rus
Issue #7-8/2021
I.V.Yaminskiy, A.I.Akhmetova, S.A.Senotrusova
Super-resolution microscopy in biomedicine and biology
Super-resolution microscopy in biomedicine and biology
DOI: 10.22184/1993-8578.2021.14.7-8.424.428
The optical microscope is one of the most common and simple tools for studying objects on the microscale, especially in the biological sciences, biomedicine, and chemistry. A significant limitation for use of the optical microscopy is the optical diffraction limit, which is about 250 nm in white light at a wavelength of about 550 nm and a microscope numerical aperture of 1.0. This resolution is often insufficient for imaging tasks of cell organelles, tissue, viral and bacterial particles. A microsphere placed on the sample surface can overcome this limitation and visualise structures as small as 25 nm.
The optical microscope is one of the most common and simple tools for studying objects on the microscale, especially in the biological sciences, biomedicine, and chemistry. A significant limitation for use of the optical microscopy is the optical diffraction limit, which is about 250 nm in white light at a wavelength of about 550 nm and a microscope numerical aperture of 1.0. This resolution is often insufficient for imaging tasks of cell organelles, tissue, viral and bacterial particles. A microsphere placed on the sample surface can overcome this limitation and visualise structures as small as 25 nm.
Теги: biomedicine biotechnologies microsphere super-resolution microscopy viral and bacterial particles вирусные и бактериальные частицы микроскопия сверхвысокого разрешения микросфера
INTRODUCTION
The resolution limit in optics was discovered by the German physicist Ernst Abbe in 1873, when he determined the minimum distance between two objects using the formula:
d =λ / (2NA), (1)
where d is the minimum distance between two structural elements, λ is the wavelength of illumination and NA is the numerical aperture of the lens used [1]. The Abbe diffraction limit predicts the size of the objects distinguishable in an optical microscope lens. The physical reason for the diffraction limit arises from exponentially evanescenting near-field waves, which carry high resolution information about an object and cannot propagate in the far field [2]. This fact limits the imaging of nanoscale structures.
In medicine and biology, there is an enormous need for an instrument that can produce optical images at high resolution that exceeds the diffraction limit. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are often used to image viral and bacterial particles at very high resolution (<10 nm) in a vacuum (see Fig.1), but they are not suitable for studying living objects or virus-cell interactions.
RESEARCH METHODS
Fluorescence optical microscopy is a method of imaging cellular structures, bacteria and viruses at resolutions up to 6 nm [3]. A disadvantage of fluorescence optical imaging is photobleaching, which limits the maximum exposure time to tens of seconds [4]. In addition, fluorescence optical microscopy techniques often require a conjugation of fluorescent molecules with the proteins of the studied subject, and only one type of stained protein can be visualised at a time, whereas in every cell there are many types of proteins of interest to researchers. A disadvantage of the scanning near-field optical microscopy is the long time it takes to acquire a complete image, making it difficult to study biological objects in dynamics.
Because of these optical microscopy limitations, attempts have been made to circumvent the diffraction limit by converting near-field evanescent waves into propagating waves that reach the far field. The possibility of transmitting information from the near field to the far field, where it is collected using a conventional optical microscope is made possible by placing nanoscale lenses [5], polymer microdroplets [6] or dielectric microspheres [7] on the surface of the sample. The use of microspheres with a high refractive index (n> 1.8), that are completely immersed in the liquid or in elastomeric plates, has also been proposed [8]. The use of immersion liquids has made it possible to obtain images of cells, subcellular structures and proteins.
In [9], a 100 µm diameter BaTiO3 lens and an optical microscopy unit were used to image adenovirus particles with a diameter of 75 nm. Microlenses have been used to visualize actin [10], to detect fluorescently stained centrioles, mitochondria, chromosomes, and to study the effect of doxycycline treatment on the expression of mitochondrial-encoded protein in a mouse liver cell line [11].
In [12], optical imaging of objects at a lateral resolution of 25 nm (∼λ / 17) under 408 nm illumination is reported by combining fused quartz and polystyrene microspheres using a conventional scanning laser confocal microscope.
A disadvantage of the microlens microscopy is the limited field of view, which cannot exceed the diameter of the lens or other object placed on the sample. Another limiting factor is that the microspheres are randomly placed on the surface, so only a certain portion of the objects can be visualised and the position of the lenses on the sample is difficult to change.
However, the work is already underway to create installations for continuous scanning of a large view field of the studied space. To create ultra-high resolution images over large areas, a microsphere is mounted on the frame attached to the microscope objective, which automatically scans the sample step by step [13].
