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Abstract
Conventional flow virometry methods are limited by their reliance on fluorescent labeling and molecular-biological specificity, making them unsuitable for universal detection of morphologically diverse virus particles across their entire size range (20–300 nm with some representatives reaching 1.4 μm). We propose an alternative approach based on laser aerosol spectrometry and optoelectronic aerosol counting, which enables size-based discrimination and morphological characterization of viral particles without requiring genetic modification or staining. This method exploits the Stokes radius as a fundamental descriptor to differentiate viruses by their hydrodynamic properties and geometric parameters. Using aerosol spectrometers, we demonstrate that viral particles and their aggregates can be detected and classified through multimodal size distribution analysis. We discuss the theoretical foundations for extending this methodology to macromolecular levels, connecting viral particle detection to fundamental concepts in molecular evolution, including the divergent evolution of semantides and episemantides. Based on the contemporary molecular genetics concepts, which propose a divergent origin of enzymatic function evolution, typically coupled with genome expression (beginning with proto-ribozymes combining RNA and enzyme functions), it is appropriate to pose the question of whether viral functions and the corresponding biochemical agent forms can be separated using laser-assisted analysis of the particle size/dispersive parameters, which provided transcription and translation processes. In this connection, fundamental significance is possessed by the separation of viruses and native genetic code carriers as semantides, on the one hand, and episemantides as small molecules and individual enzymes and allosteric regulators, on the other hand, in accordance with their dimensions. A minimal cell size (which can be measured by different laser methods and belongs to the submicron range) is determined by the compact localization of the cell components providing genome expression functions. It is impossible to further reduce it, except for the possibility of the function combination in a single structure. Thus, laser-assisted measurement techniques can be used for solving the fundamental problems of protobiology and synthetic biology, in particular, for the sufficiency validation of various protocells and artificial cells. The existence of a minimum size of a functional cellular structure with expressible genetic material can be interpreted only from the standpoint of evolutionary cytometry. The problem of detection of self-assembly processes in model prebiological environment / prebiotic media also can be addressed by laser optical biophysical techniques within the framework of coevolution of semantides, asemanties, and episemantides from nanosized to microsized assemblies and condensates due to self-assembly processes and non-covalent interactions.
Keywords: Aerosol spectrometry; Flow virometry; Viral detection; Size distribution; Stokes radius; Optoelectronic counters
Optical Virometry Techniques
In recent years, alongside optical cytometry based on the analysis of fluorescent (or cell-scattered [1,2]) light signals, a complex of virometric methods has gained considerable efficacy, notably flow virometry [3,4], analogous to flow cytometry (FACS, FCM). This suite of virometric technologies is currently positioned as a unique means of analyzing infectious profiles [5], antigen spectra [6], and kinetic analysis of viral infection, as well as conformational kinetics and comparative analysis of response chemistry relative to normal reaction parameters [7]. Consequently, emphasis has been placed predominantly on the molecular-biological aspect rather than on ultra morphological and dimensional (dispersive) distinctions among viruses.
Furthermore, in accordance with current policy at the United States National Institutes of Health, emphasis has been placed upon viral particle enumeration, particularly concerning HIV-1, the retrovirus responsible for the AIDS pandemic and belonging to the subfamily Lentiviridae [8]. This represents an extremely taxonomically restricted subset of relevant analytes and samples, with correspondingly limited nomenclature of viral variations [9-11]. Consequently, one cannot reasonably speak of universality of this method in diagnostic virology. A universal method cannot be founded upon selective, molecular-biologically specific carriers, particularly for supramolecular systems exhibiting high specificity of complementary binding, such as viral genetic mechanisms. It is in principle impossible to create a system equivalently recognizing all viral types—whether DNA or RNA viruses; possessing single-stranded or double-stranded DNA genomes, or single-stranded or double-stranded RNA genomes; accounting for all capsomer morphologies (protein subunits/protomers of viral capsids); considering the probability of detecting both unencapsulated viruses and those bearing xenogenic lipid supercapsids in samples—without contradicting the phyletic diversity of viral molecular taxonomy.
It should be noted that selective fluorescent methods of flow virometry likewise cannot serve as a universal means of detecting geometrically distinct viruses, owing to the indistinguishability of their signals at sub-wavelength viral dimensions, excepting PALM/STORM-type microfluorimetric methods. Viruses with non-identical scattering diagrams according to geometric criteria—that is, possessing different morphological, or more precisely, geometric capsid types (helical, icosahedral, elongated, and complex)—can be distinguished only within the framework of diffraction fingerprinting approximation, concordant with the corresponding viral geometry, calibrated using test particles adequate in both geometry and metrology (dimensions) to the virus studied. Ideally, such particles should possess automorphism to the virus (for instance, a virus of identical morphology but inactivated or non-pathogenic), or be homeomorphic to it. A morphism failing to establish bijective correspondence between the structure of the investigated and calibration virus/particle, for "morpho-physical" reasons, is insufficiently adequate for identification. Yet even within the framework of category morphism correspondence, whilst maintaining identical symmetry groups in calibration and flow virometry-measured particles, the possibility of dual approximation of individual structures may, in principle, constitute a source of artefacts. A classical example: rotavirus, possessing icosahedral symmetry yet effectively approximated by a spherical pattern, owing to its large number of capsomers (pentons and hexons) tiling the surface (pertailing [12]). The "transition of quantity into quality" upon introducing additional capsomers into the system typically affects the diffraction pattern also because of the objective increase in particle dimensions relative to the wavelength employed, since, with invariant capsomer dimensions determined by protein particle size, increasing the number of capsomers on the surface necessarily entails approximation of form towards greater smoothness (analogous to approximations employed in mesh methods in scientific visualization and three-dimensional graphics).
