rus
Issue #5/2021
I.V.Yaminskiy, A.I.Akhmetova, S.A.Senotrusova, Z.Wang, Yu.Bing, B.S.Luk’yanchuk, E.V.Barmina, A.V.Simakin, G.A.Shafeev
New solution for bionanoscopy based on the optical microlens technology
New solution for bionanoscopy based on the optical microlens technology
DOI: 10.22184/1993-8578.2021.14.5.292.297
The microlens microscopy is a relatively new and promising solution to overcome the optical microscopy diffraction limit. It is possible to obtain the optical images with a resolution of tens nanometers using the spheres made of barium titanate. Moreover, combining the probe and microlens microscopy makes it possible to register a wide range of physical and biochemical parameters of the studied samples spectra. The great advantage of this method is a possibility to study a biomaterial either using labels and markers or not, which is essential. This is unattainable by many other conventional research methods. The use of a copper-vapour laser in an optical scheme, makes it possible to study biological objects at low light intensity.
The microlens microscopy is a relatively new and promising solution to overcome the optical microscopy diffraction limit. It is possible to obtain the optical images with a resolution of tens nanometers using the spheres made of barium titanate. Moreover, combining the probe and microlens microscopy makes it possible to register a wide range of physical and biochemical parameters of the studied samples spectra. The great advantage of this method is a possibility to study a biomaterial either using labels and markers or not, which is essential. This is unattainable by many other conventional research methods. The use of a copper-vapour laser in an optical scheme, makes it possible to study biological objects at low light intensity.
Теги: biological objects bionanoscopy nanoscale nanoworld optical microscope probe microscope биологические объекты бионаноскопия зондовый микроскоп наномасштаб наномир оптический микроскоп
New solution for bionanoscopy based on the optical microlens technology
INTRODUCTION
In order to obtain an image of the object passing the diffraction limit, it is necessary to include a microlens in a microscope optical scheme. Nowadays, it is realized in the traditional transmitted and reflected light optical microscopy [1–3], interference microscopy [4], fluorescent microscopy [5], confocal microscopy [6] and Raman microscopy [7].
The microlens microscopy offers new opportunities to watch the live nature processes with a resolution of tens nanometers. In this case, in contrast to super resolution optical microscopy based on the fluorescent marks, the microlens microscopy does not need the markers and dyes at all. However, when brightness and contrast of an image become sufficiently high, the objects are illuminated by the optical irradiation of high intensity. It is not always acceptable when observing biological objects. There are a number of processes in nature which are necessary to observe at low radiation level. In this paper, we present an innovative solution to realize this possibility combining the microlens microscopy and a copper-vapour laser in one optical scheme.
The idea is to pass the light reflected from a biological object through the laser active media and receive a multiple brightness gain. Due to this fact we obtain a bright image using a low level gently irradiation of the biological object. Application of an additional microlens permits to obtain an optical image that is not limited by the diffraction limit. Besides, the microlens itself acts as an optical element that converts the decaying waves in the near zone into the propagating waves in the far zone.
Earlier we conducted measurements of the microchip topography using Zeiss AxioScope 40 optical microscope with the 0.9 aperture and microlens made of titanate barium to obtain images of visible pattern of 150 nm [8]. The geometric and optical parameters of the visualization imaging system produce a significant impact on the image quality to be obtained. It is determined that the visualization system field of view is proportional to the square root of the microlens diameter. For example, using the 25 μm microlens and light wavelength of 500 nm the diameter of the field of view is about 5 μm [9].
