill, J. Justin and N. Brenner (1964), A Differential Scanning Colorimeter for Quantitative Differential Thermal Analysis. Anal. Chem. 36, 1233. Westinghouse Electric Corp. Tech. Bull. (May 1964), A Scanning Electron Microscope. Box 8606, Pittsburgh, 99-362. Wilkins Instrument Co. (1964), An Indium Capillary for Introduction of Samples into Heated Zone of Column. Previews and Reviews for Gas Chromatography, Box 313, Walnut Creek, Calif. Ziegler, J., C. Hayes and D. Webb (1964), An Automatic Particle Sedimentation Analyzer. Science 145, 51.

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357 2. ATOMIC SPECTROSCOPY Atomic spectroscopy would be able to analyze the atmosphere and sur- face of Mars for most of the elements present. Of the 103 elements presently known, all but six have observed spectra. The great majority of these elements has lines in the easily available regions of the spectrum. The sensitivity of detection varies according to the accessibility of the most sensitive lines to observation, but a good working estimate of sensi- tivity for most of the elements is between 0.1 and 0.01 micrograms. In methods, such as those using the arc, where one commonly uses a milli- gram or so of sample, this is a ratio of 10,000 to 1 (of matrix to detecta- bility limit). In spark methods, where the sample is at most 100 micro- grams, the ratio is 1000 to 1 and with laser excitation where the sample is between Vi and 1 microgram the ratio is from 10 to 1, to 100 to 1. In cases where the sample is 50 micrograms or less, there is usually no inter- ference of one element with another, or rather, there are almost always some sensitive lines of an element that do not interfere with lines of other elements. There are several methods to record the spectrum which would be then transmitted to Earth. One possible method is to record the spectrum on photographic film although the problems of developing the film may be severe. This film is then read at fixed intervals (5 or 10 microns) and the distance and intensity transmitted. These data can be used as input to a computer which constructs the entire spectrum, finds the peak position of the lines and by comparison with standard wavelengths gives the wave- length of every line on the plate. The corresponding relative intensity is also obtained. A second method is to have a number of photomultipliers fixed on pre- selected lines. The phototubes are read out in order and an intensity for a given position recorded. In large commercial installations 50 to 70 tubes have been used. However, if more than 15 tubes are used, maintenance of optical alignment is difficult. A third method which has not been very extensively investigated is that of using TV cameras to record the spectrum. Considerable work would be required but this method is promising. Sampling is one of the greatest problems in spectrochemical analysis. In the laboratory, much handling of the sample is usually required to pre- pare it for analysis. In most cases the sample is weighed and transferred to electrodes, but a standard volume sample could also be used. The spectrum is excited by passing electrical discharges between the electrodes. Recently, lasers have been used to excite spectra. In this case the sample does not have to be placed in an electrically conducting electrode; it has

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416 MARTIAN LANDINGS: UNMANNED that a systematic electron optical survey can be carried out on an ultrathin section only 100A in thickness and not more than a few square microns in area, which is several orders of magnitude smaller than the requisite light microscopy samples of several hundred thousands A thickness and scores of square microns in area. This is particularly relevant to the problem of being able to sample not only the atmosphere and surface of Mars, but perhaps more importantly, the subsurface and deeper strata of Martian soil, where we may expect to find the most valuable clues on the evolution of life. An electron microscope equipped with a miniaturized ultramicrotome incorporating a diamond knife [Ferndndez-Mordn, 1962; 1964; 1953] or a diamond drill for production of ultrathin sections, which can be read off directly with the attached electron microscope, would serve all of these required functions in a practical and efficient way. As will be described later on, these systems would not in any way replace, but in fact ideally supplement, the light microscope systems now contemplated. In the following, a brief outline will be given of the distinctive methodo- logical features of electron microscopy in terms of the relative interest that this approach should attract in dealing with the basic problems. Only the essential engineering details will be presented of the specific project em- bodying a miniaturized electron microscope with coupled preparative de- vices for sampling, preparing, sorting, and telemetering of specimen data. Finally, some of the promising approaches in the development and imple- mentation of these proposals will be surveyed, with particular emphasis on the priority assigned to this project which merits a determined effort. Miniaturized Electron Microscope—Vidicon Systems Methodologically, electron microscopy and related electron optical tech- niques are of unique operational value in exobiology and space investiga- tions in general, for the reasons discussed below. The conditions under which a sample is examined in electron micros- copy—high vacuum, electron-beam and ion irradiation, thin specimens (100-1,000 A), and in some instances low temperatures, are very similar to conditions encountered in space. Successful application of the high spatial resolution of the order of 6 to 8 A reproducibly attainable with modern electron microscopes, now permits direct visualization of structural organization down to the molecular level, and in favorable cases of the array of atoms in crystalline lattices. In fact, this unique possibility of obtaining data, which is directly related to a lim- ited number of atoms or molecules only, and is thus well above the level of statistical uncertainty commonly associated with indirect analytical meth- ods, is of particular importance in the study of the structure and composi-