It is well established that viral dimensions vary by orders of magnitude across different taxonomic groups: from 20 to 300 nm (although certain representatives of the family Filoviridae with negative-sense single-stranded RNA reach lengths of 1.4 μm with diameters of 0.08 μm, whilst Pandoravirus sp. and Pithovirus sp. exhibit genome compaction levels that physically permit extension far beyond micron boundaries—1.0–0.5 μm and 1.5–0.5 μm, respectively). This necessitates qualitatively distinct metrology and analytical principles for these qualitatively different groups. Notwithstanding the widespread simplification involving extrapolation of Mie light-scattering theory interpretations across virtually the entire particle size range, including Fraunhofer diffraction as a special case, it is well established that Mie theory is inapplicable at small diffraction parameters. Computational capabilities afforded by Mie theory permit application of a single (universally interpretable, within the aforementioned extrapolating misconception) method/calculation algorithm for additive dispersity spectra—that is, particle sizes. In fact, however, this means that a portion of the sample will be miscalculated, since it is scarcely possible (unless dealing with monodisperse calibration media—for instance, specialized latexes lacking heteroscedastic distribution)—to anticipate in advance the concentrations of individual particles and the contribution of dispersive fractions (or sizes of ultrastructural and microstructural biomaterial units, differing both in static geometry and in dynamic morphometric parameters, which under normal reaction conditions represent non-stationary correlates of general protoplasmic reactivity and excitation propagation therein [13,14]). The qualimetric weighting criterion in such problems is difficult to compute, owing to differences in particle geometries ("form factors"). One might assert that "if all particles in a sample exceed the light wavelength, the Fraunhofer portion of Mie theory dominates in Mie theory calculations of particle sizes"; yet, considering anisometric particles (including viruses—such as Filoviridae with extraordinary elongation coefficients: up to 1.4 to 0.08), one cannot objectively express the numerical magnitude of the above dominance (particularly without accounting for orientation) in most cases. Evidently, since machine-solvable abstract models applicable to distribution calculations are developed upon the assumption of spherical, that is, simplest, particle form, the particle size distribution obtained from analysis actually represents a distribution of "equivalent spherical particles" rather than actual analyte particles (a classical electrodynamics problem solved in 1908 by Gustav Mie through expansion of the electromagnetic field in spherical harmonics).
For viruses differing in morphology and genetic or evolutionary position, geometric deviations from sphericity will be distinct; consequently, equivalent measurements will prove differentially effective for systematically different viruses. One cannot therefore speak about a universal "functional virometry" technology, since the dimensions of even the most prevalent or clinically relevant viral nomenclature (and thus diffraction limits and applicability boundaries of various mathematical approaches, including those differing from Mie theory) vary extremely, whilst functional activity of virulently distinct forms, as a rule, correlates comparatively weakly with the averaged morphology computed during data processing from the flow detector (even accepting that viral measurements are conducted not on dispersive substrates but in an abstract empty medium devoid of adsorbing agents [15]), and cannot be revealed in this manner. Regarding insufficient information for functional identification, one should also note that Mie theory requires knowledge of the sample's refractive and absorption coefficients (obviously non-identical for different potential viruses) and of the "dispersive medium," which in the native case likewise exhibits certain uncertainty, since samples must be analyzed from various natural environments.
Evidently, in the active medium analysis, the possibilities for staining viruses—particularly inactivated ones (for instance, by preliminary heating to 80–90 °C for SyBR green-I staining [16,17]), not fixed upon dispersive particles (nanogold, etc.) or in agaroze immobilizing beads [18], not genetically modified for fluorescent protein expression following cellular penetration [19,20], and so forth—are typically substantially limited. Particularly challenging is identifying, analyzing, and measuring in the natural infection transmission environment infectious viral agents transmitted via aerosol/airborne-droplet routes. Among the most well-known, widespread, and readily differentiable diseases (easily attributable to specific viruses) with pronounced etiology, this encompasses an extremely significant stratum of infections, beginning with upper respiratory tract infections (URTI), encompassing respiratory syncytial viral infection, rhinoviral and adenoviral infections requiring pathognomonic identification via virometry, and extending to influenza/parainfluenza, measles, mumps, adenoviral infection, and so forth. Viral determination without cultivation can be realized only in the abiogenic transmission environment. Therefore, for infections transmitted via airborne-droplet (aerosol) routes, implementation of a method enabling analysis with particle capture in the natural atmosphere is necessary. Simultaneously, in physiological aerosols, droplet size may substantially exceed viral dimensions themselves; however, standard virometry recognizes viral aggregation effects, through which "viral aggregometry" is realized on standard flow cytometers (FACS), enabling detection of HSV-1 and certain other specifically or "physiochemically" aggregating viral particles with aggregate sizes exceeding the 300–500 nm threshold applicable to most cytometers with non-super-resolution optics and factory software settings. Consequently, were there a sufficiently broad-range measurement method with calibration to specific geometric prototypes, providing size determination discreteness sufficient for determining viral particle types, aggregation, and other particles within statistical deviation limits attributable to anisotropic particle orientation variation during "gasometry" atmosphere passage through the optical flow volume, followed by partial viral deposition upon selective "sieves" for subsequent size determination validation, then the technique enabling such aerosol viral dispersive-morphological analysis would, in principle, address the majority of typical infectious epidemic/pandemic disease cases transmitted via airborne-droplet routes.
Diffusion Aerosol Spectrometers and Their Applications Proximal to Virometry
Industrially manufactured virometry systems of this designation do not exist. Owing to the sub-wavelength dimensions of most viruses, biomedical engineers typically do not emphasize such problems, regarding them as inherently intractable or prohibitively expensive, though this does not necessarily reflect the reality of functional devices. (For instance, the so-called "super-resolution microscope" of Skrynnik, positioned in 2015 as a means of achieving such visualization of sub-wavelength bio objects without dyes [21], despite extreme financing by Russian standards at the level of Russian Science Foundation and "Skolkovo" grants amounting to approximately 3 million rubles for optical installation components alone, has to date failed to deliver ultrastructural or cytometric super-resolution.) However, this does not imply that the very ideology of such measurements lacks legitimacy. Already in the 1970s, the USSR developed aerosol spectrometry systems for laser-resonator particle measurements [22], including the capability to calculate their hydrodynamic radius [23] (Stokes-Einstein radius; in spherical approximation; though similar calculations are possible for non-spherical particles according to Kunkel [24]), multiplexed and probabilistic-statistical analytical studies [25,26]. Laser aerosol and hydrosol counters were applied to analyze condensation nucleus formation, comparable in size [27] to the aforementioned viral aggregates, and formation of submicron aerosols [28,29] in natural environments, likewise comparable in dispersity to large viruses and viral aggregates. The simplicity of calibrating photoelectric, essentially photoelectronic counters (regulated by photomultiplier power supply) [30] and the possibility of investigating typical hygienic problems (for instance, measuring filter bypass coefficients—the ratio of air contamination at filter outlet to inlet contamination; or analogous filter coefficients determining the ratio of air dustiness at outlet to inlet [31]), for physical and ergonomic reasons, render rational the use of such systems as prototypes for a distinct class of virometers.
