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The different ways of GLC determination of trace impurities using phase equilibria of the objects under investigation (head-space analysis and determination of impurities in a gas using the data of their content in the liquid saturated with this gas) are discussed and compared. The limits of application and the possible source of errors of each of these methods are discussed. Examples for application of the method of equilibrium concentration for GLC analysis of impurities in food, waste water, air and biological objects are given.

For causal analysis of morphogenetic systems a device has been developed (Fig. 1) which achieves a well-defined and stable temperature gradient up to 80° C/mm inside small objectsin vivo. The technique requires that the objects are embedded in aqueous agar of 3% (Difco, Detroit/Michigan). By means of an inverted microscope time-lapse cinematography of the objects can be carried out simultaneously.

An environmental chamber with associated control equipment for the study of possible effects of electric fields on biological objects (small mammals) is described in detail. Temperature is controlled within 0.4‡ C, relative humidity within ± 1.5 % and pressure below or above ambient is regulated to ± 1.0 mm Hg.Sound and electrical noise absorption by the chamber insulation is better than 90%.

The difficulties in sectioning frozen biological objects for electron microscopic investigations are overcome by Steere's freezing-etching method. In order to test this method and to open up a wide field of application, the new freezing-ultramicrotome has been designed. The apparatus consists of the combination of an ultramicrotome with freezing-drying and shadow-casting installations in the same vacuum container. The preliminary results show, on the one hand, the practicability of all preparational steps and, on the other, that it is possible to resolve internal structures of cell organelles and even macromolecular patterns.

A method for the determination of the mass of as small biological objects as single cell structures is described. The method is based upon absorption measurements of soft filtered continuous x-rays. From adsorption data the mass can be calculated. The complete theory of the method is given. An x-ray equipment for the mass determination has been constructed and is described in detail. A number of biological applications are presented.

In December 1926, Mr. W. V. Mayneord read an extremely interesting paper before the Institute, entitled “An X-ray Study of the Crystal Structure of some Biological Objects,” in the course of which he demonstrated how, by powder methods of X-ray analysis, it was possible to ascertain the nature of the crystal formation of pearls and other calcareous structures found in nature. It is to be hoped that it will be of interest to deal with the subject from a more practical standpoint, giving a brief description of the X-ray methods which are at present in daily use for the testing of pearls. We find in no less an authority than the fourteenth (1929) edition of the Encyclopedia Britannica the statement that “The only method of ascertaining which variety (of pearls) is ‘cultured’ and which ‘natural’ is to cut the pearl and examine the cross section.” We hope to show that this statement is entirely untrue. Without going at all fully into the still vexed question of the origin of pearls, a short description of the ...

Summary A simple and inexpensive apparatus for freeze-drying histological specimensis described. Its performance has been tested by measuring three variables: weight and temperature of the block during drying, and rise of pressure in the system with pumps off. The latter measurement has been found to be a practicable and reliable means of determining the end of the drying process. The dependence of drying rate on pressure in the system has been investigated in the cases of pure ice and artificial and biological objects. In this investigation, the histological appearance of the tissues, thoughgood after the procedure finally adopted, has not been used as a criterion of dryness.

The great importance of the pretreatment of biological objects for quantitative microradiography is discussed. Freeze-drying was proved a suitable method in obtaining dehydrated sections without any measurable change in the dry substance. The different steps of the freeze-drying procedure are analyzed with special reference to their effects on mass determinations. The most important errors are the effects of shrinkage and incomplete drying, their magnitude being discussed. For microradiographic determination of dry mass per unit volume the thickness of the freeze-dried tissue sections must be measured. Different methods for thickness determination are discussed and for freeze-dried sections a procedure for focusing in a microscope proved appropriate, the error in a single determination being less than 1 μm.

Summary A simple photoelectric method has been described for the analysis of birefringence in light-scattering biological objects. The sensitivity and range of the method can be varied at the discretion of the investigator. Under the conditions reported here the smallest detectable retardation was about 10−4 radian (0.1 A). Birefringence fluctuations with a period of the order of 1 sec could easily be analyzed.

