\u003ctitle\u003eHigh-resolution light microscopy of nanoforms\u003c/title\u003e

High resolution light microscopy of nanoforms

Vitaly Vodyanoy*, Oleg Pustovyy, and Arnold Vainrub Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn

University, Auburn Alabama 36849

ABSTRACT

We developed a high resolution light imaging system. Diffraction gratings with 100 nm width lines as well as less than 100 nm size features of different-shaped objects are clearly visible on a calibrated microscope test slide (Vainrub et al., Optics Letters, 2006, 31, 2855). The two-point resolution increase results from a known narrowing of the central diffraction peak for the annular aperture. Better visibility and advanced contrast of the smallest features in the image are due to enhancement of high spatial frequencies in the optical transfer function. The imaging system is portable, low energy, and battery operated. It has been adapted to use in both transmitting and reflecting light. It is particularly applicable for motile nanoform systems where structure and functions can be depicted in real time. We have isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1-2nm metallic nanoclusters containing 40–300 atoms. Proteons are capable of spontaneous assembling into higher nanoform systems assuming structure of complicated topology. The arrangement of complex proteon system mimics the structure of a small biological cell. It has structures that imitate membrane and nucleolus or nuclei. Some of these nanoforms are motile. They interact and divide. Complex nanoform systems can spontaneously reduce to simple proteons. The physical properties of these nanoforms could shed some light on the properties of early life forms or forms at extreme conditions. Keywords: Imaging system, transfer function, nucleating centers, proteons, life forms, motility, division

1. INTRODUCTION In the middle of 18th century scientists observed and described particles that they found in human blood plasma and other liquids. These observations were carefully described and analyzed by professor of anatomy at University College in London, H. Charlton Bastian 1. Scientists of that time had at their disposal surprisingly high resolution microscopes and described and photographed very small biological objects 2. In the beginning of 19th century, French microbiologist Antoine Bechamp described small particles he observed in human and animal blood by using a high-resolution optical microscope. He called those small particles “microzymas.” He wrote, “microzymas, anatomical elements, are living beings of a special order without analogue”3. In 1925, Gunter Enderlein also described small particles he called “protits” that he found in blood. The size of these particles was estimated to be about 0.01 micron. He found that under certain conditions “protits” could evolve into other forms and sizes 4. The discovery of small particles in blood was later repeated by Wilhelm Reich 5, Royal Rife 6, and Gaston Naessens 7. These seemingly identical forms described by different authors were not characterized to such a degree that allowed identifying them as the same or as different entities. Only recently by applying methods of biochemistry and molecular biology, was it possible to achieve a better description and characterization of nanoforms isolated from blood 8, 9, 10, 11. Kajander claimed to discover small particles named “nanobacteria” in cell culture sera and blood. Nanobacteria of 0.1 – 0.5 micron in size claimed to be cultured at 37 °C with a doubling time of about one to five days. All researchers mentioned above and working with nanoforms in blood claimed their special physiological and physicochemical properties, including strong influence on human health and certain importance in the origin of life. Thus, a better method of cultivation, better characterization and classification of the particles in blood, and the uniform and fast method of production of the particles is important.

2. METHODS 2.1 Light microscopy The system produces the highly oblique hollow cone of light (N.A. 1.2-1.4). Coupled with a high aperture microscope objective with iris this system provides two different regimes of illumination. When the iris is closed so that no direct

*[email protected]; phone 1 334-844-5405; fax 1 334-844-5388

Instruments, Methods, and Missions for Astrobiology Xedited by Richard B. Hoover, Gilbert V. Levin, Alexei Y. Rozanov, Paul C. W. Davies