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Analytical Methods for Landers 417 tion of submicroscopic particles, such as those of interstellar matter with diameters of a few tenths of a micron down to 10 to 100 A. Moreover, electron optical and related microprobe analytical techniques are at present the only adequate methods for examination of gaseous and solid com- ponents as they occur in the extremely high dispersion, ultrahigh vacuum and low temperatures encountered in outer space, and in certain planetary environments. The special techniques that have been developed for electron microscope studies of specimens are actually readily adaptable to examination of extraterrestrial matter, because we are dealing essentially with the same problems. Thus, the use of thin graphite or single-crystal, stable coherent films, of plastic films, replication, shadow-casting, etc., are suitable for these purposes. In this way, and despite the numerous unsolved technical prob- lems, it should be possible to obtain and examine, as well as perform chemical analysis (electron microprobe analysis, electron diffraction, corre- lated physico-chemical and mass-spectrometric analysis, etc.) of material from space, and from lunar and planetary probes in a way that cannot be achieved by any other known technique. It should be pointed out that most of the relevant fine structures of the specimen materials would be well below the resolving power of present light and x-ray microscopes. Electron microscopy and related electron optical devices are essentially extensions of television techniques. They can, therefore, be readily adapted directly to television cameras such as those used on Surveyor or Ranger spacecraft adapted for use in the exploration of Mars. A television survey camera essentially contains the same basic elements (electron source, electromagnetic focusing lenses, vidicon tubes, etc.) as an electron micro- scope. It would only be necessary to add certain components in order to convert such a television survey camera into a simple type of electron microscope which would considerably extend the range of resolution of such a camera from the present meter range to the sub-micron range. In fact, the proposed use of miniaturized electron microscopes represents a logical supplement and extension of the "Vidicon Microscopes" for plane- tary exploration as suggested by Dr. Joshua Lederberg of Stanford Uni- versity, and now being developed in Dr. Gerald Soffen's laboratory at the Jet Propulsion Laboratory of the California Institute of Technology [Quimby, 1964]. Even a simple miniaturized electron microscope (cou- pled directly and preferably in the same vacuum system with the television survey camera) would have a resolving power of the order of a few hundred Angstrom units, which is well below the useful resolving power (of about 3,000 to 2,000 A) of the best light microscopes presently available. More- over, instead of using a vidicon tube which works on a photoconductive principle, the electron image of such an electron microscope can be con-

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418 MARTIAN LANDINGS: UNMANNED verted directly by suitable image in ten s. tiers [Maine and Casslett, 1961] into electrical signals which, when highly amplified and converted to a fre- quency-modulated signal, can be sent and received on Earth. Alternatively, as discussed further on, the electron microscope image could be recorded directly on special high-resolution and ultrathin photographic emulsions, suitably demagnified by a factor of several thousand times. In this highly condensed form the electron microscope image could be either read out and telemetered back to Earth, or conceivably sent back directly in suitably packaged form for retrieval on Earth (see supplement to this report). It is of course possible and actually advisable to combine the automatic light microscope system proposed by Lederberg and his associates with this type of miniaturized electron microscope system, in order to achieve a mutually supplementary, step-wise analytical processing of the samples. In many ways both systems complement each other and can be used either combined or singly in appropriately designed planetary missions. Although all present commercial electron microscopes are considerably larger, weighing over a ton with attached power supply cabinets, it is con- sidered feasible to reduce substantially the size and weight of microscopes with advanced techniques. Thus, by appropriate scaling down of the lenses, including the possible use of permanent magnet lenses which do not require a separate lens power supply, it should be possible to reduce the column to about 1 foot length or perhaps even less; and to combine this with appropriately miniaturized high-voltage pointed-filament electron sources and controlled power supplies. It should be pointed out that there have already been several successful attempts to produce commercially small, extremely compact electron microscopes, such as the RCA type EMT permanent-magnet electron microscope. The small table-model microscope designed by Reisner and Dornfeld [1950] has permanent magnet lenses and attains a resolution of about 100A. Electron microscope lenses have a very small useful numerical aperture (of the order of 100th radian), and their depth of field is therefore extremely large compared with that of light microscopes of high resolution where the numerical aperture is large (1 to 1.6). This large depth of field of electron microscopes is of particular interest because it makes possible the recording of "stereoscopic" views of objects having considerable extent in the axial direction. This would also considerably facilitate recording of sharp images by remotely controlled servo systems. General Design Features of a Simple Miniaturized Electron Microscope The schematic diagram (Figure 1) illustrates some of the general design features which can be incorporated into a miniaturized electron microscope