The Diffusion Aerosol Spectrometer (DAS Model 2702) can operate in monitoring mode, covering the size range from 3 to 200 nm, implementing not only measurement of total nanoparticle concentration and size distribution or "ultrafine particles" in this range, but also environmental parameters (air or carrier gas temperature, pressure, and humidity). Consequently, given that most viral sizes range from 20 nm to 300 nm, no fundamental restrictions exist for measurement across a broad range of most prevalent viruses. Regarding residuals beyond the 200 nm boundary, as well as extreme cases of micron aggregates (Pandoravirus sp., Pithovirus sp., Filoviridae, etc.), the model 2702-M includes the capability to be equipped with a submicron particle measurement module covering the range from 0.2 to 10 μm. Moreover, for any physically permissible particles, particle enlargement is realizable (via an enlargement module to optically active size). The applicability potential of aerosol and hydrosol optical counting systems, which has generated substantial resonance since the 1970s [32], can be implemented in submicron-range virometry under any physically descriptive environmental conditions favoring or inhibiting viral material proliferation: submicron particles from arid zones and aerosol parameter variations in high-altitude conditions (with corresponding atmospheric conditions, including distinct pressure and insolation—that is, natural source energy irradiance)—were measured on such equipment in the 1980s [33,34].
Moreover, laser aerosol spectrometry is likewise applicable to the problem of viral genetic material survival in cosmic conditions, as well as to the related problems of space biology/astrobiology and so-called "cosmic abiogenesis" based on particles [35,36], or virus-mediated panspermia (or astrosol transport [37,38]) and other problems of gas-phase exobiology [39,40] (typically based on gas-phase and aerosol organic chemistry reaction principles [41,42]). Thus, in the 1980s, Zhulanov aerosol counters operated on Venus aboard the Soviet spacecraft VEGA-1 and VEGA-2 [43], through which the cloud layer structure and size distribution of Venusian atmospheric aerosols [44,45] therein were studied, and the mechanism of cloud layer formation [46] was investigated. From an engineering perspective, this task encompassed creation of a specialized photoelectric converter and signal transmitter [47]. Therefore, analysis of dispersity and size spectra of organic aerosols, to which, by additive chemistry (consisting predominantly of organogenic elements), bioaerosols [48,49] belong (including those containing nucleic acids—as do viruses [50]), using laser aerosol spectrometers is self-evident both in native (terrestrial) and in xenobiotic/exobiological monitoring conditions. At present, it is evident that laser radiation utilization may prove useful in this method not solely as a source of metrological information regarding particle/viral Stokes radius, but also as a source of desorption-ionization for laser-mediated (particularly LAMMA-like, MALDI/ELDI-like, etc.) mass spectrometry of such particles, effectively replacing pyrolytic viral infection measurement methods previously applied at larger sample scales [51]. Thus, from the perspective of verifying viral concepts of abiogenesis and evolution (corresponding member of the Russian Academy of Sciences V.I. Agol [52] and others), laser aerosol spectrometry likewise constitutes an excellent instrumental tool.
Expanded Possibilities of Optical Laser Biochemical / Biophysical Techniques Based upon Evolutionarily-Divergent Analysis of Semantides and Episemantides
Based on the contemporary molecular genetics concepts, which propose a divergent origin of enzymatic function evolution, typically coupled with genome expression (beginning with proto-ribozymes combining RNA and enzyme functions), it is appropriate to pose the question of separating the functions and the corresponding biochemical agents using laser-assisted analysis of the particle size/dispersive parameters, which provided transcription and translation processes [53,54]. In this connection, fundamental significance is possessed by the separation of viruses (incapable of autonomous replication, yet not constituting "cellular machinery" / episemantides for servicing replication of other elements – the so-called primary and secondary "semantides" [55]; because “Episemantides are molecules, precursors of which often are taken up from the environment and modified, built up, or combined, through enzymatic action, into other molecular units. Semantides are informational macromolecules” [56]) and the native genetic code carriers—"semantides," on the one hand, and semantides and episemantides as small molecules and individual enzymes and allosteric regulators, on the other hand, in accordance with their dimensions. There is an effective minimum cell size, lying in the submicron range [57,58], based upon the compact localization of the minimum components providing genome expression functions. It is impossible to reduce this size further, excepting the possibilities of the function combination in a single structure (such as peptide nucleic acid combining "semantide"/nucleic acid and "episemantide"/peptide structural elements [59]).
The existence of a minimum size of a functional cellular structure with expressible genetic material can be interpreted only from the standpoint of evolutionary cytometry, providing separation of certain semantides and episemantides (by Stokes radius and ellipticity descriptors). Considering the approximately half-century debates [60,61] regarding the genetic code origin and evolution from non-genetic precursors, as well as an intensive development of the "viral world" or “virus world” [62,63] concept, representing an important step in this evolution, occurring not as "autonomous viral genome evolution," but as coevolution of self-assembling capsids, viruses proper, or capsidless elements [64], it is logical to analyze, using optical virometry methods (considering not only DNA and RNA viruses, but also their possible analogues/precursors from xenonucleic acids [65], existing in the "XNA world" [66] period preceding the "RNA world"), structures participating in the divergent evolution of the organized elements with different sizes and semantide/episemantide natures and sterically linked thereto (such as PNA-like viral particle analogues, capable of cooperative self-assembly [67]).
