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05.02.00 МАШИНОСТРОЕНИЕ
05.02.05 РОБОТЫ, МЕХАТРОНИКА И РОБОТОТЕХНИЧЕСКИЕ СИСТЕМЫ
MULTI-ANGLE GONIOMETRIC COMPUTER-ASSISTED LAB-ON-A-CHIP READING SYSTEM STAGE FOR VACUUM-GAS CHAMBERS BASED ON ANALYTICAL SCANNING ELECTRON MICROSCOPY PLATFORM (GONIOMETRIC CLEM CHAMBERS)1
Gradov Oleg V., research fellow, Photobionics Laboratory (0412), Department of Dynamics of Biological and Chemical Processes, N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences; senior researcher / senior research fellow, Laboratory of Biological Effects of Nanostructures (005), V.L. Talrose Institute for Energy Problems of Chemical Physics (Russian Academy of Sciences). Moscow, Russia. E-mail: gradov@chph.ras.ru; gradov@center.chph.ras.ru
Abstract. The article describes a multi-angle mechanical system, applicable for research at different angles and in different coordinate systems of analytical chips and biological samples. Currently, the system undergoes improvement and automation in two scientific institutes of the Russian Academy of Sciences. In the nearest future, it is planned to perform multiplexing and establish colocalization of the registograms of various variables obtained on this installation.
Key words: goniometer; CLEM; ESEM; correlative light-electron microscopy; environmental scanning electron microscopy; lens-less microscopy; on-chip imaging; lab-on-a-chip; multi-angle laser light scattering; MALLS; multi-angle laser light scattering; MALS.
КОМПЬЮТЕРИЗИРОВАННАЯ СИСТЕМА ДЛЯ МНОГОУГЛОВЫХ ИЗМЕРЕНИЙ ПРИ СЧИТЫВАНИИ АНАЛИТИЧЕСКИХ ЧИПОВ НА ПЛАТФОРМЕ КОРРЕЛЯЦИОННОЙ ЭЛЕКТРОННО-ОПТИЧЕСКОЙ МИКРОСКОПИИ (CLEM) В КОНТРОЛИРУЕМЫХ ФИЗИКО-ХИМИЧЕСКИХ УСЛОВИЯХ В КОЛОННЕ С ВАКУУМИРОВАНИЕМ И НАПУСКОМ ГАЗА (ESEM)2
Градов Олег Валерьевич, ИХФ РАН, ИНЭПХФ РАН (научный сотрудник лаборатории 0412 Института химической физики им. Н.Н. Семенова РАН; старший научный сотрудник лаборатории 005 Института энергетических проблем химической физики РАН им. В.Л. Тальрозе). Москва, Россия. E-mail: gradov@chph.ras.ru; gradov@center.chph.ras.ru Аннотация. В данной работе описывается компьютеризированная система с многоосным гониометрическим столиком для многоугловых измерений при считывании аналитических чипов на платформе корреляционной электронно-оптической микроскопии (CLEM - Correlative Light-Electron Microscopy) при контролируемых физико-химических условиях измерений / контролируемой атмосфере в колонне TESLA с вакуумированием рабочего объёма и напуском газа (ESEM - Environmental Scanning Electron Microscopy). Предварительно аргументируется необходимость реализации многоугловой аналитики, причем как для оптических, так и для корпускулярных методов измерений на любой конструктивно-эквивалентной установке. Приводятся аргументы, свидетельствующие о возможности использования подобной геометрии установки не только для
1 This work was partly presented on the X Russian-German Conference on Biomedical Engineering (LETI University, 2014), MIT - Skoltech Biomedical Conference: «Towards Therapies of the Future» (2014), International Conference and Trade fair on Laser Technology (Orlando, Florida, USA, 2015), «The Cell Cultures of Marine and Freshwater Animals» conference (Institute of Marine Biology, 2015), ADFLIM school (FRC «Fundamentals of Biotechnology» of the Russian Academy of Sciences, 2016), Russian International inference on cryoelectron microscopy (Moscow State University, 2017).
2 Части работы были представлены на: 10-й Российско-Германской конференции по биомедицинской инженерии (2014), конференциях «Towards Therapies of the Future» (2014), «The Cell Cultures of Marine and Freshwater Animals» (2015), выставке-конференции по лазерным технологиям в Орландо, Флорида («International Conference and Trade fair on Laser Technology»), школе по прогрессивным методам флуоресцентной визуализации ADFLIM (2016) и всероссийской конференции по криоэлектронной микроскопии (2017).