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light enters the objective after passing through the object, only refracted, scattered, or diffracted light goes in the objective. If the iris is open in such a way to allow the direct entrance of light into objective the front lens of the objective is illuminated by the annular light produced by the empty cone of light entering the objective. In this case the mixed illumination is produced that combines the darkfield and oblique hollow cone brightfield illuminations. The cardioid condenser is an integral part of the illumination system so that the system comprises a collimation lenses and a first surface mirror that focus light onto the annular entrance slit of the condenser. As a part of the illumination system, the condenser is pre-aligned and therefore additional alignment is unnecessary. The illumination system is positioned in Olympus BX51 microscope by replacing a regular brightfield condenser. The illumination system is connected with a light source (EXFO120, Photonic Solution) by a liquid lightguide. The objective used for this work is infinity corrected objective HCX PL APO 100×/1.40-0.70, oil, iris from Leica. The image is magnified with a zoom intermediate lens (2×-U-CA, Olympus), a homebuilt 40× relay lens, and captured by a Peltier-cooled camera (AxioCam HRc, Zeiss) and Dimension 8200 Dell computer. The microscope is placed onto a vibration isolation platform (TMS, Peabody, MA). Life images were recorded with Sony DXC-33 Video camera and Mac OS X Computer 12. 2.2 Electron microscopy Transmission electron microscopy (TEM) was performed using a JEOL JEM 2010 instrument at 200 kV. Bright field imaging (BF) provided an overview of the microstructure of the sample. Detailed structures were characterized by selected area diffraction (SAD) and nanobeam diffraction, the latter producing a narrow (<50-nm diameter) yet nearly parallel beam that produced SAD-like patterns. DF (dark field) was employed to determine which microstructural features gave rise to diffraction maxima. Care was taken to avoid excessive beam-induced sample heating 13. 2.3 Environmental chamber A homemade closed system environmental chamber is used to carry out long-term observation and recording of live cell images and motile nanoforms. The chamber has a modular design and includes two optical surfaces of microscope slide and cover slip isolated by a Teflon gasket. The cell chamber is positioned between the metal chamber cover and the heater on the surface of the plastic holder attached to a microscope stage. The chamber has entrance ports for perfusion with liquids and gases, and temperature and chemical sensors (pH, CO2, K, and Na), and an exit port connected to a buffer cavity for prevention of clogs and bubbles. The height and volume of optical cavity is controlled by geometrical parameters of the Teflon gasket. The maximum height of the cavity accessible for focusing at 100X at high imaging apertures is 150 microns. The chamber was perfused by a gravity flow or peristaltic pump (Warner 720 Compact Peristaltic Pump). A small volume of liquid with additives was delivered with a manual injection using a syringe. Temperature control was provided by two RH-2 heating elements connected with Warner TC-324B Heater Controller with accuracy better than ± 1.0 oC. For the gravity perfusion valve control, the Warner VC-66MCS Mini-Valve System was utilized. Each valve was individually controlled by a manual switch or an external digital (TTL) signal. Harvard CO2 Gas/pH Controller was used for maintaining levels of CO2 and pH. 2.4 Nanoforms 2.4.1 Proteons from blood. Freshly drawn blood (30 µl) from healthy male subjects (human, rabbit, or dog) was diluted to 1 ml with purified water (Direct QTM, Millipore, 17 MOhm) in a 1.5-ml plastic tube and centrifuged at 13,000 × g for 5 min at room temperature. The plasma supernatant was used to prepare both proteons and PNCs. To produce proteons, the supernatant was transferred to a 4-ml glass vial with a plastic cap with a Teflon liner and subjected to 120°C at 140 kPa for 2 h. The PNC suspension (filtrate) was obtained by filtering the plasma supernatant consecutively through 30 and 5kDa Centricon filters (Millipore). Proteon samples for TEM were fixed with 3% glutaraldehyde, dehydrated with ethanol and embedded in Durcupan ASM resin (Fluka). Ten microliters of proteon suspension were placed in the environmental chamber for a long-term observation and recording. 2.4.2 Cell-like nanoforms produced from erythrocytes.

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Freshly drawn blood (1 ml) from dog was subjected to osmotic shock by adding (29 ml) purified autoclaved water. Samples of 5 µl microliters were completely sealed between microscope slide and cover slip and placed in dark at 5 oC for 26 days. The samples were observed and recorded for one hour everyday for 26 days by high resolution light microscope.