The appropriateness of employing the Stokes radius descriptor for characterizing episemantides and tertiary semantides—proteins—directly follows from dynamic light scattering principles and enumeration of various episemantide and tertiary semantide classes whose Stokes radii have been measured and calculated since the 1960s:
a) Enzymes: glutamate dehydrogenases (L-glutamate: NAD oxidoreductase 1.4.1.2) [68], grape catechol oxidases [69], etc.
b) Hormones: pituitary hormones [70], radioiodinated iodothyronines [71]
c) Vitamins: for instance, vitamin B₁₂ versus transcobalamin [72], including radiocobalt-labelled variants for parallel gel filtration on sephadexes [73,74]
d) Globular proteins—spheroproteins (for instance, milk proteins) [75,76]
e) Antibodies/immunoglobulins [77]
f) Secreted proteins, plasma membrane transmembrane proteins, and lysosomal proteins maturing in the Golgi apparatus [78]
g) Ion channels: for instance, GABAA receptor—ligand-dependent channels, also known as benzodiazepine receptors [79]
h) Neuroenzymes (for instance, acetylcholinesterase [80]) and neurotransmitter receptors (for instance, muscarinic receptor—a serpentine receptor effecting signal transmission through heterotrimeric G-proteins [81])
i) Supramolecular biocoordination metalorganic chemistry or bioinorganic chemistry and metallomics agents: for instance, chelators—metallothioneins [82], the highly conserved calcium-binding protein calmodulin [83].
Viral dimensions have obviously likewise been measured. Qualitative correspondence was observed between the data obtained through dynamic light scattering [84] and the data obtained using chromatography (employing an agarose gel) [85]. Strictly speaking, from the cross-validation perspective, for most cases of obtaining ambiguous data of this type, independent methods—that is, operating on different physical principles—for measuring hydrodynamic radius should be employed. In this regard, it is appropriate to indicate the existence of such measurement methods for this descriptor as capillary electrophoresis, time-dependent gradient electrophoresis [86,87], related methods based upon isoelectric point (isoelectric focusing and analogous methods) [88]; gel filtration [89] on inorganic and organic supports (from conventional sephadexes to silicon-containing porous gels [90,91]) or size-exclusion chromatography [92]; time-of-flight methods (predominantly for nanostructures such as fullerenes [93,94]); diverse dialysis variants [95], including under external field action. The only problem is that most of the above methods do not permit assessment of the molecular asymmetry at the measurement stage [96]. This can prove critical: an elongated molecule, from a hydrodynamic perspective, will behave as a spherical molecule of larger radius and correspondingly greater mass. Friction force depends upon the moving particle's surface area; therefore, upon increasing the surface area, friction force as the physical criterion of the above methods will not remain constant upon compression of a given molecule (for instance, in certain methods employing porous supports). Yet without fulfilling the equivalence condition of molecular form and density, mass-based interpolation cannot replace the radius metric fixed through gel filtration. Additionally, since conformation contribution is fixed also from the environment—solvent (for instance, molecular hydration may occur, which is true for virtually all biorelevant episemantide ions not smaller than 5–6 ångströms), comparing optoelectronic hydrodynamic radius measurement data with chromatography and electrochemical method data is actually impossible without observing equivalences regarding the medium properties (and in thin-layer methods—the substrate properties). Polarization per se can likewise affect symmetry/asymmetry.
Environmental Effects and Self-Organization of Prebiotic Synthesis Products as Features for Laser-Assisted Measurements
Owing to the reactivity of biomacromolecule parameters (whether primary or secondary, or tertiary semantides, as well as episemantides) towards the environmental factors, the experimental method with programmable environmental conditions can likewise be given the character of an experiment modeling analogous environmental changes in protobiological or early chemical-evolutionary conditions. Therefore, it is appropriate, in this connection, to note that for variable environmental conditions, it is more important to employ the monitoring methods (not static point measurement) of hydrodynamic radius of a series of biorelevant or model/biomimetic molecules not for the structures, but for the processes. For instance, from the perspective of analyzing prebiological particle self-organization (particularly underlying self-organizing viral structure or particle formation, as well as magnetic hybrid "metalorganic" viral particles [97-99]), investigation of the particle aggregation and dissociation, characterized through hydrodynamic radius measurement methods [100,101], is appropriate. From the membrane-mimetic perspective, particularly evolutionary membrane-mimetics and evolutionary membrane biomimetics, one can consider the data on bioavailable particle transport processes through membranes and membrane models. Whilst work in this direction has proceeded exclusively in biomedical and veterinary applications, employing dialyzable sheep erythrocytes [102,103] and Bruch's membranes (vitreous plates of eyes) [104], the biochemical and cytological reasons for investigating arbitrary membranes using various labels introducing minimal contribution to the measurement results [105] appear evident, at least since the early 1980s. It may also be appropriate to employ supramolecular self-assembly principles as methods for demarcating the target agents and that portion of the signal which, not constituting noise in the strict sense, predominates over those target signals identifiable with viruses. For instance, in nanotechnological synthesis of artificial viruses [106,107], self-assembly principles are frequently employed [108]. Self-assembly of supramolecular polymeric viral mimetics (for example, of tobacco mosaic virus mimetic [109]) is known, and self-assembly of synthetic viral capsid structures based on a 24-mer peptide is also possible [110]. The founder of such work is considered to be a corresponding member of the USSR Academy of Sciences B.F. Poglazov, who conducted relevant work on the artificial viral particle reconstruction at the Institute of Molecular Biology of the USSR Academy of Sciences, at the Department of Biochemistry of the Biological-Soil Faculty of Moscow State University, and at the Interfaculty Problem Research Laboratory of Molecular Biology and Bioorganic Chemistry of Moscow State University (now known as the A.N. Belozersky Research Institute of Physical-Chemical Biology of Moscow State University). However, as a consequence of self-organization, self-assembling viral particles, beginning at the nanosize level, frequently exhibit structural polymorphism [111], similar to that observed for calibration particles in laser-aerosol-spectrometric investigations. However, owing to the identity of self-organization mechanisms, it is substantially less than the scatter of mechanically obtained particles (for example, latex particles or other calibration particles).
Most likely, these phenomena and artifacts, as well as ordinary viruses, will be studied not only by cryo-electron microscopy and AFM, but also by super-resolution optical and fluorescence microscopy [112-123].
Conclusion
The very statistical proximity of the self-assembling structure sizes may in certain cases be a descriptor of their biological, biogenic, abiogenic or biomimetic origin – and a descriptor of the chemical evolution stages for prebiotic synthesis and self-assembly. Residuals or heteroscedasticity indicators of the sample, based upon the optical signal registration data, also may constitute criteria for distinction from the reference standard biomaterial (or model abiogenic / prebiotic or biomimetic / synthetic biological material).