Methods for recording true volume holograms in photographic silver halide emulsions have been developed. The layers are swollen in an immersion of water or organic solvents to about the sevenfold of their dry thickness. After exposure and processing, the extended thickness is stabilized by forming a hardened polymer in or on the swollen layer. These thick holograms in the highly sensitive silver halide layers are needed where exposure restrictions disqualified other thick recording materials as in holography of biological objects and in holographic high-speed cinematography.

A small accelerator which produces high energy (14.7 MeV) neutrons by the H3 (H2, n) He4 reaction has been utilized for two basic biomedical studies. First, fast neutron radiography has been explored as a technique for imaging biological objects. The primary objective was to test the sensitivity of the technique by measuring the differential attenuation coefficient necessary for object identification as a function of the sample thickness. Second, fast neutron radiotherapy experiments were undertaken. Animals with induced brain tumors were treated to observe the relationship between dose fractionation and cell repair. The results are encouraging and indicate the feasibility of further studies with a small accelerator.

Modern views are presented on the primary physicochemical stage of the mechanism of the biological action of ionizing radiations and on certain laws of their action on biological objects, determined by the space and time distribution of the radiation energy in the organism.

1. The total interelectrode impedance (TII) is a nonlinear function of many variables and can characterize the functional condition of the organism.

Photomontages of cloud chamber pictures with microphotographs of organisms have been used to study the penetration and distribution of high-energy radiations in biological objects. Because of the difference in density between gases and tissues, the author suggests that the bubble chamber be employed using liquids rather than gases for recording tracks of high-energy radiations.

The technical set-up and the electric circuit diagram of an apparatus is described that has been used for investigating active ional transport processes across biological membranes. The apparatus, essentially a Wheatstone resistance bridge circuit, works by the following principle: If a current is supplied by a battery outside and sent through the membrane there exists one and only one value of the current (under steady transport conditions) for which the membrane has an apparent resistance of zero. It can be shown that the value of that current is exactly the same as the value of the hypothetical short-circuiting current which would flow — carried by active ions only — if both sides of the membrane were directly connected by an ideally good conducting lead. With the help of Ling-Gerard capillaries measurements could be done even across very small membrane areas within biological objects.

Durch den Zusatz von je 4% Saccharose, Harnstoff, Acetamid und Propionamid zu 0,666% Agar-Gel bleiben die kolloidchemischen Eigenschaften des Gels nahezu unverandert, dagegen wird die Ausbreitung der Serumprotein-Fraktionen wesentlich modifiziert. Obige polare Substanzen sind im Sinne eines steigenden Verteilungskoeffizienten (Olphase/wasrige Phase) angefuhrt. Als Testproteine dienen Serumalbumin, γ-Globulin, α1-Lipoprotein und α2-Makroglobulin. Die Ergebnisse der Modellversuche werden verglichen mit den Erfahrungen am biologischen Objekt. It has been proved that by the addition of 4% sucrose, urea, acetamide, and propionamide to 0,666% agar-gels, the colloidochemical properties are not changed, but the spreading by diffusion of serumprotein fractions is significantly altered. The polar substances which have been added are listed above in an increasing sequence of their partition coefficient (oil phase/water phase). Serumalbumin, γ-globulin, α1-lipoprotein, and α2-macroglobulin have been used as test-proteins. The results of these model-reactions are compared with corresponding experiences on biological objects.

It is assumed that the recovery factor is some function of the fraction of the total number of cells which are undamaged by n quantum “hits”: R f(S∕N). The fraction S∕N is assumed to be given by the probability distribution expression: which has been tested by extensive experiments on various biological objects. The fraction S∕N is determined after each dose in the protracted dose technic, and the recovery factor calculated on the simple assumption that it is directly proportional to S∕N. Cumulative dose curves are given.