3. RESULTS AND DISCUSSIONS 3.1 Images of light microscope The design of our light microscopy system (see Sec. 2.1) was optimized for nanoforms, which typically have low optical density and small (compared to the light wavelength) size starting at several tens of nanometers. Two goals were approached. The first goal is simply a detection of a tiny object tolerating that its real size, shape and morphology are not truly imaged because of insufficient resolving power of microscope. The best detection threshold of about 20 nm was achieved in dark field mode using cardioid annular condenser depicted in Fig. 1A 12. In this regime studies of nanoparticles motion, interaction, collision, organization/dissociation, and replication have been accomplished (Sections 3.2 and 3.3).

1

2

3

1

2

3

4 4

5 5

A B

1

2

3

1

2

3

4 4

5 5

A B

Fig. 1. Cardioid annular condenser. The illumination regime is installed by the iris opening: A – dark-field, B – bright field.

1-condenser, 2-sample, 3-objective, 4-annular diaphragm, 5-iris. The second challenging goal is not only detection, but imaging of nanoforms correctly showing the shape, internal structure and their evolution in vivo and in real time. Obviously, this type of microscopy provides enormous additional information on structure and interactions on sub-nanoform length scale. It demands both the extreme resolving power and high contrast for visibility of the smallest details. This was achieved with the same microscope illumination system, but using the bright field (Fig. 1B) or mixed (comparable intensities of dark- and bright-field contributions to the image) illumination. Practically, the illumination regime changes from dark field through mixed field to bright field mode simply by the objective lenses’ iris opening (Fig. 1); no other adjustments are needed. Fig. 2 demonstrates the sectoral pattern from the Richardson test slide imaged at λ = 450 nm in bright field mode with our annual condenser illumination system (A) and standard Olympus bright field condenser N.A. 1.4/0.9 (B). The sectors edges are clearly shaper and small arcs are better visible with annular condenser showing advantages of our illumination system. The arcs of the smallest central ring are ~ 110 nm wide.

A B

Fig.2. Bright field image of the sectoral test pattern. (A) home-built annular, (B) standard Olympus illumination system. Additional examples of test pattern images using our annular condenser are shown in Fig.3. The vertical/horizontal bar set consists of opaque lines with the width of 81 nm spaced at 81 nm and thus the period is 162 nm. The image was taken at λ = 405 nm. For the imaging system with N.A. 1.4 the Abbe limit for the pitch of resolved periodic structure is

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LAbbe = λ/(2 NA) = 405/2.8=145 nm. Therefore, the imaged period of 162 nm is in complete accord with the Abbe theory.

Fig. 3. Images of vertical/horizontal bar set (left) at λ = 405 nm and various shapes (right) at λ = 546 nm recorded using the home-built annular illumination system.

For non-periodic shapes in Fig. 3 (right part) imaged at λ = 546 nm the features with the size of ~ 100 nm are clearly visible. The two-point Rayleigh resolution criterion predicts lRayleigh =1.22 λ/(NAobjective + NAcondenser)=238 nm. Thus with the annular condenser we observed the features about twice less than lRayleigh . To understand the result we calculated the image of two illuminated pin-holes separated by the distance d. We utilized the standard diffraction theory of microscope14 that for the case of circular aperture gives the image intensity along the line connecting two incoherently lighting points

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For brevity, we express here the distance x and two-point spacing d=2a in dimensionless units. Here J1(x) is the Bessel function of the first order. In case of the annular aperture with the inner to outer radius ratio ε (for our experiments ε =0.86) each term in Eq. 1 and Eq. 2 of the structure J1(x)/x should be replaced by