RESEARCH METHODS
In this paper, we present the optical scheme consisting of a combination of microlens and laser projecting microscope based on copper-vapour laser optical amplifier (LOA) [10]. The optical scheme is shown in Fig.1. The active element super luminous density radiation of the average power about a few tens milliwatts is directed onto a test object (chromium coating on glass or a microchip with nanotopography) using the object lens objective with a NA = 0.65 numeric aperture. The characteristic dimension of the field of view is 10–20 µm. After reflection from the object, the radiation comes back into the active medium and its brightness is amplified by approximately one thousand times in a single-pass. Depending on the object reflectivity, we obtain a light beam that carries the enlarged object image with an average power output of already 1 watt and more. This device allows you to achieve a bright object image on a 1 square meter screen at a low power at the low power density on the object itself. This is the reflection LOA scheme. The optical scheme of a laser projective microscope based on the copper-vapour laser active medium was firstly realized in [11]. This scheme was applied to visualize the French snail neurons structure changes during its electrical pulses generation [12]. An image of a calibration grid was obtained with the use of this device and a 12 µm dia. microsphere.
The focus distance of the 12 µm dia. microsphere at the refraction index 1.9 is determined by the formula [13]:
. (1)
The focus distance f is measured from the center of the microlens, n – refraction index. According to the formula (1), the focus is located at a distance of 0.3 µm from the microlens surface.
Further increase of the device resolution ability is possible due to using the atomic force microscopy. Presently we have simulated the optimal alignment of the scheme consisted of the traditional optical microscope, microlens optics, precision displacement system, and probe microscope.
Calculation of the microlens resolution ability has also been made [14]. Metrological calibration of the bionanoscopy platform was carried out by making parallel measurements using a probe microscope.
The high-performing digital controller (based on FPGA – field programmable gate array – Xilinx Spartan 6) was used to synchronize the optical and probe measurements. The digital data collection system, which also controls parameters and has an adaptive feedback based on FemtoScan Online software, has been developed [15–16].
The substantative result was achieved by combining the probe and microlens microscopy in a single device to obtain a high response time of microseconds fractions, wide field view of hundreds of micrometers, and efficient manipulations with the biological objects.
The use of the device in biological research opens new unique opportunities for the direct observation of live nature molecular mechanisms, study of the viral infection development of a screening of drugs, etc.
CONCLUSIONS
The significant advantage in the biological objects observation is provided by including a copper-vapour laser into the optical scheme. The visualization of these objects can be carried out at low illumination. In traditional microlens optics, when the required contrast is to be achieved, the light intensity reaches high values due to the photonic jet formation. In case of biological objects this phenomenon can lead to their damage or even combustion. It is possible to obtain a high contrast at low sample illumination using a copper-vapour laser in the optical system. It produces a good effect on the observation process of the biological objects in vivo at high spatial resolution on the 1/4–1/8 wavelength.
Integral combination of the microlens technology, an optical amplifier based on a copper-vapour laser and an atomic force microscope permits to obtain the highest parameters to observe the biological object not only in air, but also in liquids. The reachable technical parameters are:
Prospective models of combined microscopy are: a high-speed FemtoScan X atomic force microscope and a copper-vapour laser as an optical amplifier.
ACKNOWLEDGEMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005. ■
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
INTRODUCTION
In order to obtain an image of the object passing the diffraction limit, it is necessary to include a microlens in a microscope optical scheme. Nowadays, it is realized in the traditional transmitted and reflected light optical microscopy [1–3], interference microscopy [4], fluorescent microscopy [5], confocal microscopy [6] and Raman microscopy [7].
The microlens microscopy offers new opportunities to watch the live nature processes with a resolution of tens nanometers. In this case, in contrast to super resolution optical microscopy based on the fluorescent marks, the microlens microscopy does not need the markers and dyes at all. However, when brightness and contrast of an image become sufficiently high, the objects are illuminated by the optical irradiation of high intensity. It is not always acceptable when observing biological objects. There are a number of processes in nature which are necessary to observe at low radiation level. In this paper, we present an innovative solution to realize this possibility combining the microlens microscopy and a copper-vapour laser in one optical scheme.
The idea is to pass the light reflected from a biological object through the laser active media and receive a multiple brightness gain. Due to this fact we obtain a bright image using a low level gently irradiation of the biological object. Application of an additional microlens permits to obtain an optical image that is not limited by the diffraction limit. Besides, the microlens itself acts as an optical element that converts the decaying waves in the near zone into the propagating waves in the far zone.