многоугловых измерений аналитических чипов (либо с использованием встроенных КМОП- или ПЗС-матриц - детекторов систем проекционной безлинзовой микроскопии на чипе в центральной зоне гониометрического стола в контакте с образцами, либо на месте детектора проходящих электронов TED), но и в исследованиях природных сред - в частности, минеральных структур и структур биоминерализации при различных физико-геохимических условиях их формирования и замещения, с различным текстурно-пространственным разрешением. Предложен принцип комплементарных координат, в которых фиксируемые дескрипторы, значения переменных, по которым производится идентификация объектов / их структурных компонент или компартментов, проецируются на согласованную с геометрией объекта (с соответствующим размерам его структурных компонент разбиением) сетку, а исследование распределения переменных происходит в нескольких системах координат, колокализованных друг с другом. В качестве кастомизированного для данной установки программного решения приводится репрезентация, использующая одновременно эйлеровы угловые координаты и кватернионы.
Ключевые слова: гониометры; корреляционная электронно-оптическая микроскопия; электронная микроскопия в контролируемой атмосфере; безлинзовая микроскопия; имэджинг на чипе; лаборатории на чипе; многоугловое лазерное рассеяние (MALLS); многоугловое рассеяние света (MALS).
Introduction
At present, angular selectivity is a necessity realized as a prerequisite for a metrological result in numerous branches of physical, physicochemical and biological research. For example, angular selective transmittance measurements of thin films [1-7] require goniometric installations with a wide spectral range or operating in a monochromatic mode. In principle, angular selective transmittance does not exhaust all the possibilities of angular measurements of the optical characteristics of films and coatings. The work on multi-parametric analysis of the optical properties of thin films in the goniometric mode has begun not less than twenty years ago [8]. The coatings of glass sheets and slides may also be measured by a glossmeter and reflection meter with angular resolution.
In this case, the emittance control at the sample preparation stage can lead to qualitatively different «glazing parametrics» at different angles due to the spectral dependence of the refraction and the fact that the transmission coefficient of the film on the glass is a function of the direction of incidence [9-11].
At the moment, researchers rely on several variations of the theory of angular selectivity for such coatings; however from the practical point of view the Le Bellac-Niklasson-Gran-quist method seems to be the most pragmatic [12; 13]. The authors of this method (the founders of the approach together with Smith) carried out not only theoretical, but also experimental studies in this field, illustrated the optimal convergence between the theory and experiment, and efficiently worked on «fitting» the experiment based on the objective physical prerequisites.
Another field in need of angular measurements is the solar energy conversion that depends on the optimal orientation of solar cells towards the source of radiation / the sun (the he-liostats were previously used for this purpose), or the so called «angular-selective photovoltaics» [14-18] (including translucent [15] or «bidirectional», that is, transparent or light absorbing in both directions [19; 20]), as well as solar photothermal power engineering and thermovoltaics [21-26] that have similar tasks. The solution to the problem of angular selectivity is usually reduced to two basic versions of the technical transformation either based on filters / coatings / applied films and other materials with programmable properties (applicable to structural refractometry and spectrochemistry) [27-31], or based on a system of optical diffraction elements, calculated as kinoforms by computer optics technologies [32; 33].
The latter is an engineering approach, not requiring goniometric operations at the material control stage; so we do not focus on it in the future. The materials for the first approach can differ in key descriptors, because of and as a result of which their
reactivity will vary and depend on the chemistry of the coating, in particular, beyond the limits computable with modern tools of the chemical similarity theory (QSAR model). In this regard, their metrological properties should be analysed at various regimes during goniometric and spectral scanning, as well as under changing environmental conditions.
These tasks are justified once we take into account the non-unified variety of chemical / physico-chemical parameters of the target media:
a) anisotropic polymer films synthesized, in particular, by a photochemical method [34], and therefore possessing emergent characteristics in the optical region;
b) cholesteric liquid crystals exhibiting angular dependencies of optical and other parameters under the action of an electric field, but intact with respect to the target signal level under normal conditions [35];
c) single one-dimensional anisotropic photonic crystals and their ensembles that possess angular selectivity only under conditions of cooperative optical behavior [36];
d) thin films with anisotropy of polarization, birefringence and angular «azimuth transmittance» [37], etc.
