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23
XXIII Congress of I.P. Pavlov Physiology Society
Software Development for Custom-Made Two-Photon System for Implications in Neuroscience
Maxim Doronin*, Alexander Popov*, Sergey Makovkin, Yulia Dembitskaya, Alexey Semyanov
Institute of Neuroscience, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia * Presenting e-mail: [email protected], [email protected]
Two-photon microscopy is one of the main and quickly developing imaging techniques in neuroscience. Therefore, scientists need to regularly modify their systems according to changing in experimental needs. This task is not always possible with commercially available microscopes. Earlier we have reported how to build a custom-made laser scanning microscope (LSM) that can be easily modified by the user for a specific task in in vivo and in vitro. Here we describe our custom-made software - AMAScan (MATLAB) to control LSMs in a flexible and user-friendly manner.
Modern imaging techniques with high spatial and temporal resolution provide key tools for studying the brain functioning significantly advance neuroscience field. Developing new tools and upgrading available ones is crucial for further progress [1]. Thus, we developed custom-made two-photon microscope that took into account specific needs of various types of experimental approaches in vitro and in vivo. This system allowed us to significantly reduce the cost and increase the quality of obtained images by implementing the BM3D filtering algorithm for the noise reduction [1, 2]. We optimized the optical path in order to minimize the excitation lose and tissue damage in combination with increased efficiency of the signal detection.
Notably, this system can be easily modified for cultures, brain slices and in vivo recordings from behaving animals. For the first two types of samples, we minimized the distance between the objective and the condenser in order to optimize the quality of images for morphometry and line-scan imaging. In contrast, for in vivo procedures on behaving animals, the condenser can be removed and distances can be easily adjusted to fit in a platform for virtual reality and a floating sphere where an animal can run.
Specifically we focused on the software development realizing its crucial role for different research paradigms. In order to improve the quality of obtained images we optimized not only the technical (physical) characteristics of the microscope [3-5], but also the software. The system is fully controlled via three types of connections: 1) digital-to-analog converter (DAC), 2) analog-to-digital (ADC) converter (National Instruments boards), 3) the USB, RS232 connections to control the microscope components (e.g.). Importantly, we implemented existing well-optimized mathematical algorithms to control the scanning mirrors and the detection process [6]. Despite that, numerous types of the software are available on the market (software (Scanlmage, Micro-Manager, YouScope, etc.), in this system we developed a custom software (Matlab, LabView, Python), so we could adjust to the hardware in task-specific manner. Here we created a few sub-programs for each element to be controlled by the software (e.g. manipulators, objective, lasers, shutters, scanners, acousto-optic modulator (AOM), detector, etc.). After maintaining the control over each element we combined and optimized those sub-programs to the single program that controls and synchronize their work. Currently we are optimizing the interface in a quickly adjustable and user-friendly way. Particularly, we developed a graphical interface that is controlling the system and collects images in various scan modes (2D, 3D, line scan mode). For the maintenance purposes, the software interface can be also accessed as a command line tool.
In order to simplify the routine laser alignment process in the software we implemented the option to identify the laser positions where you can monitor it and perform the alignment in relative coordinates where (0; 0) represents the center and the optimal laser position. In this position, the laser beam should pass through the centers of all optical elements. The second procedure to optimize the scanning before the experiments can be also easily performed in the software. It is implemented as a a circular scanning that allows to measure aberrations under the objective at the focal plane. These manipulations are implemented in the "Beam Calibration Setup" service mode and significantly simplify the routine adjustment procedure.
To summarize, here we described the development of the software to control and improve custom-made two-photon systems. This approach significantly reduces the costs, increases efficiency, flexibility and improves the quality of obtained data from various types of samples including cultures, brain slices and in vivo recordings on behaving animals.
This study was supported by the research grant of RFBR foundation No. 16-34-00961 MO^_a. References
1. Danielyan A. [u gp.]. Denoising of two-photon fluorescence images with Block-Matching 3D Filtering. // Methods (San Diego, Calif.). 2014.
2. Danielyan A., Katkovnik V., Egiazarian K. BM3D frames and variational image deblurring // IEEE Transactions on Image Processing. 2012. № 4 (21). C. 1715-1728.
3. Doronin M., Popov A. Design and building of modern two-photon laser scanning microscope for using in neurosci-
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XXIII Congress of I.P. Pavlov Physiology Society ence // MATEC Web of Conferences. 2016. (77). C. 11002.
