. This article aims to ensure continuity between classical methods of ultraviolet microscopy or /and micromanipulation using ultra violet microbeam and lens -less ultraviolet microscopy and microbeam exposure of cells and ti s- sues. Considering the history of the development of the method and the poss i- bility of working with different methods of mechanical scanning, the authors propose to use mirror galvanometers and an electromechanical scanning sy s- tem in the mechanical engineering of lensless microbeam installations. These installations make it possible to provide both scanning with an ultraviolet m i- crobeam to obtain a line scan image, a nd precision micromanipulation at the level of individual cells or individual organelles (in the case of large cells). We propose to extrapolate mathematical models previously developed for galvanic mirrors of light -beam oscilloscopes for microbeam scannin g systems, position - sensitive micromanipulation, and real -time microphotometric cell analysis. Keywords: ultraviolet microbeam , mirror galvanometer , galvanometric sca n- ners, radiopuncture microscopy, lens -less FRAP, lens -less FLIP . 1 Introduction. 1.1 The History and Emergence of Chakhotin UV Beam Method As Chakhotin’s pioneering works about exposing cells [1 -3], organelles [4,5] and cytoplasmic colloids providing phase physiochemical cell morphology adaptabi l- ity [6, 7] to UV emission showed, the ultraviolet micro beam is an efficient and acc u- rate tool for solving experimental cytology and morphology problems [8]. The author originally called it “UV radiopuncture methods” [2,9]. However, in one of his papers (in Compt. Rend. Soc. Biol., 1920) he calls it “radiopunct ure microscopie”. Perhaps he meant that the UV irradiation effect and the microscopic observation of cell changes in UV microbeams are simultaneous. Chakhotin’s “radiopuncture” should not be confused with the radium radiopuncture method that was common in the same period. Subsequently, other researchers working with UV irradiation in experimental morphology called it the same, including Anne Dürken [10]. She researched UV 2018 Chakhotin seminar, INEPCP RAS, Biophysical instrumentation group 2 irradiation effects on newt sensory system morphogenesis. These studies were init iat- ed by Bernhard Dürken [11], the German experimental zoomorphology founder, in the first half of the 20th century. It turned out that the UV irradiation applications are much more extensive than Chakhotin believed in his first works. a b Fig. 2: S. Chak hotin with his microbeam setup s: a) in the laboratory of the Max Planck Inst i- tute in Germany, 1931 ; b) in the Laboratory of Prof. Pietro Di Mattei at the Pharmaco- logical Inst itute in Rome, 1957 (from the Posudin’s book) . 3 3 In the 1930s he demonstrated that a UV beam could induce parthenogenesis in invertebrate (and the morphogenesis, as a result) [12 -14]. About at the same time, his studies found the correlation between the biomorphology changes induced by the beam (as much as UV beams were applicable to ult ramicroscopy of colloid particles or cytoplasm organoids [6,7] and ultramorphology) and the functional or physiolog i- cal changes. This enabled the microbeam to be applied to moprhophysiological and functional morphology r esearch with great accuracy for that period (Fig. 1 - Fig. 3 ). a b c Fig. 2 . S. Chakhotin demonstrating his microbeam experiments: a) in Heide lberg, 1929; b ,c) in Mo scow, 1960th. These works include daphn ia heart rate suppression by the UV beam [15], the UV microbeam effects on myonemes (myonemes are similar to myofibrils, and they also have a smooth or striated thready muscle structure) of Campanularia [17] in 2018 Chakhotin seminar, INEPCP RAS, Biophysical instrumentation group 4 subcellular level, and other UV beam micro op erations on protozoans [18]. Chakhotin’s experiment skills were unparalleled because even nowadays the daphnia heart rate suppression problem cannot be solved without interferometric autodyne detection tools [16]. Micro manipulations with individual cell p opulations were also introduced [19]. Fig. 3 . S. Chakhotin experimental system for cell micro -irradiation . Chakhotin was working at Wilhelm Röntgen's labs for a long time. He ma n- aged to handle the methodology of physical interpretation of experimental results, that is why his physiological -morphological results were always accompanied by physical-chemical and biophysical explanations. In that way, he was ahead of the research conducted in the first third of the 20th century, because biophysical mo r- pholo gy as a separate field emerged only in the last quarter of the century. Intern a- tionally, the emergence is commonly attributed to the Techniques of Biochemical and Biophysical Morphology monograph by David M. Glick, Robert M. Rosenbaum (Editors) pu blished i n the 1970s. The cytoplasm reaction in Chakhotin’s experiments to UV irr adiation was associated with pH and membrane permeability [20] following the phys ical reductionism principles. The cell hydrostatic reaction was described in 5 5 terms of the imbalance bet ween the cytoplasm and the vacuolar system of the cell [21]. The method proposed by S. Chakhotin subsequently was adopted in various biology areas and turned into a multi -discipline tool. A UV micro -beam scanning TV system developed in 1962 [22] enabled d igital processing and storing of acquired morphological data. Later, as semiconductors replaced vacuum tubes in 1970s, such systems became simpler and more affordable [23], which contributed to their exte n- sive adoption. While the method was mesoscopic in terms of the exposure magnitude and beam detection , mostly tissue and histochemical effects were studied. Partic ular- ly, in 1960s experimental exposures of muscles [24] and tissue cultures [25] to the microbeam were common. For the same reason, mesoscopic ex periments with rel a- tively large Ciliata Euplotes species continued. For instance, the UV beam effect on cell morphogenesis was studied [26]. Fig. 4 . Contemporary computerized version of the UV microbeam setup based on ZEISS OPTON photomicroscope with o ptical waveguide spectrometer, UV microspectrometer, ZEISS OPTON micromanipulator and computer -assisted data acquisition system. Semenov Institute of Chemical Physics, Gradov’s multiparametric microscopy infrastructure, 2018. Still, as early as at the end of 1950s, the research began to be more and more focused on multi -beam applications for cytogenetic, karyomorphological, and mitotic studies, and for the associated experimental morphology studies. The effects and contribution of nucleoli on the mitosis p rocess were studied with an UV micro -beam in 1958 [27]; in the 1960s the research was continued and verified by another team [28]; later they switched to cell membrane stud ies not related to genetics [29 ]. The UV micro -beam effects on nucleoli were studied through 1970s, and 1980s [30, 31 ] (see Fig. 4, Fig. 5 ). 2018 Chakhotin seminar, INEPCP RAS, Biophysical instrumentation group 6 Since the 1960s spindle apparatus microbeam effect studies were undertaken but in the first works the additive physical methods allowed to register only optical changes, for example, the spindle app aratus birefringent effects [32]. Subsequently, the studies in this field became more extensive. In 1970, cytomorphological effects were considered and interpreted as “false anaphase”, true anaphase delay, and the spindle apparatus destructuion / disorgani zation [33]. Then, in the 1980s the spindle apparatus extension under the UV beam was observed [34]. After introducing the novel methods for working with single microtubules in vitro , the microtubule asy m- metric behavior and destruction under UV microbeam w as registered [35]. The action spectrum of the beam for this case [36] was studied, and then the forces emerging from the spindle mitotic dynamics were assessed [37]. Some research teams conti n- ued to study protozoa [34,38,39] in the 1970 -1980s, but more of them focused on histological test objects (tissue sections, bacterial cultures, grafts in isolated cha m- bers, etc.) However, it was not related to the additive effects on these objects, but due to the equipment that enabled working with the relatively smal l cells. Fig. 5 . Ultraviolet (micro)beam setup prototype based on nitrogen laser ILGI -503 and scanning microspectrophotometer MSPhP (Inst. En. Prob. Chem. Phys., 2016). At the end of the 1980s, technically feasible UV microbeam (almost DIY) equipmen t based on commercially available components [22] became common. Cheap UV lasers with diffe rent wavelengths, for example, nitrogen or helium – cadmium ones, enabled to study chromatin photodegradation and genetic material photoreactivation [40] in a single e xperiment in a static object using the same e xper- imental facility. The first laser UV microbeams entered the microscopic practice in the early 1970s [41]. The above prerequisites made it possible to operate with ex- tremely narrow, highly coherent beams and resulted in UV subcellular microdi ssec- tion intr oduced by S. Chakhotin. This method was expected to boost in the 1990s. 7 7 However, the non -coherent UV equipment was still used during the massive chang e- over to lasers (even in 1991, the techn ical support docume ntation for lamp -based microbeam experiments using monochromators and filters was still pu blished). This is due to the fact that lamp -based microbeam sources with monochr omators possess a wider wav elength range than the laser ones, and hence, lead to the v arious biological effects under different optical parameters of the microbeam [42]. At the beginning of the 21st century, more compact designs based on laser diodes and DPSSL, as well as comp act gas lasers were implemented (O.V. Gradov , GEOKHI RAS , 2010 – Fig. 6a; Andrew Skrynnik , 2017, based on inverted phase -contrast microsope by the Gradov’s idea, INEPCP RAS , Gradov’s group – Fig. 6b ). a b Fig. 6. Contemporary Russian compact micr obeam setups for field and (auto -)mobile lab conditions: a) combined laser diode / DPSSL compact microscope for hydrobi o- chemical / geobiochemical and hydrobiological microbeam investigations (GEOKHI RAS, Oleg Gradov, 2010); b) HeNe laser microbeam setup ba sed on inverted phase - contrast micr osope (INEPCP RAS, Andrew Skrynnik , 2017 ). The research trends of the 1990s resulting from the emergence of the above UV lasers and laser systems should be briefly reviewed. First of all, the studies of microbeam effects on the microtubules and genetic material were continued. In pa r- ticular, Soviet researcher Uzbekov’s team explored the postmitotic nucleolus reco n- struction in cell culture during centrosome microbeam photoinactivation [43]. The centrosome defect in mitosis induced by the microbeam [44] was considered as a special case of the mitotic system dysfunction under UV microbeam irradiation of the spindle apparatus [45]. The same studies were carried out outside of the USSR. For 2018 Chakhotin seminar, INEPCP RAS, Biophysical instrumentation group 8 example, Forer’s team studied epithel ium cells and spermatocytes, but they were focused on UV effects on kinetochore considering it as a protein structure that fa s- tened the spindle apparatus fibers to the chromosome during the mitosis [46,47,48,49]. a b c d Fig. 7 . Computerized multi -module multi -wavelength UV microbeam setup with two possible cooling pathways (O.V. Gradov, Research Center of Neurology, Brain R e- search Department, Laboratory of Neural Brain Structure, Moscow, 2012 -2013). These studies continue the cytogenetic works in the UV microbeam method which were successfully performed by the Sovi et scientists in it in the 1970s [50]. They also developed the interphasic control methods with UV microbeams. These methods started to be developed in the 1960s through Amenta’s team classic studies on the UV microbeam irradiation on Triturus viridescens granule and dysgranule cells [51,52]. Such works are beyond the molecular -genetic trend in the UV m i- crobeam method development that was presented mostly by the immunological and virological papers [53], and rather belong to experimental ultramorphology. Th e possibility to detect irradiated areas after the cell treatment with uranyl acetate e na- bles to map the irradiated sites and cellular compartments depending on their sens i- tivity or resistance. This method was common in the 1990s [54]. In our group, prot o- 9 9 types of systems for synchronous manipulations on cells and microphotometry or luminometry were created (see Fig. 7, Fig. 8) . In particular, similar experiments were planned in gas microchambers (Fig. 9) , which, however, did not happen due to the budget def icit and staff leaving. Fig. 8 . Prototype of the ultraviolet microscope and ultraviolet microbeam setup in the external fields in the Faraday cage rag (based on mobile luminescence / fluore scence microscope MLD, multi -wavelength laser photometer FM -01an d photomult iplier- assisted quantometer, which also was used in the prototype UV setup on the Fig. 5). Project initiator: F. Orekhov (Gradov’s Biophysical instrumentation group, 2017). UV microbeam irradiation according to the Chakhotin’s experimental met h- odology is a modern high -tech experimental approach based on the effectively f o- cused laser systems. It is compatible with the probe and atomic -force microscopy [55], lab -on-a-chip techniques [56], optical tweezers and manipulators [57], systems for synchr onous chemical and morphological control of the free radical species [58], immunofluorescence methods and tools for radiobiological / photobiological co m- parative studies [59]. The microbeam applications to the experimental morphological studies have been e xpanding. In the 1970 -1980s the microbeam effects were macr o- scopic or mesoscopic with the morphological results achieved by the direct effects on the organism or its embryo [60 -62], while nowadays the same results are achieved with a “molecular resolution” by genetic switches or changes in the membrane ion channel operation. In neuromorphology, where UV microbeams were used for co n- trol and inhibition of the cell and tissue differentiation since the 1980s [63], an opt o- genetic trend has emerged which is based on the optical stimulation of the light -gated ion channels embedded into the cell membrane (e.g., opsins) with the genetic eng i- neering methods used to express such channels. There are known several methods of pulse near -ultraviolet (UV -A) irradiation for optog enetic research [64,65] and the related areas [66] (consider the reference list in the review paper [67] for more i n- 2018 Chakhotin seminar, INEPCP RAS, Biophysical instrumentation group 10 formation on this). This novel tool for genetic research drastically differs from the laser UV beam induced DNA introduction into plant cells popular in the 1980s [68], as well as from chromosome / kariogenetic experiments with UV microbeams in animal cells conducted at the same period [69]. Fig. 9 . Project of UV experimental setup with gaseous chamber for experime ntal investigations in coacervate morphogenesis with microspectral and electrophysical monitoring with Fourier transform based signal analysis and spectral morphometry (Gradov’s Biophys ical instrumentation group, 2016 ; F. Orekhov, 2015 -2016; this project was cancelled by the mass spectrometric lab management in 2016). 