For the standard optical range and household applications all the characteristics can be calculated in advance with applied mathematical tools (for example, for window coatings) [38-41]. On the other hand, in case of spectral ranges beyond the visible region this task becomes complicated. The problem is in applications that are adapted for daylight [42] (since bi-directional transmission and its applications [19; 20; 43] are meant for the user who does not need transparency in the UV range) in order to cut off an unnecessary part of the spectrum and recycle its energy to preserve visibility in the area perceived by the human eye. At the same time, from the point of view of the physics of wave processes, the angular sensitivity of the distribution is inherent to the broadest range of agents interacting with matter from cold neutrons [44] and X-rays [45] to the THz wave bands (for sensing metamaterials) [46] and further. The principles for the constructing of the radiation patterns, directograms and scattering indicatrix (and other descriptors) are relevant for all these areas, and the applicability of the radio technology principles associated with antennas and radiation patterns begins even from the optical region [47; 48].
The effectiveness of coatings with respect to the angular-selective performances of obliquely deposited birefringent thin film, from the standpoint of the corpuscular-physical analogy (the introduction of the variational method in solving the problems of transport and diffusion of particles), considering the radiation flux with angular resolution as the scattering
particles indicatrix (for example - neutrons [44]), is then interpreted for single particles in terms of value and conjugate function (as in case of reactor physics - the value of neutrons).
It must besaidthatthecorpuscular analogy in theinterpretation of goniometric measurements and, as a consequence, the explication of the elements of the mathematical apparatus and data processing facilities for the heuristic-valuable analysis of angular measurements, definitely makes sense, since terms and principles for determining angular selectivity are relevant for a multitude of nuclear-physical or corpuscular-physical projects. Among the projects of the megascience level is the last (currently ongoing) KATRIN experiment (Karlsruhe Tritium Neutrino Experiment) for which an angular selective electron gun (ASEG [50-52], also known as angular selective electron source - ASES [53] or / and angular selective photoelectron source for KATRIN spectrometers [54; 55]) was manufactured, where the photoelectron source uses ultraviolet rather than X-ray radiation to provide photoemission [56].
Ideally, it would be expedient to create an installation that allows to measure and also reconstruct three-dimensionally the spatial distributions of descriptors in different spectral ranges (for hyperspectral optical, corpuscular, quasi-optical and radio-optical wide-range analysis) with angular specificity / selectivity (which is equivalent) - with angular resolution. The advantage of such system engineering is obvious: for metrology of the comparative («ratiometric») and correlation (starting from the system of coincidence analysis and ending with analog or computer clustering) analysis of the measurement data and establishment of the colocalized zones (Regions of Interest, or ROI) with certain parameters in different variables or descriptors or in different spectral ranges - the «heterospectral» correlation analysis [57] seems to be the optimal solution. Using two-dimensional heterospectral correlation analysis of wide-angle X-ray scattering and infrared spectroscopy [57], it is possible to identify structural and conformational composition of compounds, and, based on the dynamic and kinetic approaches, selectively identify specific chemical interactions, including weak intermolecular interactions in polymer systems. The dimension can be enhanced from 2D to 4D, as well as visualized, if the dimensionality of data is increased by using non-orthogonal coordinate systems.
«What can the multi-angle measurement of a complete nomenclature of variables or some, although not exhaustive, but relevant multifield set of variables, provide for a technical analyst working in these ranges? » This is the most important question posed by specialists who refuse to use multi-angle techniques for the reason of their comparative complexity. We will not dwell on the usefulness of multi-angle measurements in general, since this question was qualitatively covered above, but will offer examples in which the feasibility of multi-angle complex analysis of matter becomes obvious. Let us assume that there is an arbitrary sample of a complex nondeterministic composition on a planar substrate (chip) containing ROI (compartments) with initial or introduced (in the indicator format) carriers of physical and chemical properties that allow to differentiate these compartments from each other. At the same time, the analytical signal from the ROI is in different regions of the full spectrum / is received on different channels from physically non-identical converters with different integration times, sampling frequencies, etc. An example of such a problem is the analysis of localization and colocalization of luminescent, magnetic or radioisotope-labeled regions with the sample structure determination by diffraction
methods for a geological sample containing a magnetically oriented compound (magnetic spinel of the magnetite type [58-62] with the localized isotopic labels3 in some ROI and luminescence of the individual ROIs also typical for many spinels [74-80]). In this case, in the presence of decaying isotopes and the corresponding ionizing radiation, the luminescence can not be attributed to the presence of the corresponding analytes since the release of the ionizing radiation energy in the surrounding rock is accompanied by the mechanisms of radioluminescence rather than photoluminescence [81-89], and the elements formed in the decay process affect the intensity of the analytical signal, including luminescent properties, because they are acting like quenchers or optical filters (including the cases when thin surface oxide films are formed).