Earlier we conducted measurements of the microchip topography using Zeiss AxioScope 40 optical microscope with the 0.9 aperture and microlens made of titanate barium to obtain images of visible pattern of 150 nm [8]. The geometric and optical parameters of the visualization imaging system produce a significant impact on the image quality to be obtained. It is determined that the visualization system field of view is proportional to the square root of the microlens diameter. For example, using the 25 μm microlens and light wavelength of 500 nm the diameter of the field of view is about 5 μm [9].
RESEARCH METHODS
In this paper, we present the optical scheme consisting of a combination of microlens and laser projecting microscope based on copper-vapour laser optical amplifier (LOA) [10]. The optical scheme is shown in Fig.1. The active element super luminous density radiation of the average power about a few tens milliwatts is directed onto a test object (chromium coating on glass or a microchip with nanotopography) using the object lens objective with a NA = 0.65 numeric aperture. The characteristic dimension of the field of view is 10–20 µm. After reflection from the object, the radiation comes back into the active medium and its brightness is amplified by approximately one thousand times in a single-pass. Depending on the object reflectivity, we obtain a light beam that carries the enlarged object image with an average power output of already 1 watt and more. This device allows you to achieve a bright object image on a 1 square meter screen at a low power at the low power density on the object itself. This is the reflection LOA scheme. The optical scheme of a laser projective microscope based on the copper-vapour laser active medium was firstly realized in [11]. This scheme was applied to visualize the French snail neurons structure changes during its electrical pulses generation [12]. An image of a calibration grid was obtained with the use of this device and a 12 µm dia. microsphere.
The focus distance of the 12 µm dia. microsphere at the refraction index 1.9 is determined by the formula [13]:
. (1)
The focus distance f is measured from the center of the microlens, n – refraction index. According to the formula (1), the focus is located at a distance of 0.3 µm from the microlens surface.
Further increase of the device resolution ability is possible due to using the atomic force microscopy. Presently we have simulated the optimal alignment of the scheme consisted of the traditional optical microscope, microlens optics, precision displacement system, and probe microscope.
Calculation of the microlens resolution ability has also been made [14]. Metrological calibration of the bionanoscopy platform was carried out by making parallel measurements using a probe microscope.
The high-performing digital controller (based on FPGA – field programmable gate array – Xilinx Spartan 6) was used to synchronize the optical and probe measurements. The digital data collection system, which also controls parameters and has an adaptive feedback based on FemtoScan Online software, has been developed [15–16].
The substantative result was achieved by combining the probe and microlens microscopy in a single device to obtain a high response time of microseconds fractions, wide field view of hundreds of micrometers, and efficient manipulations with the biological objects.
The use of the device in biological research opens new unique opportunities for the direct observation of live nature molecular mechanisms, study of the viral infection development of a screening of drugs, etc.
CONCLUSIONS
The significant advantage in the biological objects observation is provided by including a copper-vapour laser into the optical scheme. The visualization of these objects can be carried out at low illumination. In traditional microlens optics, when the required contrast is to be achieved, the light intensity reaches high values due to the photonic jet formation. In case of biological objects this phenomenon can lead to their damage or even combustion. It is possible to obtain a high contrast at low sample illumination using a copper-vapour laser in the optical system. It produces a good effect on the observation process of the biological objects in vivo at high spatial resolution on the 1/4–1/8 wavelength.
Integral combination of the microlens technology, an optical amplifier based on a copper-vapour laser and an atomic force microscope permits to obtain the highest parameters to observe the biological object not only in air, but also in liquids. The reachable technical parameters are:
- optical resolution of 25–50 nm,
- spatial resolution of atomic force microscopy in three coordinates X-Y-Z is 1–1–0.1 nm,
- temporary resolution of atomic force microscopy of 1 ms,
- microlens microscopy temporary resolution of 1 ms.
Prospective models of combined microscopy are: a high-speed FemtoScan X atomic force microscope and a copper-vapour laser as an optical amplifier.
ACKNOWLEDGEMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005. ■
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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