)()(

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xxJ

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The results for circular and annular illumination are shown in Fig.4 and demonstrate clearly up to two times increase in two-point resolution for the annular case. The presented theoretical analysis shows up to two times advantage in two-point resolution of the annular compared to the circular illumination system with the same numerical aperture. It provides the physical explanation of improvement of the visibility and contrast of small objects using the annular imaging systems sometimes regarded as mysterious and contradicting to the Abbe-Rayleigh theory of microscope. As shown, this is a confusion. Our results are in complete accord with the classical diffraction theory of microscope developed by Abbe and Rayleigh still in the end of 19th century. Development of the Fourier optics theory (started in the middle of 20th century) gives deeper insight into physics of imaging with the different amplitude and phase aperture masks15. Although the Fourier approach is beyond the scope of this communication, we want to note that it clearly explains the improvement in visibility of small details as a result of the enhanced high spatial frequencies in the optical transfer function of the annular versus circular aperture imaging system.

4 µm 0

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A B

-8 -4 0 4 80.000

0.004

0.008

0.012Annular incoherent (dmin= 2.3)

Inte

nsity

x = kaw

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nsity

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nsity

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0.2

0.3Circular coherent (dmin= 4.6)

Inte

nsity

x = kaw Fig. 4. Annular vs. circular condenser in bright-field mode: Two-point resolution. A, B – annular illumination;

C, D – circular illumination. The spacing dmin of resolved two points for annular incoherent illumination 2.3 (A) is twice less than for circular coherent illumination 4.6 (D).

3.2 Proteons Proteons are the small ball-like bodies are easily observed in a fresh sample of blood of any healthy human or animal when using a high-resolution dark-field microscope. They are seen like small bright balls of different sizes, estimated in a range of 50-250 nm (Figure 5, label 1). In the dark-field view healthy erythrocytes look like bright rigs of about 7 microns in diameter (Figure 5, label 2). They move slowly and randomly in the microscope view. Neutrophils, the

Fig. 5. Darkfield image of human blood. 1-proteon; 2-erythrocyte; 3-neutrophil.