References
- Maltsev VP, Chernyshev AV, Semyanov KA, Soini E (1996) Absolute real-time measurement of particle size distribution with the flying light-scattering indicatrix method. Applied optics 35(18): 3275-3280.
- Shvalov AN, Surovtsev IV, Chernyshev AV, Soini JT, Maltsev VP (1999) Particle classification from light scattering with the scanning flow cytometer. Cytometry: The Journal of the International Society for Analytical Cytology 37(3): 215-220.
- Zamora JLR, Aguilar HC (2018) Flow virometry as a tool to study viruses. Methods 134: 87-97.
- Zicari S, Arakelyan A, Fitzgerald W, Zaitseva E, Chernomordik LV, et al. (2016) Evaluation of the maturation of individual Dengue virions with flow virometry. Virology 488: 20-27.
- Gaudin R, Barteneva NS (2015) Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles. Nature communications 6: 6022.
- Arakelyan A, Fitzgerald W, Zicari S, Vagida M, Grivel JC, et al. (2017) Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles. Journal of visualized experiments 119.https://pubmed.ncbi.nlm.nih.gov/28190041/
- Landowski M, Dabundo J, Liu Q, Nicola AV, Aguilar HC (2014) Nipah Virion Entry Kinetics, Composition, and Conformational Changes Determined by Enzymatic VLPs and New Flow Virometry Tools. Journal of virology 88(24): 14197-14206.
- Bonar MM, Tilton JC (2017) High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry. Virology 505: 80-90.
- DeSantis MC, Kim JH, Song H, Klasse PJ, Cheng W (2016) Quantitative correlation between infectivity and Gp120 density on HIV-1 virions revealed by optical trapping virometry. Journal of Biological Chemistry 291(25): 13088-13097.
- Arakelyan A, Fitzgerald W, King D, Souza BDV, Grivel ZSJC, et al. (2016) Some HIV-1 virions are more equal than others: mosaics of envs on individual HIV virions as evaluated with flow virometry. JAIDS Journal of Acquired Immune Deficiency Syndromes 71: 68.
- Arakelyan A, Fitzgerald W, King DF, Rogers P, Cheeseman HM, et al. (2017) Flow virometry analysis of envelope glycoprotein conformations on individual HIV virions. Scientific reports 7(1): 948.
- Schroeder M (2009) Fractals, chaos, power laws. – Dover, Mineola pp. 430.
- Nasonov DN (1962) Local Reaction of Protoplasm and Propagating Excitation. In: (2nd), — Moscow; Leningrad: Publishing House of the USSR Academy of Sciences.
- Nasonov DN (1962) Local reaction of protoplasm and gradual excitation. National Science Foundation (USA), Washington, by the Israel Program for Scientific Translations; [available from the Office of Technical Services, U.S. Dept. of Commerce, Washington], pp. 425.
- Lippé R (2018) Flow virometry: a powerful tool to functionally characterize viruses. Journal of virology 92(3): e01765-17.
- Marie D, Brussaard CPD, Thyrhaug R, Bratbak G, Vaulot D (1999) Enumeration of marine viruses in culture and natural samples by flow cytometry. Applied Environmental Microbiology 65: 45–52.
- Brussaard CP (2004) Optimization of procedures for counting viruses by flow cytometry. Applied Environmental Microbiology 70: 1506-1513.
- Rappé MS, Giovannoni SJ (2003) The uncultured microbial majority. Annual Revues in Microbiology 57: 369–394.
- Loret S, El Bilali N, Lippé R (2012) Analysis of herpes simplex virus type I nuclear particles by flow cytometry. Cytometry A 81: 950-959.
- El Bilali N, Duron J, Gingras D, Lippé R (2017) Quantitative evaluation of protein heterogeneity within herpes simplex virus 1 particles. Journal of Virology 91.
- Skrynnik A (2015) Laser scanning biological microscope for non-invasive studies of the cell structures without fluorescent dyes. Super-resution in different dimensions. OJSC Human Stem Cell Institute, Moscow, Russia pp. 87.
- Zhulanov YV, Sadovskii BF, Petryanov IV (1975) Use of a laser resonator for raising the sensitivity of laser-type aerosol spectrometers. Soviet Physics Doklady 20: 810-812.
- Zhulanov YV, Sadovskii BF, Petryanov IV (1978) Automatic method for determining the stokes size of aerosol-particles. Colloid Journal of the USSR 40(4): 637-641.
- Kunkel WB (1948) Magnitude and character of errors produced by shape factors in Stokes' law estimates of particle radius. Journal of Applied Physics 19(11): 1056-1058.
- Zhulanov YV, Lushnikov AA, Nevskiy IA (1985) Unique Features of Multiple Counting of Particles by Photoelectric Counters. Izvestiya, Atmospheric and Oceanic Phys 21(11): 885-889.
- Zhulanov YV, Lushnikov AA, Nevskiy IA (1986) Probability Method of Analyzing Dispersed Systems. Izvestiya: Atmospheric and oceanic physics 22: 39.
- Zhulanov IV, Petrianov IV (1978) Sadovskii BF Photoelectric laser spectrometer for large condensation nuclei. Academy of Sciences of the USSR, Atmospheric and Oceanic Physics 14: 520-526.
- Zhulanov YV, Sadovskii BF, Nikitin ON, Petryanov IV (2021) Investigation of the maritime submicron aerosols. Doklady Akademii Nauk SSSR 242(4): 800-803.
- Zhulanov YV, Petryanov IV (1980) (The) Mechanism of generation of marine aerosols. Doklady Earth Sciences pp. 250.
- Zhulanov YV (1979) Sedimentation method for calibrating photoelectric aerosol counters. Measurement Techniques 22(9): 1138-1139.
- Karneeva NY, Zhulanov YV, Belov SV, Pavlikhin GP, Krasovitskaya KA (1981) Investigation of the fraction coefficients of overshooting by a laser aerosol spectrometer (Abstracts of Deposited Papers). Journal of Engineering Physics 41(3).
- Zhulanov YV, Sadovskiy BF, Petryanov IV (1978) Potential of optical analysis of aerosol systems. Doklady Earth Sciences 238.
