rus
Issue #2/2024
A.I.Akhmetova, A.D.Terentev, S.A.Senotrusova, T.O.Sovetnikov, D.I.Yaminsky, V.V.Popov, I.V.Yaminsky
DIFFRACTION GRATING AS A MEANS OF METROLOGICAL SUPPORT OF MICROSCOPY
DIFFRACTION GRATING AS A MEANS OF METROLOGICAL SUPPORT OF MICROSCOPY
DOI: 10.22184/1993-8578.2024.17.2.128.133
Abstract. Visualization of biomedical samples in their natural environment at micro- and nanoscale is crucial for studying fundamental principles of biosystems functioning with complex interactions. The study of dynamic biological processes requires a microscopic system with multiple measurement capabilities, high spatial and temporal resolution, versatile visualization environments and local manipulation options. Scanning capillary microscopy and microlens microscopy are promising tools for these tasks, but correct operation of either technique is impossible without metrological support. This paper demonstrates the possibility of using a diffraction grating sample for these purposes.
Abstract. Visualization of biomedical samples in their natural environment at micro- and nanoscale is crucial for studying fundamental principles of biosystems functioning with complex interactions. The study of dynamic biological processes requires a microscopic system with multiple measurement capabilities, high spatial and temporal resolution, versatile visualization environments and local manipulation options. Scanning capillary microscopy and microlens microscopy are promising tools for these tasks, but correct operation of either technique is impossible without metrological support. This paper demonstrates the possibility of using a diffraction grating sample for these purposes.
Теги: дифракционная решетка живые системы метрология микролинзовая микроскопия сканирующая капиллярная микроскопия
INTRODUCTION
Microscopy techniques relevant to biomedical research include optical microscopy, cryoelectron microscopy and scanning probe microscopy. Different types of microscopy techniques have their own resolution capabilities, experimental conditions: working environment and sample preparation steps. Depending on the experimental task, appropriate methods can be chosen, but each of the above methods requires a standard for calibration and selection of measurement conditions for optimal resolution.
One such reference can be a diffraction grating. A diffraction grating is a regularly spaced pattern of slits or protrusions applied to a surface. Such a structured pattern can have different periods and heights of protrusions, which makes the grating an ideal calibration standard.
In this work, diffraction gratings with different periods were used to evaluate the atomic force accuracy, scanning capillary and optical microlens microscopes. Scanning a diffraction grating sample allows not only to calibrate the scale bar in optical microscopy when using microlenses, but also to adjust certain scanning parameters in atomic force microscopy, to select individual parameters of capillary stretching for capillary microscopy.
MATERIALS AND RESEARCH METHODS
The sample was a relief diffraction grating on metallised thermoplastic with known period and structures' dimensions. The diffraction grating obtained by impression of the relief from a nickel die on thermoplastic applied on photopolymer was used. Thermal plastic was applied on a 35 µm PET film.
To overcome the limitation in optical microscopy, microlenses can be used to significantly improve the image qualities [1, 2]. Barium titanate microspheres immersed in silicone oil and polymethylacrylate (PMA) microspheres in air were used to observe the magnified image in optical microscopy. Observations were made on a Zeiss Scope.A1 optical microscope in reflected light mode. Zeiss 50x/0.55 DIC, Zeiss 100x/0.75 DIC objectives were used. The microlenses were placed on top of the sample surface.
Scanning capillary microscopy was performed on the FemtoScan Xi and FemtoScan Aion microscope.
Atomic force microscopy was performed on the FemtoScan multifunctional microscope.
RESULTS AND DISCUSSIONS
Two kinds of microlenses made of barium titanate in silicone oil and of polymethacrylate in air were used for optical images of a diffraction grating with a grating period of 800 nm and a slit width of 300 nm.
Images with barium titanate lenses are presented in Fig.1, the magnification is ~2.4, the lens magnification is 50x. The fringes are clearly distinguishable on the image, although the central part of the microlens looks slightly illuminated.
In Fig.2, the diffraction grating image was visualised using polymethacrylate microspheres. The period of the grating is 800 nm, slit width is 300 nm. The magnification is ~2.1. The objective lens magnification is 100x.
When studying the same grating on an atomic force microscope, the bandwidth value was slightly overestimated, because during scanning the cantilever smoothes sharp vertical transitions of protrusions and due to this there is a widening effect.
The diffraction grating with a period of 1.7 μm and a bandwidth of 0.5 μm was examined on a FemtoScan Xi scanning capillary microscope (Fig.4). A capillary with an orifice diameter of about 60 nm was used, and the oscillation amplitude and convergence rate were 300 nm and 15 nm/ms, respectively.
The period and bandwidth of obtained results of SCM measurements agree with the stated values. Separately, it is worth noting the achieved better display of vertical walls of diffraction bands in comparison with the AFM probe, which gives broadening of the vertical relief.
The grating sample with a period of 1.0 μm and a bandwidth of 0.4 μm was also used as a calibration measure for the metrology of the FemtoScan Aion microscope (Fig.5). The obtained images helped to calibrate the lateral course of the piezo motion.