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most abundant white blood cells, are about 7-15 microns, are often surrounded by erythrocytes, and they may remain stationary or move at certain directions using its membrane pseudopodia (Figure 5, label 3). One can easily see a few nuclei of the neutrophil and the remaining interior of the cell is filled with small granules which are practically indistinguishable from proteons visible outside the cell. In many cases neutrophils are in contact with damaged or infected erythrocytes. Proteons are usually moving fast and seemingly random. Proteons move in the space between blood cells, they seldom collide with each other and with erythrocytes, but quite often make contact with neutrophils, and some times they appear to go in and out of neutrophils. When a blood sample becomes aged, the morphology and number of the proteons change. Proteons appear bigger in diameter, and other forms looking like small motile rods and long motionless fiber-like structures appear, blocking the movement of proteons, erythrocytes and neutrophils. Some proteons become visible inside erythrocytes. Small round proteons move rapidly and large spore-like particles of about 500 nm appear attached to the internal surface of the erythrocyte membrane. We observed proteons in fresh blood of all samples collected from human, pig, rabbit, cat, dog, rat, and fish. Similar proteon-like structures are observed in the sap of many plants we observed. The method of fast production of proteons is founded in experiments of samples isolated from blood and subjected to elevated temperature and pressure. The fresh full blood sample contains approximately 3×108 proteons/ml which are visible with a darkfield microscope. After dilution and centrifugation, the sample contained ~9×106 proteons/ml. Before the high temperature and pressure treatment, the sample looked clear and light pink. After the temperature treatment, the sample becomes brown in color, not transparent, and slightly opalescent. There is a tremendous increase in number of suspended and moving particles which is approximately 3×1011 proteons/ml. If this number is adjusted for the initial dilution of the blood sample, it will become ~ 1×1013 proteons/ml. Thus, within two hours the number of proteons increased by approximately 5 orders of magnitude. If we assume that the population of proteons grows exponentially, then the number of proteons (N) at any given moment of time (t) can be described by the equation: N=N0ebt; where N0 is the initial number of particles, b is the growth coefficient. If we substitute N, N0, and t with the experimental values found in the experiment we can estimate that the growth coefficient (b) for this case is 5.2 1/hour, and proteons population is doubled every 8 minutes. This is a very conservative estimate because the exponential component of growth takes a much shorter time than the 2 hours used for these calculations. The time during which the population of proteons is doubled (8 minutes) is far smaller compared to the few days or over a week observed by using other methods 16, 17, 18. Thus, the growth occurs at least a 1000 times faster than that of other methods. There are a few forms of proteons observed by the optical darkfield, the transmission electron microscope (TEM), and scanning electron microscope (SEM). Type 1 proteons are largely presented by small particles of spherical shape of 50-250 nm. (Figure 6, A, B). The size distribution of particles is not even on the surface, and particles of same diameter are confined in elongated sacs (Figure 6 A). In the light darkfield in the liquid samples, proteons look like fast moving bright dots. TEM images show small proteons as dark discs (Figure 6 A, B). Figure 6 A shows small proteons in the area of about 100 square microns, and Figure 6 B depicts proteons in a magnified small area of about 0.7 square microns. Another form of proteons (type 2), coconut-shape particles of about 1-5 microns with one or many nuclei are also observed by TEM (Figure 6, C, D). A characteristic feature of this proteon is a cell-like appearance. The proteon has a very distinctive external membrane-like structure (Figure 6, C). This membrane-like structure, however, looks very different from the bacterial wall or the mammalian cell membrane. This external structure looks like a curly fibrous shell of 10-12 nm thick. Some fiber-like structures and a few nuclei are visible inside the proteon. More distinguished nuclei are visible inside the proteon shown in Figure 6, D. The type 2 proteon observed with SEM is shown in Figure 6 E. The large coconut-shape proteon with a large opening is positioned on a bed covered with small type 1 proteons. We believe that proteons viewed by the electron microscopy are not artifacts of sample preparation, because similar images were obtained by two different EM techniques and by the light microscopy where samples of proteons were not subjected to any physical or chemical treatments. Figure 6 F shows an example of a type 2 proteon obtained by high resolution light microscopy. The oval shaped sac contains a large number of particles of the type 1 proteons. The particles are being expelled through the opening in the left side of the sac similar to what was observed in many SEM photographs of the type 2 proteon (Figure 6, E). One more representative structure was always observed with practically all samples of proteons. The fibrous or filamentous structures were observed by all techniques.

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5 microns

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Fig. 6. Micrographs of proteons. TEM of thin section of type 1 (A, B) and type 2 (C, D) proteons. E – SEM of type 2 proteon. F –

darkfield light microscope micrograph of type 2 proteon. G –TIM of thin section of fibrous surfaces with proteons. These structures are presented in a great variety of shapes and forms. They are seen as shapeless bundles filling the space between the proteons and they are structured into the outer shells and inside of large proteons (Figure 6 C, D). Figure 6 G shows cross-sections of fibrous surfaces, which look like small beads on thin-coiled fiber threads. Type 1 proteons of different sizes look lined along these threads and showing a certain affinity to the fibrous surfaces.

500 nm

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100 200 300 400 500 600

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Fig. 7. Size dependence of proteon movement. A-Size distribution of proteons of type B. frame of the series of photographs made

at the interval of 10 milliseconds. C. Proteon displacement during the interval of 10 milliseconds as a function of the proteon radius.