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Analytical Methods for Landers 419 SINGLE CRYSTAL POINTED FILAMENT 1 TO, (with S«fvo-motor) 1 o VIDICON TUBE OR IMAGC MTENSIFKI) Figure 1, Schematic diagram of miniaturized electron microscope for exobiology studies. I for detection of extraterrestrial life. The microscope column (with a length of 6 to 12 inches approximately) comprises a pointed filament source (of tungsten, or preferably rhenium for longer life) with a Schottky type of gun for T-F emission at accelerating voltages of 10 to 50 kV. One objective lens of the permanent magnet, standard electromagnetic, electrostatic or superconducting type is shown here. However, it may be necessary to use an objective and projector gap in series to obtain higher magnifications. The image is recorded either directly onto a vidicon tube with fluorescent screen, or onto an image intensifier of the solid state or scanned type [Maine and Cosslett, 1950]. Alternatively, the special type of ultrathin photographic films on tapes could be used for recording of the image either at high magnification or after appropriate dcmagnilication. The whole column assembly can be made very rigid and attached directly onto the television survey camera, or be a supplementary element to the vidicon microscopes currently being designed at the Jet Propulsion Laboratory. At present, two basic approaches are being pursued in trying to solve the difficult specimen mounting and preparation problems: (1) There is a possibility of effecting a continuous scan of ultrathin specimens (fine particles in aerosol suspensions, atmosphere samples, etc.) by sandwiching them between vacuum-tight, 200 to 1,000 A thick, single-

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420 MARTIAN LANDINGS: UNMANNED crystal mica, graphite, aluminum or beryllium windows. These windows would be transparent to electrons and, in this case, the microscope itself would be constantly kept under high vacuum which does not have to be broken in order to examine the specimens. This would also permit a continuous sampling of wet or partially hydrated specimens, and the use of very fine "ultratape" reels of indefinite length. The envisaged miniature objective pole pieces with a bore of 1 mm or less would be ideal for this approach. In the rarefied atmosphere of Mars this type of approach to specimen examination may prove to be particularly useful. A variant would involve the use of an impaction plate (as in the case of the vidicon microscope being designed in Dr. Soffen's laboratory [Quimby, 1964]) which can be periodically cleaned by suitable electro- static filtering devices. (2) Specimens could be collected on a traveling ultrathin ribbon of tan- talum or rhenium foil about 100 to 1,000 A thick and about 1 mm wide, with appropriate reinforcements and suitably etched slits or holes (1 to 20 microns in diameter). These thin metal tapes can be fed automatically into the microscope after having been exposed to the environment, in the same way as the transparent tape first proposed by Lederberg et al. [1961] or by the Gulliver biochemical probe device being developed by Dr. Nor- man H. Horowitz of California Institute of Technology and by Dr. Gilbert V. Levin of Haselton Laboratories, Inc. [Quimby, 1964]. The advantage of these rhenium or tantalum foils would be that no direct specimen sub- strate film is required, and that they can be re-used many times by appro- priate heating to destroy old specimens. This practice of heating to incan- descence in a vacuum has proved to be very effective in eliminating con- tamination. The "ultratapes" would be mounted on shields which can be operated by remotely controlled servo systems. A similar type of reel could also act as a substrate for the ultrathin, very fine-grained photographic emulsions used in recording electron microscope micro-images as described later. The miniaturized electron microscope would be equipped with an ap- propriately miniaturized ultramicrotome (e.g., of the Fernandez-Moran type with "V-shaped diamond or sapphire bearings without lubrication" equipped with a diamond knife [Ferndndez-Mordn, 1953]) for automatic production of ultrathin sections of the specimens which would be auto- matically fed to the microtape specimen reel and examined directly by electron microscopy. It is also interesting to consider the possibility of using a similar device to produce an "aerosol" of very fine particles, by using a diamond knife revolving at high speeds, of even the hardest mate- rials. These particles could then be examined in the previously described impact device. Alternatively, a miniaturized diamond drill with a servo-