CONCLUSIONS
Imaging techniques that allow to determine the physicochemical and mechanical properties with high temporal and spatial resolution in liquids will always be needed. That is why it is so relevant to use different standards for calibrating instruments, which can show advantage of one or another imaging method. The diffraction grating is a good standard for both optical microscopy and probe microscopy. The essential advantages of the diffraction grating are due to the following factors. The geometric relief of the grating is tied to the wavelength of the light forming the grating pattern. As a result, high metrological accuracy of the grating is achieved. The grating is formed on transparent plastic, which ensures its convenient use when positioning the probe microscope on an inverted optical microscope. This enables full optical observation of both the grating itself and the sample located both in immediate vicinity of the grating and on it. Additional important advantages of the grating are availability and low cost.
ACKNOWLEDGMENTS
The work was carried out under the state order with the financial support of the Physical Department of the Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by Advanced Technologies Center (www.nanoscopy.ru).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
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.
Microscopy techniques relevant to biomedical research include optical microscopy, cryoelectron microscopy and scanning probe microscopy. Different types of microscopy techniques have their own resolution capabilities, experimental conditions: working environment and sample preparation steps. Depending on the experimental task, appropriate methods can be chosen, but each of the above methods requires a standard for calibration and selection of measurement conditions for optimal resolution.
One such reference can be a diffraction grating. A diffraction grating is a regularly spaced pattern of slits or protrusions applied to a surface. Such a structured pattern can have different periods and heights of protrusions, which makes the grating an ideal calibration standard.
In this work, diffraction gratings with different periods were used to evaluate the atomic force accuracy, scanning capillary and optical microlens microscopes. Scanning a diffraction grating sample allows not only to calibrate the scale bar in optical microscopy when using microlenses, but also to adjust certain scanning parameters in atomic force microscopy, to select individual parameters of capillary stretching for capillary microscopy.
MATERIALS AND RESEARCH METHODS
The sample was a relief diffraction grating on metallised thermoplastic with known period and structures' dimensions. The diffraction grating obtained by impression of the relief from a nickel die on thermoplastic applied on photopolymer was used. Thermal plastic was applied on a 35 µm PET film.
To overcome the limitation in optical microscopy, microlenses can be used to significantly improve the image qualities [1, 2]. Barium titanate microspheres immersed in silicone oil and polymethylacrylate (PMA) microspheres in air were used to observe the magnified image in optical microscopy. Observations were made on a Zeiss Scope.A1 optical microscope in reflected light mode. Zeiss 50x/0.55 DIC, Zeiss 100x/0.75 DIC objectives were used. The microlenses were placed on top of the sample surface.
Scanning capillary microscopy was performed on the FemtoScan Xi and FemtoScan Aion microscope.
Atomic force microscopy was performed on the FemtoScan multifunctional microscope.
RESULTS AND DISCUSSIONS
Two kinds of microlenses made of barium titanate in silicone oil and of polymethacrylate in air were used for optical images of a diffraction grating with a grating period of 800 nm and a slit width of 300 nm.
Images with barium titanate lenses are presented in Fig.1, the magnification is ~2.4, the lens magnification is 50x. The fringes are clearly distinguishable on the image, although the central part of the microlens looks slightly illuminated.
In Fig.2, the diffraction grating image was visualised using polymethacrylate microspheres. The period of the grating is 800 nm, slit width is 300 nm. The magnification is ~2.1. The objective lens magnification is 100x.
When studying the same grating on an atomic force microscope, the bandwidth value was slightly overestimated, because during scanning the cantilever smoothes sharp vertical transitions of protrusions and due to this there is a widening effect.
The diffraction grating with a period of 1.7 μm and a bandwidth of 0.5 μm was examined on a FemtoScan Xi scanning capillary microscope (Fig.4). A capillary with an orifice diameter of about 60 nm was used, and the oscillation amplitude and convergence rate were 300 nm and 15 nm/ms, respectively.
The period and bandwidth of obtained results of SCM measurements agree with the stated values. Separately, it is worth noting the achieved better display of vertical walls of diffraction bands in comparison with the AFM probe, which gives broadening of the vertical relief.
The grating sample with a period of 1.0 μm and a bandwidth of 0.4 μm was also used as a calibration measure for the metrology of the FemtoScan Aion microscope (Fig.5). The obtained images helped to calibrate the lateral course of the piezo motion.
CONCLUSIONS
Imaging techniques that allow to determine the physicochemical and mechanical properties with high temporal and spatial resolution in liquids will always be needed. That is why it is so relevant to use different standards for calibrating instruments, which can show advantage of one or another imaging method. The diffraction grating is a good standard for both optical microscopy and probe microscopy. The essential advantages of the diffraction grating are due to the following factors. The geometric relief of the grating is tied to the wavelength of the light forming the grating pattern. As a result, high metrological accuracy of the grating is achieved. The grating is formed on transparent plastic, which ensures its convenient use when positioning the probe microscope on an inverted optical microscope. This enables full optical observation of both the grating itself and the sample located both in immediate vicinity of the grating and on it. Additional important advantages of the grating are availability and low cost.
ACKNOWLEDGMENTS
The work was carried out under the state order with the financial support of the Physical Department of the Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by Advanced Technologies Center (www.nanoscopy.ru).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
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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