An analysis of TEM images reveled the size distribution of proteons with a maximum about 150 nm (Figure 7, A). To estimate the speed dependence on the radius of proteons in water solutions at the room temperature, the time lapse photography of moving proteons was carried out with a high-resolution dark-field microscope. Figure 7 B shows a single frame of the series of photographs made at the interval of 10 milliseconds. Figure 7 C depicts experimental data showing proteon displacement during the interval of 10 milliseconds as a function of the proteon radius. Einstein derived the equation predicting the Brownian displacement (X) of the spherical particles of radius r suspended in a fluid of viscosity η and temperature T, after an observation period t 19: X=(kTt/3πr)1/2. According to the equation, the particle displacement should decrease with the increase of the radius (solid line in Figure 7 C). Quite the reverse is observed in the experiments: the displacement is increased when the proteon radius is increased. This discrepancy indicates that other moving forces besides the Brownian diffusion effect the movement of proteons 20. We observed proliferation of proteons in blood plasma during continuous long term experiments in the environmental chamber under high resolution light microscope. Early in the proliferation process when diluted blood plasma, obtained as it was described in the Method section, was placed in the chamber, just a few motile proteons were observed by light microscope. The rest of the space around these proteons looked clear and void. As time passed there was no change in the number of proteons in the observation field, but the average size of proteons slightly increased. The clarity of solution was progressively reduced and an empty space was gradually filled with milky clouds that slowly turned into myriads of tiny faintly visible particles. These were not produced by division of original proteons those numbers at this moment were not changed but rather originated from large number of small particles invisible by light microscope and became observable when their size reached the limit of detection of the microscope system. Gradually tiny particles grow bigger and completely fill the observation field. Following this state proteons started to combine in groups, aggregate, and make large type 2 proteons. The fact of appearance of large number of small proteons at certain stage of proteon proliferation and kinetics of proliferation suggested that proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs) 13, 18. There are certain requirements the seeds should meet in order to serve as nuclei for proteins and their fragments: (1) Small, a few nanomiters or smaller; (2) Large quantity, about 1013 seeds/cm3 of blood or tissue; (3) Abundant source for replenishing; (4) Binding sites and strong affinity to proteins; (5) Variability to bind different proteins; (6) Stability to resist extreme physical and biochemical conditions. After searching among many organic and inorganic candidates for the seeds, we have found just one entity so far to meet these requirements. We suggest that metal clusters 21 play a role in the seeds (PNCs) in proteons. The most remarkable property of metal clusters is that they provide a binding template for folding and aggregation of proteins 22, 23. Studies of the principles of protein folding suggest that close packing of secondary structures prefers some geometrical patterns 24, 25. Analysis of different classes of symmetry of protein folding revealed that the number of different protein folds, i.e. the number of different arrangements of major secondary structures and/or chain topologies, is 327. Of these folds, approximately 30% belong to mixed classes. Therefore number of unique topological configurations is only 327×0.7=228.9≅230 26. It may be not a coincidence that totals of 230 unique space groups describe all possible crystal symmetries27. By methods of TEM and SAD we found that that human