- Zhulanov YV, Zagaynov L, Yu S, Nevskiy IA, Stulov D (1986) Highly dispersed submicron atmospheric aerosols of the arid zone]. Izvestiya Akademii Nauk SSSR, Fizika Atmosfery I Okeana 22: 29-40.
- Zagajnov V, Zhulanov Y, Lushnikov A, Stulov L, Osidze I, et al. (1987) Diurnal-variations of the atmospheric aerosol parameters of mountain regions. Izvestiya Akademii Nauk SSSR. Fizika Atmosfery I Okeana 23(12): 1323-1329.
- Donaldson DJ, Tervahattu H, Tuck AF, Vaida V (2004) Organic aerosols and the origin of life: An hypothesis. Origins of Life and Evolution of the Biosphere 34(1-2): 57-67.
- Tervahattu H, Tuck A, Vaida V (2004) Chemistry in prebiotic aerosols: a mechanism for the origin of life. In: Cellular Origin and Life in Extreme Habitats and Astrobiology 6: 153-165.
- Targulian VO, Mergelov NS, Goryachkin SV (2017) Soil-like bodies on Mars. Eurasian soil science 50(2): 185-197.
- Certini G, Scalenghe R, Amundson R (2009) A view of extraterrestrial soils. European journal of soil science 60(6): 1078-1092.
- McKay CP, Stoker CR, Morris J, Conley G, Schwartz D (1986) Space station gas-grain simulation facility: Application to exobiology. Advances in Space Research 6(12): 195-206.
- Coll P, Coscia D, Smith N, Gazeau MC, Ramırez SI, et al. (1999) Experimental laboratory simulation of Titan’s atmosphere: aerosols and gas phase. Planetary and Space Science 47(10-11): 1331-1340.
- Raulin F (1997) Titan's organic chemistry and exobiology. Huygens: Science, Payload and Mission 1177: 219.
- Raulin F, Coll P, Smith N, Benilan Y, Bruston P, et al. (1999) New insights into Titan's organic chemistry in the gas and aerosol phases. Advances in Space Research 24(4): 453-460.
- Zhulanov YV, Mukhin LM, Nenarokov DF (1986) Aerosol Counts in Venus Clouds-Preliminary VEGA-1 and VEGA-2 Density Profiles h= 63-47-KM. Soviet Astronomy Letters 12: 49-52.
- Zhulanov YV, Mukhin LM, Nenarokov DF (1987) Structure of the cloud layer in the Venusian atmosphere (the Vega project). Doklady Earth Sciences 292.
- Zhulanov YV, Mukhin LM, Nenarokov DF (1987) Particle-size distributions in the cloud layer of the Venusian atmosphere (the Vega experiment). Doklady Earth Sciences 295.
- Zhulanov YV, Mukhin LM, Nenarokov DF (1987) Mechanisms generating the cloud layer in the Venusian atmosphere. Doklady Earth Sciences 295.
- Zhulanov YV (1988) Photoelectric transducer for analysis of aerosols in the cloud layer of the atmosphere of Venus.Colloid Journal USSR (English Translation) 50(2): 221-228.
- Lee BU (2011) Life comes from the air: a short review on bioaerosol control. Aerosol and Air Quality Research 11(7): 921-927.
- Zhen H, Han T, Fennell DE, Mainelis G (2014) A systematic comparison of four bioaerosol generators: Affect on culturability and cell membrane integrity when aerosolizing Escherichia coli bacteria. Journal of Aerosol Science 70: 67-79.
- King MD, McFarland AR (2012) Bioaerosol sampling with a wetted wall cyclone: cell culturability and DNA integrity of Escherichia coli bacteria. Aerosol Science and Technology 46(1): 82-93.
- Madonna AJ, Voorhees KJ, Hadfield TL, Hilyard EJ (1999) Investigation of cell culture media infected with viruses by pyrolysis mass spectrometry: Implications for bioaerosol detection. Journal of the American Society for Mass Spectrometry 10(6): 502-511.
- Agol VI (1976) An aspect of the origin and evolution of viruses. Origins of life 7(2): 119-132.
- Yoshikuni Y, Ferrin TE, Keasling JD (2006) Designed divergent evolution of enzyme function. Nature 440(7087): 1078.
- Gerlt JA, Babbitt PC (2001) Divergent evolution of enzymatic function: mechanistically diverse superfamilies and functionally distinct suprafamilies. Annual review of biochemistry 70(1): 209-246.
- Zuckerkandl E, Pauling L (1965) Molecules as documents of evolutionary history. Journal of theoretical biology 8(2): 357-366.
- Zuckerkandl E (1979) Controller node complexity: a measure of the degree of gene coordination. Journal of molecular evolution 14(4): 311-321.
- Forster AC, Church GM (2006) Towards synthesis of a minimal cell. Molecular systems biology 2: 45.
- Porcar M, Danchin A, de Lorenzo V, Dos Santos VA, Krasnogor N, et al. (2011) The ten grand challenges of synthetic life. Systems and synthetic biology 5(1-2): 1-9.
- Uhlmann E, Peyman A, Breipohl G, Will DW (1998) PNA: synthetic polyamide nucleic acids with unusual binding properties.Angewandte Chemie International Edition 37(20): 2796-2823.
- Woese CR (1965) On the evolution of the genetic code. Proceedings of the National Academy of Sciences of the United States of America 54(6): 1546.
- Jukes TH (1973) Possibilities for the evolution of the genetic code from a preceding form. Nature 246(5427): 22.
- Koonin EV, Senkevich TG, Dolja VV (2006) The ancient Virus World and evolution of cells. Biology direct 1(1): 29.
- Kristensen DM, Mushegian AR, Dolja VV, Koonin EV (2010) New dimensions of the virus world discovered through metagenomics. Trends in microbiology 18(1): 11-19.
- Koonin EV, Dolja VV (2014) Virus world as an evolutionary network of viruses and capsidless selfish elements. Microbiology and Molecular Biology Reviews 78(2): 278-303.
- Hunter P (2013) XNA marks the spot: What can we learn about the origins of life and the treatment of disease through artificial nucleic acids? EMBO reports 14(5): 410-413.