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Analytical Methods for Landers 421 controlled motor would be used to prepare samples of the different strata of Martian soil. All of these specimen preparation devices would auto- matically convert the sample into appropriate ultrathin sections or frag- mented specimens suitable for electron microscopy. In analogy with the ingenious design first proposed by Lederberg et al. [1961], one could combine ultrathin sectioning and ultrasonic dissociation of the specimens with a suitable centrifugal or ultracentrifugal separator. It should be pos- sible to miniaturize these separators to permit use of density gradient techniques which would be of key value in sampling the environment and in obtaining specimens of the size range of virus particles and larger macro- molecules (DNA, nucleo-proteins, etc.). The required high ultracentrifu- gal forces could perhaps be generated by a modified design of the Beams magnetic suspension ultracentrifuges suitably scaled down for this purpose. In its simplest form, such a miniaturized electron microscope should not be more difficult to operate than are any of the other contemplated devices for detection of extraterrestrial life. Since all of its components are of rugged construction and are readily adaptable to the already tested tele- vision cameras for spacecraft, these devices can be expected to perform reliably, without the need for sophisticated techniques. This point deserves particular emphasis, since it is generally accepted that high resolution electron microscopy does indeed require considerable sophistication and experience in experimental technique. Here we would not be dealing with high resolution electon microscopy, but rather with electron microscopy in an intermediate range of resolution which could yield a vast fund of useful information which is well beyond the limits of light microscopy. Moreover, this type of electron microscope can serve as a prototype for the special demagnification electron microscope, described in Appendix II, which could be used to condense information on ultrathin films for sub- sequent retrieval. Design Features of More Advanced Miniaturized High-Resolution Electron Microscopes With present advances in the generation of stable superconducting elec- tromagnetic fields and progress in low-temperature electron microscopy, etc., it is conceivable that the contemplated design of new types of high resolution "cryo-electron microscopes" immersed in a liquid helium cryo- stat using superconducting electromagnetic lenses [Fernandez-Mordn, 1953; 1962; 1964; 1965] and related types of miniaturized electron micro- scope systems may find direct application in the examination in situ of lunar and planetary matter by electron optical techniques. It is readily conceivable that such a cryo-electron microscope could be

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422 MARTIAN LANDINGS: UNMANNED miniaturized. By being of smaller size and invested with a far greater resolving power, with useful magnifications on the order of 105 to 108 times that of a light microscope, it would permit a greater range of applications, and thus supplement and extend the usefulness on a planet of the presently contemplated automatic light microscope systems proposed by Lederberg and associates. By further development of the concepts embodied in our earlier low- temperature electron microscopy techniques [Ferndndez-Mordn, 1962; 1964] it has been possible to design a new type of miniaturized high resolution electron microscope totally immersed in liquid helium. These "cryoelectron microscopes" operating at temperatures of 1 to 4°K would embody the following significant features: (1) Highly stable superconducting electromagnetic lenses, with very ripple-free magnetic fields when operated in the persistent current mode; (2) Operation in ultrahigh vacuum and low temperatures resulting in decisive advantages of minimized specimen contamination, specimen dam- age and thermal noise; (3) Improved single-crystal pointed filament sources, with optimum conditions for both low voltage (i.e., 1 to 10 kV) and high voltage electron microscopy. In addition, the use of high efficiency viewing (single-crystal fluorescent screens, fiber optics, etc.) and recording devices operating at optimum low temperature would make it possible to use high speed cine- matography and stroboscopic recording (e.g., obtained through pulsed T-F emission from pointed filament sources) for attainment of high temporal resolution combined with high spatial resolution. The described combination of optimized instrumental design parameters, operative under conditions of minimized specimen perturbation represents one of the most promising coherent experimental approaches towards attainment of the theoretical resolution limit (about 2 A) in direct exami- nation of organic and biological structures. At present an instrument of the type shown in Figure 1 is currently being developed at our laboratories in the University of Chicago, as part of a comprehensive research program in the field of low-temperature electron microscopy [Ferndndez-Mordn, 1965]. Potential Applications of Electron Microscopy in Combination with Other Techniques for the Detection of Extraterrestrial Life Based on the extensive background of classic terrestrial biological observations, both with the light and electron microscopes, the direct ob- servation of structural detail at the various levels of dimensional hierarchies lends considerable support to the morphological approach. While it is