4. Doronin M., Popov A. Development and design of up-to-date laser scanning two-photon microscope using in neuroscience // Proceedings of SPIE - The International Society for Optical Engineering. 2017. (10250). C. 102501M.
5. Popov A., Doronin M. Design, upgrading and creation of the state-of-the-art two-photon laser microscope view of features used in neuroscience // 2016 6th International Workshop on Computer Science and Engineering, WCSE 2016. 2016. C. 673-677.
6. Svoboda K., Yasuda R. Principles of two-photon excitation microscopy and its applications to neuroscience. // Neuron. 2006. № 6 (50). C. 823-39.
Network Ca2+-Cell Activity Field CA3 Hippocampal Slices of Rat Early and Late Postnatal Development
Mitaeva Y.I.1, Mozherov A.M.1, Mukhina I.V.1'2
1 Lobachevsky State University of Nizhni Novgorod, Nizhny Novgorod, Russian Federation;
2 Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russian Federation.
Information processing in the brain - is the result of the constant interaction between two cellular networks: the neuronal and the glial. Hippocampus - the structure of the central nervous system, which is involved in the mechanisms of emotion and memory consolidation. The hippocampus has a certain topology distribution of cellular elements, which provides the many cellular networks. One of them is the network of neurons in the CA3 field. This network receives inputs from cells of the entorhinal cortex and the dentate gyrus, in addition CA3 pyramidal neurons form the connection between themselves and interneurons, forming a closed network that operates in conditions of acute slice and generates spontaneous Ca2+ activity. Neuronal network interacts with the glial network, the main manifestation of activity which are Ca2+ oscillations. Therefore, to estimate the age dependence of Ca2+ activity in the cells were investigated Ca2+ oscillations in neuronal and glial networks and the interactions between them.
In this work, we investigated changes in the characteristics of Ca2+ oscillations cells of rat hippocampal CA3 field in early (P5-8, P14-16) and late (P21-25), postnatal development. Also shown the effect of temperature of perfusion solution on cells Ca2+ activity of CA3 field hippocampal slices of rats in different postnatal periods. Besides in the study was valued role of network activity in the formation of spontaneous Ca2+ oscillations cells of rat hippocampal CA3 field in early and late stages of postnatal development. Experiments were carried out on acute hippocampal slices from rats. Was used laser scanning confocal microscope Carl Zeiss LSM 510 Duoscan (Germany). Recording fluorescence kinetics were carried out in full frame (field of view of 400x400 mm), with a resolution of 512x512 pixels digital and scanning frequency of 1 Hz. Fluorescence indicators recorded in the range 500-530 nm (Oregon Green 488 BAPTA-1 AM) and 650-710 nm (Sulforhodamine 101). The fluorescence intensity (s.u.) shows the dependence of the concentration of [Ca2+]i in time, indicating the metabolic activity of cells. Method of cross - correlation analysis was used to evaluate synchrony of Ca2+ oscillations cells of CA3 field of rat hippocampus. We chose the time interval size in 3 seconds and within this interval were found synchronous Ca2+ oscillations in all possible pairs of cells. Further, the number of synchronously occurring Ca2+ oscillations were normalized to the minimum number of Ca2+ oscillations in one of the cells analyzed pairs.
The studies have shown that the parameters of cell Ca2+ oscillations field CA3 of hippocampal slices vary depending on the period of postnatal rats. Reducing the amount of Ca2+ oscillations with age due to the formation and complexity of synaptically connected neural networks, the transition of electrical synapses in the chemical. Transitional period is 14-16 days of postnatal development, and for 21 days - there is a fully formed neural network. Electrically connected network is weakly controlled, excitement is freely distributed over the network, involving work of all cells, resulting in a high Ca2+ activity in rat hippocampal cells of younger age group. In mature hippocampal brain slices spontaneous Ca2+ activity with low due to lack of active neural network. In this case, the spontaneous Ca2+ oscillations are due mainly metabolic activity of cells has been shown in our experiments. This study showed that changes in Ca2+ activity in the cells of rat hippocampal CA3 fields occurring during postnatal development directly related to the functioning of the neural networks, and the metabolic state of the cells. Ca2+ signaling in mature brain - is a complex multicomponent process involving various receptor systems capable of mutual substitution in violation of the normal functioning of one or more of them.
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