The use of very high-energy gamma and electron radiation for the treatment of cancer in man poses the question of the eventual biological effects on the tissues involved. Does radiation of such type cause a different response qualitatively and/or quantitatively in comparison to standard x-ray exposure? Investigation of this problem is a prerequisite to the application of high-energy radiation therapy. Though many studies have been conducted (1–6), research is continued in the interest of improving treatment technic and enhancing the response of the malignant tissues to radiation. Biological effectiveness has been observed to vary with the energy of the irradiating agent (7–9, and others). Quastler (5), Haas (1), and others have demonstrated such variation in biological objects irradiated with betatron x-rays of 22 MEV and electrons of similar energy. The difference of the effects as compared with those of 400- and 200-kv x-ray exposures seems to be of an entirely quantitative nature; no qualitative change...

A given amount of ionization produced by fast neutrons has an injurious biological action equivalent to that of several times as much ionization produced by x-rays. This has been demonstrated for various biological systems by Lawrence and Lawrence (1), Zirkle and Aebersold (2) and Lawrence, Aebersold and Lawrence (3). This fact indicates that the mechanism of biological action of fast neutrons must differ in some essential way from that of x-rays, and suggests the further possibility that the neutron's superiority in destructiveness may be greater in the production of some biological effects than is the case for others. This possibility, in view of the known differences in distribution of ions produced by neutrons and x-rays, is of great interest in the study of the mechanism of biological action of ionizing radiations in general (1, 2). Moreover, it has important clinical implications. The purpose of the experiments reported below was to investigate this matter with various small biological objects which could be irradiated in large numbers both with neutrons and with x-rays. The biological effects chiefly studied were inhibition of hatching in Drosophila eggs and retardation of growth in wheat seedlings. The irradiation of these two organisms was done under the same conditions of geometry, dosage measurement, and operation of the neutron generator. Hence the results obtained with one organism are directly and accurately comparable to those obtained with the other. A third biological effect studied was inhibition of cell division in the spore of the fern Pteris longifolia. The conditions of irradiation of this organism were not exactly the same as for the other two, but the data obtained, for reasons which will be given below, should be comparable.

An account is given of a method for the preparation of thin nitrocellulose films. They are intended for use as a reference system in the determination of the mass of extremely small biological objects according to Engstrom and Lindstrom 's microradiographic method. The factors affecting the formation of a nitrocellulose film of the desired thickness are discussed. An apparatus simplyfying their preparation is described. A comparison is made between nitrocellulose films produced with the earlier method and with that suggested by the authors. It shows that the variations in weight amount to 18–20% in the former cases and to 6% in the latter.

Before initiating quantitative mass determination of biological objects in one’s own laboratory, the following prerequisites must be available: Electron Microscope Any electron microscope is suitable for quantitative work.

As already mentioned in the introduction, attempts to determine the physical mechanisms of the biological action of Em Fs and to estimate the minimum intensity at which Em Fs can act on biological objects have usually been based on a consideration of the energetic interactions of Em Fs with these objects. In some cases the center of interest has been the physicochemical processes induced in living tissues by Em Fs and in others it has been the relationship between this action and the macroscopic parameters of the biological objects.

Various measures to increase the sensitivity of the polarizing microscope for visual observation are discussed. These include choice of polarizing apparatus, reducing condenser and objective aperture, reducing lamp iris, blooming lenses, use of mica plate compensators and use of bright light sources. By such means as these it is possible to detect retardations down to 0.28 A., or 1/20,000th of a wave-length. A photographic method for measuring retardations down to the same limit in small biological objects, is also described. We are deeply indebted to Mr E. W. Taylor of Messrs Cooke, Troughton and Simms Ltd., for his great help in fitting our microscope with double Polaroids and a graduated compensator mount, as well as for blooming our lenses and making special low retardation mica plate compensators. We are also indebted to Dr L. E. R. Picken and Dr A. F. Hallimond for helpful suggestions at various stages of the work.