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Analytical Methods for Landers 423 likely that certain structural attributes may be specific expressions of life, it is recognized that many other criteria derived from biochemistry, physical chemistry, and the physical sciences are, in fact, indispensable in charac- terizing biogenic origin. With this in mind, it is interesting to note that an electron microscope can be readily used in combination with other techniques to carry out correlated biochemical and biophysical studies. Thus, by introducing cer- tain modifications in the design of the miniaturized electron microscope, the following procedures could be adopted: (1) X-ray microscopy [Engstrom and Finean, 1958], particularly in the use of microabsorption x-ray spectrometry; (2) X-ray microdiffraction, which would be of particular use in exam- ining larger bulk samples of fossil or other material. This could lead to the detection of the characteristic small-angle, x-ray diffraction pattern, for example, of collagen and other fibrous proteins which appeared to be uniquely specific for biogenic origin. (3) Different types of scanning microscopes (Nixon, Westinghouse) \"Haine and Cosslett, 1950] could be used and these would be of particular value in the rarefied Martian atmosphere, since they could permit the use of microprobe analysis outside the microscope itself. Other conceivable applications would be in connection with mass spec- trometry, micro-spectrophotometry, micro-histochemistry, micro-fluorome- try, radio-isotope biochemical probes, and devices like the Wolf Trap of Professor Wolf Vishniac for detecting the growth of microorganisms. This latter combination is of particular significance in connection with the detec- tion of submicroscopic organisms like viruses, which may be potentially dangerous to man. It is, for example, conceivable that single-cell cultures could be kept frozen in special microchambers, then infected or otherwise placed in contact with extraterrestrial material and the subsequent changes in the thawed-out cell observed both by light and electron microscopy as well as by biochemical techniques. In fact, the Wolf Trap optics lend them- selves readily to adaptation to electron optical techniques. All of these analytical systems complement each other and should be used in combination in appropriately designed planetary missions. Elec- tron microscopy is particularly suitable for the study of certain features of biological systems when adequately supplemented by the results of parallel biophysical and biochemical investigations. One of the most challenging problems that can be approached with electron microscopy in combination with x-ray diffraction techniques would be the detection on Mars of the presence of highly organized, repetitive, periodic but asymmetric fibrous and other anisodiametric structures at the macromolecular level. These have been hitherto found on Earth to be characteristic of life, although

