Optical Ultrastructural Virometry and Its Limitations
The identification of the standard sizes of viral, aggregative and other particles using the virion flow analysis by the light-scattering and fluorescence spectrum requires preliminary calibration of the measuring instrument. Calibration should be carried out according to specific geometric prototypes with specific dimensions. However, the size of viruses varies over a wide range of lengths ranging from nano- to submicrometer sizes. This leads to measurement uncertainty for a wide range of viruses. The software for widely implemented optical instruments is based on a simplified interpretation of the Mie scattering theory, extending it to almost the entire range of particle sizes, even submicrometer ones. The review examines the metrological problems of optical virometry and offers solutions to the issues of instrument calibration.
Along with optical cytometry based on the analysis of a fluorescent (or scattered by a cell [1, 2]) light signal, virometry methods have also shown their effectiveness in recent years. This became especially noticeable when using the flow virometry [3, 4], which was named similarly to flow cytometry (FACS, FCM).
The complex of virometry technologies is a unique tool for analyzing infection profiles , antigenic spectra , kinetic analysis of virus infection, as well as conformational kinetics and comparative analysis of response chemistry, in comparison with the reaction norm . The analysis is mainly focused on the biomolecular aspect, and not on the ultramorphological and size (disperse) differences of viruses.
It should be noted that the USA National Institutes of Health, as part of their current policy, are focusing on the countable number of viruses. This is especially true for studies of the HIV‑1 virus, which is responsible for the AIDS pandemic (HIV‑1 belongs to the Lentiviridae subfamily of retroviruses ). This means that the subset of relevant analytes and drugs, as well as the nomenclature of viral variations, are extremely taxonomically limited [9–11]. Therefore, this method of diagnostic virology cannot be considered universal.
Universal method cannot be based on selective (from biomolecular perspective) carriers. This statement especially applies to supramolecular systems with high specificity of complementary fixation, including the genetic mechanisms of viruses. In principle, it is impossible to create a system capable of equivalent recognition of all types of viruses. This is the limitation of the method. (This implies a system for both DNA and RNA; with a single-stranded or double-stranded DNA genome or a single-stranded or double-stranded RNA genome; for all forms of capsomeres, i. e. – protein subunits / capsid protomers – outer viral envelopes; taking into account the probabilities of detection in samples of both viruses not coated with viral envelope and viruses with xenogenic lipid supercapsid). The statement of such universalized problem contradicts the phyletic diversity of the molecular taxonomy of viruses.
It should be emphasized that the sizes of viruses are in the range of subwavelengths. Therefore, selective fluorescence methods of flow virometry are also not universal for detecting viruses of various geometric shapes. This is due to the indistinguishability of their optical signal (exceptions are PALM / STORM and similar microfluorimetric methods).
It should be noted that the equipment with which modern core facility centers (core facilities)  are equipped has limitations. For example, the BD LSRFortessa SORP cell analyzer is optimized for the size of lymphocytes, monocytes and granulocytes. The method used in the autoMACS Pro fluorescent magnetic bioparticle counter prevents counting of single virus particles. The BD FACS Celesta multicolor flow cytometer (available with 488, 405, 650, 355, and 561 nm lasers) is not capable of measuring structures of the order of the size of viral and other genetic particles. Interference due to parasitic fluorescent signals caused by UV radiation (similar effects have already been observed when using UV microscopes) should be taken into account.
However, if viruses have a morphological difference (in adjecto – differ in the geometric type of the capsid, the protein envelope of the virus), then the light flux scattering indicatrices will also differ among themselves. When the capsids of viruses have spiral, icosahedral, oblong and complex shapes, this is reflected in the shape of the scattering indicatrix. It should also be taken into account that these differences can be constantly observed only in calibrated devices.
Calibration procedures for such measuring systems represent a separate scientific issue. Test particles that correspond in geometry and size to the viruses under study should be selected for calibration. Ideally, they should be automorphic to the virus. For example, to be a virus of the same morphology (i. e. geometry), but inactivated or non-pathogenic type, or be homeomorphic to it. The morphism that does not provide a one-to-one correspondence between the structure of the studied virus and the calibration virus (calibration particle) is not quite sufficient for identification. The reason is that the discrepancy can become a source of artifacts in the decoded diffraction pattern.