The purpose of these remarks is to present a brief description of some techniques of image processing and to offer a few illustrative examples of the ways in which image processing might contribute to studies of red cell shape. Image processing refers to the application of automatic techniques, both digital and analog, to visual images so as to transform the images or to extract quantitative information from them. While it has been known for some time that one can adequately represent a visual image by a mathematical function, it is only within the past 10–15 years that the electronic technology has made it practicable for images to be translated into numerical form and manipulated by computer programs. A number of working groups [8, 9] have already applied image processing to a variety of problems of biological and medical interest; the applications to health science appear to be particularly attractive and numerous, perhaps because so much life science research depends upon visual inspection of biological objects.

A series of dimensions of the shoulder girdle of primates has previously been chosen as being related to function in that anatomical region. Their examination by canonical analysis suggests that they do indeed reflect aspects of the use of the shoulder in locomotion in the different primates. Further analysis is here performed using the technique of neighborhood limited classification and this confirms the basic picture presented by the previous analysis. The new method also gives more detailed information about the grouping of the specimens; thus it endorses the reality of functional divisions that appear to exist in the data. And in addition the groupings reflect differences in the structure of the shoulder that correlate well with certain taxonomic subdivisions of the order. The method maintains contact with individual specimens throughout the analysis and is capable of placing them within groups, at the boundaries of groups, within the interfaces between groups, or as satellites to groups. The new method appears to have a part to play in the description of the relationships between biological objects that is complementary to that of canonical analysis. As the mathematical concepts upon which the two techniques are based differ totally, the risk that the results might be inherent in statistical assumptions is thus averted.

Viruses are biological objects which have no metabolism of their own and can replicate only with the aid of the genetic and metabolic systems of a host cell, i. e. they are “parasites at the molecular level”. If the host cell is a bacterium they are referred to as bacteriophages, or phages. Since the effects of radiation have been investigated more thoroughly in bacterial viruses than in other systems, a large proportion of the experimental material discussed in this chapter is derived from investigations on bacteriophages.

SEVERAL reports have dealt with the deleterious effects on biological objects of irradiated sugars, or foods containing them. Some people have even implied possible harmful consequences as a result of consuming these irradiated products. Because current research programmes involve the use of radiation as an agent for preserving food, some of the reports should be further investigated to establish clearly the incidence, nature and conditions in which deleterious effects are induced. We are therefore studying damage to chromosomes in the root tip of Vicia faba caused by treatment with irradiated sucrose solutions, one of the subjects included in a report by Holsten, Sugii and Steward1.

The study of biological objects in the electron microscope requires special techniques. The basic electron-microscopic methods used to investigate biological objects have been fully described elsewhere [7].

The nucleus, both during interphase and mitosis, has come to be regarded as one of the most difficult of biological objects to study by methods of electron microscopy. Apart from the nuclear envelope there are few land marks and little evidence of order such as we find in collagen or muscle. What organization there may be is distributed in depth in the whole nucleus and so defies all but the most painstaking efforts in serial thin sectioning. It is not surprising, therefore, that the problem has proved discouraging except for the most cfetermined and interested investigators. Of these we have a representative few on the program of this symposium and they will give you some measure of the progress that has been made to date. In view of the difficulties, their achievements must be regarded as impressive.

Standardization in fluorometry is still connected with difficulties. The theoretical and practical reasons for this have recently been discussed during the National Bureau of Standards conference in Washington (Conference on Accuracy). In the field of microscope fluorometry standardization is even more difficult because of the tiny volumes and amounts of substances involved and their correlation to microscopic objects to be measured. Previous attempts at the calibration of instruments and standardization of fluorescence intensities were mainly based on, (a) fluorescent glass plates or crystals for calibration of instruments, (b) biological standards such as spermatozoa (Ruch 1961), and (c) microdroplets (Rotman 1966, Rigler 1966, Jongsma et al. 1971) or microspheres. All these attempts claim to produce reproducible fluorescence signals and permit the measured intensities to be correlated with the amounts of fluorescent substances in biological objects.