Thus, the multiparametric (and multi-physical) colocalization analysis of the angular distribution of variables by definition requires the presence of a multi-angle system with the tunable discreteness sufficient for collecting and processing the signal of all variables, measured at high angular resolution (ideally, measured in a synchronous mode and in the technically limited cases - measured in a sequential mode for a number of relevant variables or for a full range of the detector modes).
This example with the low statistical occurrence of such natural samples may seem exotic for the standard user. However, it is possible to point out a number of techniques for studying natural samples in which the probes or indicators of certain physical and chemical properties are artificially introduced. For example, cells of living organisms can be simultaneously studied with radioimmunoassay or radioautographic, magnetoimmune / magnetographic (magnetic labels), luminescent / fluorescent probes (exogenous fluorophores / luminophores and intrinsic bioluminescence / chemiluminescence, for example, in case of lipid peroxidation of the cell membranes) methods.
The cell is a structure with pronounced dynamic and reactive behavior, and hence, it requires continous measurements with angular resolution, otherwise the colocalization and correlation of the descriptors will not be established due to their displacement (taking into account the time constant, in particular, identified with the diffusion time constant in Turing reaction-diffusion and morphogenetic processes). A simultaneous multiplexed measurement of the group of variables that seems to be excessive for serendypic mineral samples may well be justified and even inevitable for many complex biomedical tasks.
For the object localized on a multi-angle rotational stage (in atmospheric or artificial gas conditions, excluding vacuum since it evaporates biological samples on a substrate /chip) in chambers or at the goniometric stands for CNC-based angular measurements using stepper motors or multichannel ADCs and CAMAC crates. In the ideal case we must be able not only to measure, but also to predict the physical indicatrices for monitoring the analytical signal by modeling the geometry of the detector and the traces of particles propagating at different angles, considering the signal propagation in different media, from the atmosphere to the sample. In practice, biological objects are often replaced in the model by their water phantoms,
3 This is typical of spinels not only of geochemical (e.g. xenoliths [63; 64]), but also of cosmochemical origin (for example, so-called presolar grains, or dispersions of microscopic and ultramicroscopic minerals condensed in the pre-sun period and including isotopic signatures of nucleosynthesis from the pre-Katarchean period [65-68], or the spinels of the meteoritic origin with the well studied isotopic effects (for example, carbonaceous chondrites, such as the Murchison meteorite [69-71], Vigarano (CAI 477B) [72], Mighei (C2) [73], etc.
due to the high water content in biological objects; the similarity criteria being acceptable for the model geometry, but useless for accurate compartmentalization and the cell ultrastructure).
For the systems of processes with charged-particle propagation where the magnetic field affects the trajectories of particles, and for other such processes where particle scattering in a medium is simulated by Monte Carlo methods (including thermoluminescence [90]), the need to take angle into account is obvious. The methods of computational experiment and numerical simulation are developed for all systems (regardless of the propagation scheme) - from simulating the atmospheric propagation of muons [91; 92] to neutron physics [93] (including propagation through the soft-matter medium, in particular, through biological tissues, as well as the generation of neutrons in various angular sectors during the interaction of protons with water in biological liquids [94] (although for a photon dose, methods with angular resolution in medical physics and radiology are applied quite effectively [95])).
Therefore, considering the simulation principles with the angular resolution for all types of the above signals (except for the magnetic signal that «influences but does not trace through» the trajectory of a deterministic medium, like particles) using Monte Carlo methods, including luminescent and radioluminescent measurements described as problematic above [96, 97], it is possible to recommend the multi-angle multi-physical studies with comparative fitting to the reference model for the analysis of biophysical samples.