However, in the flow virometry, if identical symmetry groups are maintained for the calibration and measuring particles, calibration of the measuring system is possible with their help. A textbook example: a rotavirus with icosahedral symmetry is effectively approximated by a spherical pattern due to the large number of capsomeres (a capsomere is a repeating group of virion structural protein molecules that form an ordered capsid structure). In the language of fractal geometry this is called pertiling .
The developers of scanning flow spectrometers (BioUniScan, Novosibirsk) state that in order to determine the characteristics of particles, it is necessary to solve the inverse problem of light-scattering. This problem is nontrivial even for the case of observation of spherical homogeneous particles of the sublong-wavelength range. Preliminary calibration of the system is as much know-how as the control algorithm / measurement protocol.
It is well known that the sizes of viruses in different taxonomic groups differ by orders of magnitude: from 20 to 300 nm. Although some members of the Filoviridae family with negative single-stranded RNA are up to 1.4 µm in length and 0.08 µm in diameter. And in Pandoravirus sp. and Pithovirus sp., the level of genome compaction, in principle, physically allows going far beyond the micrometer boundaries 1.0–0.5 µm / 1.5–0.5 µm, respectively.
This implies a qualitative difference in metrology and analysis principles for these qualitatively different groups. Despite the widespread simplification, which amounts to extrapolation of the Mie scattering theory interpretations practically to the entire range of particle sizes (including Fraunhofer diffraction as a special case), it is well known that the Mie theory is not suited for small diffraction parameters. The computational capabilities provided by the Mie theory allow the use of one calculation method / algorithm for the additive spectrum of dispersion, that is, particle sizes.
Let us hypothesize that this extrapolation within the framework of measurements of the indicatrices of light beams that have passed through a virus flow is a mistake. This means that part of the sample with viruses will be calculated incorrectly. Let us explain this statement.
It is possible to use simplified calculations only when it comes to monodisperse calibration media, for example, special latexes. They are homoscedastic. As for media containing individual particles that differ both in static geometry and in dynamic morphometric parameters, the heteroscedasticity of the distribution appears. This leads to inefficiency of the assessments, since it is impossible to know in advance the concentration values of individual particles and the contribution to the diffraction pattern of dispersed fractions (or the sizes of ultrastructural and microstructural units of biomaterial). In dynamics, they differ from the assessments performed both according to static geometry and according to dynamic morphometric parameters. These criteria in the reaction norm are a nonstationary correlate of the general reactivity of protoplasm / viroplasm and the propagation of excitation in it [14, 15]. And the qualimetric weighting criterion is difficult to calculate in such problems due to the differences in the geometry (“form factors”) of the particles.
It is said that “if all the particles in the sample are greater than the light wavelength, then part of Fraunhofer theory dominates Mie theory when calculating the particle sizes”. However, after taking into account the anisometric particles (which include viruses – for instance, Filoviridae with an incredible “prosenchymness” coefficient, as this property would be called in the case of plant cell cytometry, according to Takhtadzhian: up to 1.4 to 0.08), an objective numerical expression of the indicated dominance is out of the question (especially, without taking into account orientation) in most realized cases.
Machine-solvable abstract models for calculation of distributions are based on the assumption that the particles have spherical shape. Therefore, the distribution of the particles by size obtained from the analysis is actually the distribution of the “equivalent spherical particles” and not of the actual analyte particles. (It is worth reminding that the classical problem of electrodynamics, solved in 1908 by Gustav Mie, is solved by decomposing the electromagnetic field into spherical harmonics).
For viruses that differ in morphology and genetic or evolutionary position, geometric deviations from the spherical shape will be different. Therefore, the effectiveness of equivalent measurements will differ for systematically different viruses. In this case, the universal technology of “functional virometry” is impossible, since the size of the nomenclature of even the most widespread or relevant viruses varies extremely. Consequently, the diffraction limits and the limits of the optima of applicability of one or another mathematical approach, including the one that differs from Mie theory, also vary.