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424 MARTIAN LANDINGS: UNMANNED admittedly no single criterion is acceptable. Thus, for example, the repeat- ing pattern of 660 A observed in collagen both by x-ray diffraction and electron microscopy has been found to hold true even for fossil specimens. Such patterns are not explicable solely by statistical combination of random asymmetric elements, but imply at least some form of replication, self- assembly and self-checking, which are quite distinct from the epitaxial growth of crystals. Although much work remains to be done in this area, it remains a fundamental problem to ascertain what morphological criteria at a given level of morphological organization are characteristic of biogenic origin. Once a given type of specimen has been thoroughly worked out and understood, it would be possible to program these microscope systems to "react" specifically to a given submicroscopic entity of predetermined con- figuration. This latter possibility would be of special interest in the case of electron microscope sensor and contamination monitoring devices. The merits of this approach are manifest when taking into consideration the example already discussed by Lederberg [1964]: assuming capture of a dust sample of about 100 milligrams, containing at most 100 micrograms of organic matter, perhaps 1 jag (about 10 nanomoles) of a particular spe- cies. Appropriate diffusion, differential ultracentrifugation techniques, etc., could be devised to separate such species and the various steps monitored by light microscopy until the enriched molecular species can be examined directly by electron microscopy. In the envisaged science of metrology, the orderly study of methods of measurement [Lederberg, 1964], the described electron microscopy, and electron optical techniques are bound to play a key operational role. Implementation of the described experimental ap- proaches appears to be well within the present capabilities of technology and applied scientific research. REFERENCES Engstrom, A. and Finean, B. (1958), Biological Ulirastructure, Academic Press, New York. Fernandez-Moran, H. (1953), A Diamond Knife for Ultrathin Sectioning. Exptl. Cell Res. 5, 255-256. Fernandez-Moran, H. (1962), New Approaches in the Study of Biological Ultrastructure by High-Resolution Electron Microscopy. In: Symposia of the International Society for Cell Biology 1, R. J. C. Harris, ed., Academic Press, London, 411-427. Fernandez-Moran, H. (1964), New Approaches in Correlative Studies of Biological Ultrastructure by High-Resolution Microscopy. Roy. Microsc. Soc. 83, 183-195.

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Analytical Methods for Landers 425 Fernandez-Moran, H. (1965), Electron Microscopy with High-Field Super- conducting Solenoid Lenses. Proc. Nat'l. Acad. Sci. (U.S.) 53, 445-451. Haine, M. E., and V. E. Cosslett (1961), The Electron Microscope, Interscience, New York. Lederberg, J. (1961), Exobiology: Experimental Approaches to Life Beyond the Earth: In: Science in Space, L. V. Berkner and H. Odishaw, eds., McGraw- Hill, New York, 407-425. Lederberg, J. (1964). Signs of Life: A Survey of the Detection Problem in Exobiology. Exobiology Summer Study 1964. Newberry, S. P., Buschmann, E. C. and Klotz, T. H. (1963), Advanced Elec- tron Beam Recording Techniques: Final Report, RADC-TDR-63-234, Rome Air Development Center, Griffiss AFB, New York. Quimby, F. H., ed. (1964), Concepts for Detection of Extraterrestrial Life, NASA SP-56, Washington, D. C. Reisner, J. H. and E. G. Dornfeld (1950), A Small Electron Microscope. /. Appl.Phys.21, 1131-1139. 18. A NOTE ON MORPHOLOGICAL CRITERIA FOR RECOGNITION OF LIFE When considering morphological criteria, by which the existence of extraterrestrial living forms may be recognized, it is necessary to concen- trate on those criteria that should be characteristic of any form of life, however it evolved and whatever its nature. It is useless to compile, even into broad classes, the forms of organisms with which we are acquainted on Earth. If pictures of earthly forms were transmitted back to us from Mars they would be recognized as living without much difficulty. I feel that it is also useless to try to set out criteria for recognizing "organisms" that are very low on the evolutionary scale and are just making the change from the chemogenic to the biogenic stage. The classification of such forms as living or non-living, on the basis of morphology, would be most difficult, even if they could be investigated in our laboratories here on Earth with the best microscopes and microtomes available. Rather, we should concen- trate on general criteria that we feel would apply to any forms of life regardless of its evolutionary history, genetics and energy transport system. The following is a list of criteria that I believe are applicable: 1. Motility—this is self explanatory. 2. Change in form—demonstration of change in form would be most compelling evidence for life. Under this heading I would include both spontaneous change such as pulsation or amoeboid movements and as changes induced by alterations in the environment as, for example, irritability, or shrinkage and expansion of microscopic particles in re-

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426 MARTIAN LANDINGS: UNMANNED sponse to change in osmotic pressure. This would be strong evidence for a semi-permeable membrane enclosing the particle and I cannot conceive of living organisms without such a membrane. 3. Complexity of Structure a. Macroscopic 1. Evidence that larger forms are comprised of discrete smaller units (cells). 2. Non-random association of the sub-units. b. Intracellular—existence of organelles, membranes, vacuoles, etc. Tests that would be useful for the recognition of some of these morpho- logical characteristics include: a. Differential staining. b. Impermeability—this would indicate the presence of a membrane surrounding a particle. c. Histochemistry—this would be a powerful method because it com- bines the morphological approach with a functional or chemical assay.