Methods and results
The authors assembled a recording system based on a gas-filled vacuum chamber from a TESLA scanning electron microscope of the 1980s and provided a mechanized movement of the sample around the centered laser source located at the top of the column where the Vennelt cylinder is usually attached. CCD cameras were positioned at the transparent side windows. Active chips with position-sensitive matrices that allow to establish localization and colocalization of various descriptors in the sample by means of mapping of the magnetic, optical and radiometric properties, were used as matrices for the active substrates [98,99]. Fixation area of the transmission electron detector (TED) was applied as a place for the additional positionsensitive meters (when using a passive chip instead the active position-sensitive chip on the rotationa stage). If necessary, additional sensors of physical characteristics without positional sensitivity can also be fixed there. The following modes for measuring / positioning samples were tested: goniometric, rotational-goniometric, linear scanning, vertical scanning («Z-scan» that changes the active focal length for a number of chips and converter systems - the so-called «focuscopic regime»). The approbation of these modes and the movement of the adapted SEM-positioner from different projections are shown in the video below:
• https://vimeo.com/273509409
• https://www.adcdirectory.com/products/multi-angle-goniometric-lab-on-a-chip-reader-stage-for-vacuum-gas-chamber--maglors--40
Discussion and prospects
As can be seen from the video materials with the time code, such positioning system ensures not only angular, but also stereometric modes, or topologies, of the experiment (both static and dynamic). Therefore, the measurements and
visualization can be significantly more heuristically valuable if the data representation and positioning control are performed in the Euler's angles (rotation of an absolutely solid body in three-dimensional Euclidean space) or, optimally, in quaternions that make it easier to combine rotations and avoid the problem of the impossibility of rotations around the axis, regardless of the completed rotations on other axes. At present, the simplest version of the installation is implemented, in which the polar coordinate system is extended to the third dimension by adding another angular coordinate. The resulting spherical coordinate system contains the polar coordinate system as a subset. This is optimal for fixation, but not optimal for dynamics; optimal for registration, but not for positioning. It should be noted that from the perspective of the signal representation, nothing prevents the use of exotic, but rational (coordinated with the dimensionality and geometry of the experiment) coordinates, for example, pentaspheric coordinates for the three-dimensional case (with the help of pentaspheric coordinates, allowing a zero element, we can supplement the 3-dimensional Euclidean space to the spherical one). In the present experiments, the concept of the complementary coordinates providing complementary visualization by the variables-descriptors in implementation within exotic coordinate systems) is considered redundant. Therefore, we consider here only quaternions and Euler angles, using also effective schemes and algorithms for their interconversion [100-104], which have been developing since the 1970s along with the space industry growth and the need for effective navigation of the autopiloted devices.
This aspect, which focused «quaternion positioning» mostly on space [106-110], avionics [111-113] and navigation [114-118], including gyroscopy and accelerometry [119], to provide dynamic positioning feedback [120], is, in fact, the reverse side of specialization (so many classical and effective modern works with academic bias are not in sufficient demand [121-126]). It also indicates the positive trend towards academic implementation of metrological multi-axis / multi-angle devices with positioning and / or registration in appropriate coordinate systems with quaternion representation.
The main inspiration of the quaternion-based approach in multi-axix / multi-angular multiphysical detection methods is the analogy between the dynamic modes of the multi-angle scanning devices and the relative movements of the scanning or tracking radars and multi-purpose direction-finding devices that can be implemented as the particular cases of scanning modes of the multi-angle devices [127-129] (if we discount the potential of high-performance tracking scanning with adaptive-implementable coordinated filter, and other exotic, but necessary options for the investigation of complex non-exotic objects).
Thus, measurement schemes for multi-angle stationary chip readers (strips, samples, etc.) with the geometric complexity level of Multi-angle Goniometric Lab-on-a-Chip Reader Stage for Vacuum-Gas Chamber (MAGLORS-4-VGC) can be improved by using visualization or data representation in non-standard coordinate systems and positioning of mechanics (ideally -multi-angle / multi-axis mechatronics) in these coordinates. Numerous conclusions of research articles, starting from 1970s, may be adapted to modern algorithms and software texts and in future form the foundation of multi-axis multi-angle software and hardware complexes controlled by a user-friendly interface (GUI) within modern operation systems. This can be used, in particular, for the development of biophysical (and physico-chemical) mapping with angular resolution in multimodal
microminiaturized devices, localized in the chamber of an electron microscope, electron diffraction and photoelectron spectrometer («biophysical mapping with the angular resolution using lab-on-a-chip») [130].
Acknowledgments
The work was started in 2016-2017 with the financial support of the Russian Foundation for Basic Research (project No. 16-32-00914). The work was partly supported by FASO (project 0082-2018-0006, registration code AAAAA18-118020890097-1) in 2018. The author thanks his students for their help in preparing the bibliography and also his colleagues from VINITI for translating some Chinese articles into Russian.
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