The functional activity of different virulent forms usually correlates relatively weakly with the averaged morphology calculated in the course of in-line processing of the data from the flow device detector. Even if it is assumed that the measurements of viruses are not carried out on dispersed media, but in an abstract empty environment with the absence of adsorbing agents, the calibration of the measuring system will still be ineffective . Regarding the information insufficiency for functional identification, it should also be noted that Mie theory requires knowledge of the refractive and absorption coefficients of the sample (different for different potential viruses) and the “dispersion medium”, which in the native case is also characterized by some uncertainty, since it is necessary to analyze samples from different environments.
It is apparent that in the case of the analysis of native media, the possibilities of staining viral particles (especially inactivated ones) are also significantly limited. This method is used for staining SyBR green-I [17, 18]); not fixed on disperse particles (nanogold, etc.) or in agarose immobilization beads ; for expression of non-genetically modified fluorescent proteins after penetration into the cell [20, 21].
It is especially difficult to identify, analyze and measure aerosol-transmissible infectious viral agents in the natural transmission environment. These, of the most famous, common and easily differentiated diseases with a pronounced etiology, include a significant number of infections, such as URTI (Upper Respiratory Tract Infections), which combines respiratory syncytial virus infection, rhinovirus and adenovirus infections; influenza / parainfluenza, measles, mumps, adenovirus infection, etc.
Determination of viruses without cultivation can be realized only in the abiogenic transmission environment. Therefore, for aerosol-transmissible infections, it is necessary to introduce a method that provides analysis with the capture of particles in the natural atmosphere.
At the same time, the droplet size in physiological aerosols usually can significantly exceed the size of the virus. However, the virus aggregation effect is also apparent in standard virometry. Therefore, the “viral aggregometry” method is implemented in standard flow cytometers (FACS). The detection of HSV‑1 and a number of other aggregating virus particles with aggregate sizes above the limit of 300–500 nm is achieved due to this method.
These limitations can be overcome by using laser aerosol spectrometers. A surge of interest in aerosol and hydrosol optical counters appeared in the 1970s . At that time, submicron particles of the torrid (arid) zone and variations of aerosol parameters were studied in high-altitude conditions with increased insolation and decreased pressure [23, 24]. Later, in the 1980s, Zhulanov’s aerosol counters operated on Venus on the Soviet spacecrafts VEGA‑1 and VEGA‑2 . They were used to study the structure of cloud layers and the size distribution of the aerosol of the Venusian atmosphere [26, 27], as well as the mechanism of the formation of cloud layers .
Zhulanov’s diffusion aerosol spectrometers measure a spectrum of sizes ranging from the size of molecular clusters to fractions of a micron, that is, they can be optimized for most virometric tasks that do not involve fluorescent tags and beads. There is a modification for monitoring particles with a size of 3–200 nm. This modification allows expanding the upper limit up to 10 microns.
Optical methods of virometry in the submicron range have a high potential in solving problems of analysis of the causes of the formation of self-organizing virus structures or particles [29, 30], magnetic hybrid “organometallic” viral particles . Moreover, laser aerosol spectrometry can be used in survival studies of viral genetic material in space conditions, as well as in related problems of space biology / astrobiology and so-called “space abiogenesis” on the basis of sol particles [32, 33] or virus-mediated panspermia (or astrosole transport [34, 35]) and other problems of gas-phase exobiology [36, 37].
Optical methods of virometry in the submicron range can be successfully implemented in any environment that favors or prevents the replication of viral material. And the issues of the selection of calibration particles in laser-aerosol-spectrometric studies are solved differently if the principles of dynamic light-scattering are used.
Gradov Oleg Valerievich, e-mail: o. firstname.lastname@example.org, Senior Researcher, Department of Dynamics of Chemical and Biological Processes, Institute of Chemical Physics named after Semenov, Moscow, Russia.
Area of interest: microfluidics, patch clamp, ESEM, CLEM, lensless microscopy, YMD, DIC, SPIM, LSFM, biophysical instrumentation
Zhulanov Yuri Vasilievich, Cand. of Scien. (Phys.-Math.), senior researcher, Karlov Institute of Physics Chemistry, Moscow, Russia.
Makaveev Pavel Yurievich, designer of aerosol spectrometers and counters, Karlov Institute of Physics Chemistry, Moscow, Russia.
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