CONCLUSIONS
Our research group is also working on improving the microlens microscopy system, in particular, we are developing a facility for combined atomic force microscopy and microlens microscopy based on the FemtoScan Xi scanning probe microscope [14]. A microlens is fixed on the cantilever for the atomic force microscopy and scanning is performed in parallel in two modes – the software displays the surface topography and the image through the microlens. Images of test specimens are obtained using an optical microscope and titanium TiO2 oxide microlenses. The distance between the red marks corresponds to approximately 100 nm (see Fig.2).
ACKNOWLEDGEMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, and RFBR, Project No. 20-32-90036. This research was carried out with financial support from the FASIE, Project No. 71108, and Agreement No. 0071108.
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.
The resolution limit in optics was discovered by the German physicist Ernst Abbe in 1873, when he determined the minimum distance between two objects using the formula:
d =λ / (2NA), (1)
where d is the minimum distance between two structural elements, λ is the wavelength of illumination and NA is the numerical aperture of the lens used [1]. The Abbe diffraction limit predicts the size of the objects distinguishable in an optical microscope lens. The physical reason for the diffraction limit arises from exponentially evanescenting near-field waves, which carry high resolution information about an object and cannot propagate in the far field [2]. This fact limits the imaging of nanoscale structures.
In medicine and biology, there is an enormous need for an instrument that can produce optical images at high resolution that exceeds the diffraction limit. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are often used to image viral and bacterial particles at very high resolution (<10 nm) in a vacuum (see Fig.1), but they are not suitable for studying living objects or virus-cell interactions.
RESEARCH METHODS
Fluorescence optical microscopy is a method of imaging cellular structures, bacteria and viruses at resolutions up to 6 nm [3]. A disadvantage of fluorescence optical imaging is photobleaching, which limits the maximum exposure time to tens of seconds [4]. In addition, fluorescence optical microscopy techniques often require a conjugation of fluorescent molecules with the proteins of the studied subject, and only one type of stained protein can be visualised at a time, whereas in every cell there are many types of proteins of interest to researchers. A disadvantage of the scanning near-field optical microscopy is the long time it takes to acquire a complete image, making it difficult to study biological objects in dynamics.
Because of these optical microscopy limitations, attempts have been made to circumvent the diffraction limit by converting near-field evanescent waves into propagating waves that reach the far field. The possibility of transmitting information from the near field to the far field, where it is collected using a conventional optical microscope is made possible by placing nanoscale lenses [5], polymer microdroplets [6] or dielectric microspheres [7] on the surface of the sample. The use of microspheres with a high refractive index (n> 1.8), that are completely immersed in the liquid or in elastomeric plates, has also been proposed [8]. The use of immersion liquids has made it possible to obtain images of cells, subcellular structures and proteins.
In [9], a 100 µm diameter BaTiO3 lens and an optical microscopy unit were used to image adenovirus particles with a diameter of 75 nm. Microlenses have been used to visualize actin [10], to detect fluorescently stained centrioles, mitochondria, chromosomes, and to study the effect of doxycycline treatment on the expression of mitochondrial-encoded protein in a mouse liver cell line [11].
In [12], optical imaging of objects at a lateral resolution of 25 nm (∼λ / 17) under 408 nm illumination is reported by combining fused quartz and polystyrene microspheres using a conventional scanning laser confocal microscope.
A disadvantage of the microlens microscopy is the limited field of view, which cannot exceed the diameter of the lens or other object placed on the sample. Another limiting factor is that the microspheres are randomly placed on the surface, so only a certain portion of the objects can be visualised and the position of the lenses on the sample is difficult to change.
However, the work is already underway to create installations for continuous scanning of a large view field of the studied space. To create ultra-high resolution images over large areas, a microsphere is mounted on the frame attached to the microscope objective, which automatically scans the sample step by step [13].
CONCLUSIONS
Our research group is also working on improving the microlens microscopy system, in particular, we are developing a facility for combined atomic force microscopy and microlens microscopy based on the FemtoScan Xi scanning probe microscope [14]. A microlens is fixed on the cantilever for the atomic force microscopy and scanning is performed in parallel in two modes – the software displays the surface topography and the image through the microlens. Images of test specimens are obtained using an optical microscope and titanium TiO2 oxide microlenses. The distance between the red marks corresponds to approximately 100 nm (see Fig.2).
ACKNOWLEDGEMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, and RFBR, Project No. 20-32-90036. This research was carried out with financial support from the FASIE, Project No. 71108, and Agreement No. 0071108.
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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