- Pinheiro VB, Holliger P (2012) The XNA world: progress towards replication and evolution of synthetic genetic polymers. Current opinion in chemical biology 16(3-4): 245-252.
- Cao M, Wang N, Zhou P, Sun Y, Wang J, et al. (2016) Virus-like supramolecular assemblies formed by cooperation of base pairing interaction and peptidic association. Science China Chemistry 59(3): 310-315.
- Page M, Godin C (1970) Determination of the Stokes radius of native and succinylated glutamate dehydrogenase on Sepharose 4B. Journal of Chromatography A 50: 66-71.
- Lerner HR, Mayer AM (1975) Stokes' radius changes of solubilized grape catechol oxidase. Phytochemistry 14(9): 1955-1957.
- Ryan RJ (1969) Stokes radius of human pituitary hormones and demonstration of dissociation of luteinizing hormone. Biochemistry 8(2): 495-501.
- Ribela MTCP, Bartolini P (1988) Stokes radius determination of radioiodinated polypeptide hormones by gel filtration. Analytical biochemistry 174(2): 693-697.
- Hippe E (1970) Changes in Stokes radius on binding of vitamin B12 to human intrinsic factor and transcobalamins. Biochimica et biophysica acta 208: 337-339.
- Hom BL (1969) Stokes radius of 57Co-labeled vitamin B12-transcobalamin I and II determined by Sephadex gel filtration. Biochimica et Biophysica Acta (BBA)-Protein Structure 175(1): 20-23.
- Olesen H, Rehfeld J, Hom BL, Hippe E (1969) Stokes radius of 57Co-labelled vitamin B12-transcobalamin I and II and 125I-labelled insulin estimated by sephadex G-2000 gel filtration in human plasma AT 37°. Biochimica et Biophysica Acta (BBA)-Protein Structure 194(1): 67-70.
- Tanev G (1973) Determination of molecular weight, diffusion constant and Stokes' radius of alphas-casein of ewe's milk. Zhivotnovudni nauki.
- Kaur S, Bhatia KL (1990) Free secretory component of buffalo milk. Part II: Stokes radius and partial specific volume. Indian Journal of Dairy Science 43(3): 411-413.
- Chandy KG, Stockley RA, Burnett D (1980) The estimation of Stokes radius for secretory immunoglobulin A, using thin-layer chromatography followed by crossed immunoelectrophoresis. Hoppe-Seyler's Zeitschrift fur physiologische Chemie 361(12): 1855-1857.
- Bendiak B, Ward LD, Simpson RJ (1993) Proteins of the Golgi apparatus: Purification to homogeneity, N-terminal sequence, and unusually large Stokes radius of the membrane-bound form of UDP-galactose: N-acetylglucosamine β1-4galactosyltransferase from rat liver. European journal of biochemistry 216(2): 405-417.
- Ray JP, Mernoff ST, Sangameswaran L, de Blas AL (1985) The stokes radius of the CHAPS-solubilized benzodiazepine receptor complex. Neurochemical research 10(9): 1221-1229.
- Bon S, Rieger F, Massoulié J (1973) Properties of elongated acetylcholinesterase molecules in solution. Stokes radius, density, and mass. European journal of biochemistry 35(2): 372.
- Kalinoski L, Potter LT (1980) Stokes radius of the muscarinic receptor protein from rat-brain. FASEB Proceedings 39(3): 1008-1008.
- Phillips JL (1983) Effect of zinc on the Stokes' radius of metallothionein. Biochemical and biophysical research communications 114(3): 998-1000.
- Sorensen BR, Shea MA (1996) Calcium binding decreases the stokes radius of calmodulin and mutants R74A, R90A, and R90G.Biophysical journal 71(6): 3407-3420.
- Oliver CJ, Shortridge KF, Belyanin G (1976) Diffusion coefficient and molecular weight of type 5 adenovirus by photon-correlation spectroscopy. Biochimica et Biophysica Acta (BBA)-General Subjects 437(2): 589-598.
- Shortridge KF, Belyavin G, Oliver CJ (1976) Estimation of type-5 adenovirus particle stokes radius by molecular-sieve chromatography on agarose-gel. Microbiology Letters 2(5): 33-37.
- Castagnola M, Rossetti DV, Cassiano L, Misiti F, Pennacchietti L, et al. (1996) Determination of peptide dissociation constants and Stokes radius at different protonation stages by capillary electrophoresis. Electrophoresis 17(12): 1925-1930.
- Rothe GM (1988) Determination of molecular mass, stokes' radius, frictional coefficient and isomer-type of non-denatured proteins by time-dependent pore gradient gel electrophoresis. Electrophoresis 9(7): 307-316.
- Felgenhauer K (1971) Determination of stokes radius and isoelectric point with microanalytical techniques. Zeitschrift fur Klinische Chemie und Klinische Biochemie 9(5): 455.
- Horiike K, Tojo H, Yamano T, Nozaki M (1983) Interpretation of the Stokes radius of macromolecules determined by gel filtration chromatography. The Journal of Biochemistry 93(1): 99-106.
- Pagé M, Godin C (1969) On the determination of the Stokes radius of glutamate dehydrogenase monomer by gel filtration of Sephadex G-200.Biochimica et biophysica acta 194(1): 329.
- Horiike K (1982) High-speed gel-filtration chromatography of proteins-evaluation and calibration of a column of porous silica-based aqueous gels based on the correlation of distribution coefficient with stokes radius. Biochemistry International 4(5): 477-483.
- Sablonniere B, Lefebvre P, Formstecher P, Dautrevaux M (1987) Improved stokes radius measurement of the glucocorticoid receptor using TSK G4000SW and TSK G3000SW high-performance size-exclusion columns: Analytical and preparative applications. Journal of Chromatography A 403: 183-196.
- Lim SK, Burba ME, Albrecht AC (1993) The Stokes law radius of the C60 radical anion in n-hexane by time-of-flight mobility measurements. Chemical physics letters 216(3-6): 405-408.
- Burba ME, Lim SK, Albrecht AC (1995) Relative electron affinity of C60 and C70 and the Stokes' law radius of the C70 radical anion in n-hexane by time-of-flight mobility measurements. The Journal of Physical Chemistry 99(31): 11839-11843.