Inhibition of vinyl polymerization by the embedding of biological specimens is due to the presence of biogenic amines. This inhibition is eliminated by acylation of the embedded objects with acid anhydrides.

It has become increasingly obvious to all biological electron microscopists that staining and shadowing techniques must be eliminated, and the rapidly advancing technology of water trapping devices demands it. If we are to view biological systems at the molecular level or in a condition approaching the physiological state, contrast must be enhanced by electron optical means, not by specimen alteration.Therefore, we have investigated Zernike type phase contrast, the principle of which is shown in Fig.1. Our phase plates consist of a carbon film carried on a 30μ objective aperture and are centrally drilled with a laser microscope made available by the American Optical Co. Because our phase plates incorporate a 30μ aperture the results are expressed as a ratio of phase contrast to 30μ aperture contrast. Fig.3 shows the early results of Dr. H. M. Johnson, previously of this laboratory.

SINKING velocity is an important ecological factor for many small biological objects such as pollen grains, airborne fruits and microplankton. Elaborate structures are often developed to increase drag and reduce sinking velocity, but their effectiveness cannot be directly investigated because of the difficulties involved in manipulation. The hydrodynamics of such small objects can be investigated, however, by using models of convenient size in highly viscous fluids. We have recently used this method in considering the feasibility of a planktonic mode of life for an extinct bivalve mollusc1 and think the method should interest planktologists, palynologists and others who do not read palaeontological literature.

THE reversible transfer of amino groups between aminodicarboxylic acids and α-ketoacids, or ‘trans-amination’, reported in a series of papers by me and my associates1, is of almost universal occurrence in biological objects. It is readily effected by most animal tissues2, and also, as shown in this laboratory and independently by Virtanen3 and Euler et al.4, by vegetable tissues and micro-organisms. Trans-amination is effected by a special group of intracellular enzymes, for which the name amino-pherases is suggested (in preference to the clumsy although more precise term aminotransportases). As trans-amination involves intermolecular oxido-reduction, the aminopherases should be classed among the desmolases. They differ in mode of action and in many properties from the formerly known enzymes of amino acid metabolism, including the I- and d-deaminases and specific glutamic and aspartic dehydrogenases.

One-of the major efforts of our laboratory is the observation of hydrated whole cell mounts in the electron microscope. Efforts to date have shown superior resolution as compared to the light microscope only in the thin portions of spread cells (Parsons et al., 1972). The thicker portions suffer from severe chromatic aberration which limits contrast and resolution even at 800kV. Thus, an image mode capable of enhancing image contrast and eliminating chromatic aberration is necessary. Furthermore, it is presently unrealistic, for the thick portions of the cell, to rely on increasing the accelerating potential. Since the selection of an image mode is a complex function of the required contrast and resolution, and the specimen under observation, we have limited ourselves to a resolution of 20.0nm as an intermediate goal, and ask what image mode will maximize the contrast.

Cine camera recordings have been made of (a) the movement of dislocations in thin metal foils, (b) contrast changes in single crystals of colloidal silver iodide. Electron diffraction pattern changes have also been recorded on cine film. Methods and apparatus are discussed together with recent attempts to film structural details in biological objects examined in the electron microscope.

THE recent proposal1 to analyse shapes of biological objects by means of orthonormal functions opens up some interesting possibilities. In the work cited, a particular set of two-dimensional orthonormal functions synthesized from Walsh2 functions which take only the values +1 and −1 was used. As is well known, however, there is a vast number of other complete sets of orthonormal functions and, in fact, they could be constructed indefinitely. There are also standard linear methods for transforming from one set of orthonormal functions to another; these procedures can be realized as computer programmes.

A modification of the Kohler method is described which facilitates the measurement of the small retardations frequently found in minute biological objects.