2 UV Microbeam Positioning and Focusing Devices Similar to Galvanometric Scanners Considering the capabilities described above, the UV microbeam method should be introduced in many areas of biology where its appli cation seems to be r a- tional. However, despite the fact that UV light sources, including lamps, are affor da- ble, it does not happen due to the inaccessibility of the other conponents of the i nstal- lation r equired. Devices for accurate UV microbeam positioning required for a wide cytological, gistological, embryological, and experimantal -morphological a pplication of UV microbeam technoque possess high cost and low commercial avai lability. A modern microbeam facility is not only the optics, but also the accurat e micro -electro - mechanical and computer -based adaptive control systems that manip ulate the beam with a high speed and micron / sub -micron accuracy. Nowadays, ga lvanometric mi r- rors / scanners [70 -72] are used in experimental biology research for these purpo ses. Their scanning rate is up to a kilohertz, and they aloow to operate with fine micro- 11 11 structures, and even perform laser transfection [73,74] or laser clo ning [75] by me m- brane fusion [76]. Fig. 10 : A typical Photo -Kymograph. The first half of the 20th century: designed by Prof. Broemser (Photo -Kymograph ion nach Prof. Broemser), Image courtesy: Jaquet J. Jaquet Apparatus: Catalogue. No. 503: a: p. 51, fig. 1022; b: p. 0052, fig. 1022a. The galvanometric scanner operates as follows: the mirror head rotates about two axes and changes the beam incident a ngle on the sample controlled by electric signal with the the computer -generated waveform. The higher the control signal fr e- quency, the higher the scanning rate. However, the higher the frequency, the more expensive the galvanometric reflector is. For this reason, in developing countries 2018 Chakhotin seminar, INEPCP RAS, Biophysical instrumentation group 12 lower scanning rates are often used. For many cytophysiological researchers, this is an ultimate obstacle preventing them from experiments with the UV microbeam. Nevertheless, bifilar / light -beam oscillographs with movable galvanometers int e- grated into the recorder cassette are commonly used. These are galvanometric sca n- ners that are controlled not by a computer over a dedicated controller but by any signal supplied to the analog input of the light -beam oscillograph. When t he se- quences of signals / pulses generated by the DAC are used as a “control program” to generate the required light beam trajectories, the galvanometer modules of the old light-beam oscillographs can be used to scan microscopic slides / tissue cultures wi th a UV beam created by a Hg UV lamp integrated into the oscillograph housing (see below ). It should be noted that the application of light -beam (or bifilar) osci llo- graphs to biomedical research and the development of dedicated measurement d evic- es for pra ctical medicine were drastically accelerated as early as at the beginning of the 20th century as the vacuum tube technology was improved, and further intens i- fied by the 1930s. In Germany, this approach was developed in the most comprehe n- sive way. As early as in 1930 the nerve electric currents (“nervenaktionsströme”) were studied with bifilar (“schleifenoszillograph”) oscillographs [77], and in 1931 the vacuum tube and light -beam registration technology were used for physiological acoustics studies [78] and acoustic analysis of breath sounds [79]. In 1932 for the first time heart tones were recorded by a light -beam oscillograph on photosensitive paper [80]. In 1935 for the first time, a light -beam oscillograph was applied to study an acoustic stimulation of the vestibular system [81], while in 1938 the same device was used to register the oscillations of vibrating vocal cords with a setup based on a sensitive photoelement [82]. In the same year, Richter published a paper that pr esent- ed the advantages of Brown tube-based oscillographs over otherbioelectric pote ntial registration methods [83]. The paper was welcomed by the medical community. Af- ter that (post hoc non est propter hoc) as early as in 1939 the first study of electr o- physiolog ical brain activity measu rement errors was published. It noted that due to the physical principles of signal registration, light -beam oscillographs were not free of electr ophysiological signal recording errors [84]. A detailed study of the available