- Lantz CA (1979) Isolation and partial physiochemical characterization of a peptic fragment (residues 307-385) of bovine serum albumin which exhibits steroid-binding activity. estimation of its stokes (molecular) radius by a novel thin-film dialysis technique. – Ph.D. Thesis, The University of North Carolina at Chapel Hill.
- Demassie S, Lachance JP (1974) Molecular filtration on sepharose 6b-empiric relationships between distribution coefficient and stokes radius on one hand and molecular-weight on other of proteins of different degrees of asymmetry. Journal of Chromatography 89(2): 251-258.
- Yang L, Liang H, Angelini TE, Butler J, Coridan R, et al. (2004) Self-assembled virus–membrane complexes. Nature materials 3(9): 615.
- Zhao Q, Chen W, Chen Y, Zhang L, Zhang J, et al. (2011) Self-assembled virus-like particles from rotavirus structural protein VP6 for targeted drug delivery. Bioconjugate chemistry 22(3): 346-352.
- Huang X, Bronstein LM, Retrum J, Dufort C, Tsvetkova I, et al. (2007) Self-assembled virus-like particles with magnetic cores. Nano letters 7(8): 2407-2416.
- Le Maire M, Arnou B, Olesen C, Georgin D, Ebel C, et al. (2008) Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca 2+–ATPase as an example. Nature protocols 3(11): 1782.
- Castagnola M, Rossetti DV, Misiti F, Cassiano L, Giardina B, et al. (1997) Modification of capillary electrophoresis selectivity in hydro-organic solutions: dissociation constants and Stokes radius measurements of peptides in water–2, 2, 2-trifluoroethanol mixtures. Journal of Chromatography A 792(1-2): 57-65.
- DeLoach JR, Andrews K (1986) Stability of membrane pores in hypotonically dialyzed erythrocytes: coencapsulation of different stokes radius probes can occur for at least 21 days in human erythrocytes. Biotechnology and applied biochemistry 8(6): 537-545.
- DeLoach JR, Andrews K (1986) Membrane pores in hypotonically dialyzed sheep erythrocytes remain open after three weeks of storage: entrapment of different stokes radius probes, [14C] sucrose and [3H] inulin. Biotechnology and applied biochemistry 8(6): 546-552.
- Santiago ZA, Marmorstein AD, Marmorstein LY (2011) Relationship of stokes radius to the rate of diffusion across Bruch's membrane. Investigative ophthalmology & visual science 52(7): 4907-4913.
- Aimoto S, Richards FM (1981) Synthesis of carriers of differing Stokes radius with activated acyl groups for use as reagents in labeling membrane proteins. Journal of Biological Chemistry 256(10): 5134-5143.
- Zuber G, Dauty E, Nothisen M, Belguise P, Behr JP (2001) Towards synthetic viruses.Advanced drug delivery reviews 52(3): 245-253.
- Mastrobattista E, Van Der Aa MA, Hennink WE, Crommelin DJ (2006) Artificial viruses: a nanotechnological approach to gene delivery. Nature reviews Drug discovery 5(2): 115.
- Lim YB, Lee E, Yoon YR, Lee MS, Lee M (2008) Filamentous artificial virus from a self-assembled discrete nanoribbon.Angewandte Chemie International Edition 47(24): 4525-4528.
- Percec V, Heck J, Lee M, Ungar G, Castillo AA (1992) Poly {2-vinyloxyethyl 3, 4, 5-tris [4-(n-dodecanyloxy) benzyloxy] benzoate}: a self-assembled supramolecular polymer similar to tobacco mosaic virus. Journal of Materials Chemistry 2(10): 1033-1039.
- Matsuura K, Watanabe K, Matsuzaki T, Sakurai K, Kimizuka N (2010) Self-assembled synthetic viral capsids from a 24-mer viral peptide fragment. Angewandte Chemie International Edition 49(3): 9662-9665.
- Nguyen HD, Brooks CL (2008) Generalized structural polymorphism in self-assembled viral particles. Nano letters 8(12): 4574-4581.
- Li L, Guo W, Yan Y, Lee S, Wang T (2013) Label-free super-resolution imaging of adenoviruses by submerged microsphere optical nanoscopy. Light: Science & Applications 2(9): e104-e104.
- Müller B, Heilemann M (2013) Shedding new light on viruses: super-resolution microscopy for studying human immunodeficiency virus. Trends in microbiology 21(10): 522-533.
- Laine RF, Albecka A, Van De Linde S, Rees EJ, Crump CM, et al. (2015) Structural analysis of herpes simplex virus by optical super-resolution imaging. Nature communications 6(1): 5980.
- Li Z, Fang C, Su Y, Liu H, Lang F, et al. (2016) Visualizing the replicating HSV-1 virus using STED super-resolution microscopy. Virology journal 13(1): 65.
- Gray RD, Beerli C, Pereira PM, Scherer KM, Samolej J, et al. (2016) VirusMapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy. Scientific reports 6(1): 29132.
- Chojnacki J, Eggeling C (2018) Super-resolution fluorescence microscopy studies of human immunodeficiency virus. Retrovirology 15(1): 41.
- Long RK, Moriarty KP, Cardoen B, Gao G, Vogl AW, et al. (2020) Super resolution microscopy and deep learning identify Zika virus reorganization of the endoplasmic reticulum. Scientific Reports 10(1): 20937.
- Domínguez GI, Massaguer PE, Cervera L, Gòdia F (2020) Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis. Biotechnology and bioengineering 117(7): 1929-1945.
- Gast M, Wondany F, Raabe B, Michaelis J, Sobek H, et al. (2020) Use of super-resolution optical microscopy to reveal direct virus binding at hybrid core–shell matrixes. Analytical chemistry 92(4): 3050-3057.
- Touizer E, Sieben C, Henriques R, Marsh M, Laine RF (2021) Application of super-resolution and advanced quantitative microscopy to the spatio-temporal analysis of influenza virus replication. Viruses 13(2): 233.
- Romero AM, Pujals S, Albertazzi L (2021) Towards a quantitative single particle characterization by super resolution microscopy: From virus structures to antivirals design. Frontiers in Bioengineering and Biotechnology 9: 647874.
- Robb NC (2022) Virus morphology: Insights from super-resolution fluorescence microscopy. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1868(4): 166347.