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blood plasma contains crystalline metallic nanoparticles of 1-2 nm in diameter. Among many metals nanoclusters of copper, zinc and iron found in large quantities. By studying proliferation of proteons in controlled condition we determined that these metal clusters are playing the role of PNCs 13. Each milliliter of human blood contains approximately 7×1013 PNCs. PNCs are comprised of 1-to 2-nm metallic nanoclusters containing 40-300 atoms. We were able to find an interesting description of forming particles in blood plasma which was of observed more than 140 years ago. H. Charlton Bastian described new particles formed in blood plasma under continuous microscope observation: “It seems almost equally certain that they did not originate from particles which were recognizable by microscopic power employed, since the fluids were at first, to all appearance, perfectly homogeneous. Either therefore, the minute particles which were seen at a later stage must have originated owing to some primitive formative process taking place in a really homogeneous organic solution, or else the fluid, seemingly homogeneous, in reality contained the most minute particles (microscopically invisible), derived in some unknown way from the previously existing protoplasmic elements” 1. Describing further particles of different sizes professor Bastian wrote: “The corpuscles also presented different aspects, the largest of them appeared to possess a cellular structure – there were slight evidences of a boundary wall, and numerous large protein granules within, more or less completely concealing a faint ovoid nuclear-looking body. This granular appearance seemed to become more and more marked as the corpuscles become larger, and the nucleus also become more and more distinct, though only appearing as a space free from granules. The corpuscles which were about 10 microns in diameter, as well as those that were of smaller size, presented none of these characters... There was no break whatever in the continuity of the series; all graduations in size could be and were measured, from mere plastide particle 250 nm in diameter, up to fully developed corpuscle… But in those corpuscles which exceeded 10 microns the protoplasm gradually become granular, and they then began to exhibit changes which appear characteristic of age and approaching degeneration.”1. Professor Bastian also described particles similar to those we observed by electron microscopy (Figure 6, A-D) the particles “had assumed a most distinctly cellular appearance – each cell containing one or perhaps two well-defined ovoidal nuclei…”1. 3.3 Cell-like nanoforms When erythrocytes are subjected to osmotic shock it triggers eryptosis, a death of erythrocytes characterized by cell shrinkage, cell membrane blebbing, phospholipid exposure at the cell surface, and loss of hemoglobin. After erythrocytes underwent osmotic shock, and placed in a sealed camera and placed in the dark at 5 oC the cells underwent very strong morphological changes. The external cell membrane ruptured in many places and resealed creating a number of closed vesicles of different shapes. All vesicles are characterized by very distinguished external membranes. In light microscope they look bright compared with other structures. The vesicle shape varies and manifests round, elongated, odd forms, and very thin filaments. Sizes of these vesicles vary from a few microns to a few hundred nanometers. Smaller vesicles at times are entrapped in the bigger ones. Almost all vesicles are in vigorous motion. They vibrate, pulse, move, and interact with each other. Hemoglobin molecules released from erythrocytes interact with proteon nucleating centers (PNCs) and produce proteons by process of nucleation and growth. First, proteons are small and barely visible with light microscope, but in time proteons become bigger. They interact with each other and create associations of two, three, and many proteons, confined by a thin membrane-like shell. Most of the proteons are engaged in robust non-Brownian movement. They vigorously move in both free spaces and being confined inside vesicles. Cell-like nanoforms and proteons are capable of spontaneous assembling into higher nanoform systems assuming structure of complicated topology. Figure 8 shows frames of video taken from different nanoforms. Figure 8, frames 1-4 show proteons of various topologies. Video records indicate that nanoforms are engaged in interactions, fusion, and division, mimicking many regular cellular activities. Figure 8.1 shows a union of three vesicles and one filamentous form that look to be in close association and move together. The filamentous construct is firmly attached to the round vesicle by a tip similar to the attachment of Borrelia burgdorferi (species of bacteria of the spirochete class of the genus Borrelia). Figure 8.2 catches a few different nanoforms. They show different number of inclusions. At the very top of the frame, a vesicle has one large “nucleolus”. Other vesicles have different number of smaller particles inside, and the movie showed very intensive movement of all particles. The vesicle on the very bottom of this frame is just about to divide. Frame of Figure 8.3 shows two small vesicles that interconnect with each other through the membrane of a larger vesicle. This construct is useful for modeling of exocytosis. In Figure 8.4 two vesicles in the upper left portion of the frame are just about to make a firm contact.

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Figure 8, frames 5-8 show assembling of proteons into a complex assembly of particles that are joined by a common “membrane”. Figure 8, frames 9-12 show dividing of an elongated filamentous proteon into small parts that mimics the process of cell division. The arrangement of complex proteon system mimics the structure of a small biological cell (Figures 6, C, D). It has structures that imitate membrane and nucleolus or nuclei. Some of these nanoforms are motile.

Fig. 8. Assembling and disassembling of nanoforms. Video frames clipped from continuous recording illustrate complex topology and behavior of nanoforms.

They interact and divide. Complex nanoform systems can spontaneously reduce to simple proteons. The physical properties of these nanoforms could shed some light on the properties of early life forms or forms at extreme conditions. They can be used to study molecular mechanisms of cell growth and proliferation. They also can also serve as carriers of protein, DNA, and other molecules in processes of tissue repair, artificial organs, and drug delivery.

ACKNOWLEDGEMENTS The work was supported by Fetzer Institute Inc., Aetos Technologies Inc., and CytoViva Technologies Inc.

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