rus
Issue #3-4/2023
S.A.Senotrusova, A.I.Akhmetova, I.V.Yaminsky
SUPERRESOLUTION OF MICROLENSES IN THE PHYSICS OF LIVING SYSTEMS
SUPERRESOLUTION OF MICROLENSES IN THE PHYSICS OF LIVING SYSTEMS
DOI: https://doi.org/10.22184/1993-8578.2023.16.3-4.168.176
The spatial resolution of any conventional optical microscope is limited by the diffraction of light waves on the objective aperture, with white light the best optical microscopes resolution is about 200 nm. One way to overcome this limitation is microlens microscopy. Microsphere microscopy is a type of label-free microscopy in which spherical microlenses are placed directly on or near the sample to produce high-resolution optical images. With label-free and real-time imaging, optical microlens microscopy shows great potential in medicine and biology.
The spatial resolution of any conventional optical microscope is limited by the diffraction of light waves on the objective aperture, with white light the best optical microscopes resolution is about 200 nm. One way to overcome this limitation is microlens microscopy. Microsphere microscopy is a type of label-free microscopy in which spherical microlenses are placed directly on or near the sample to produce high-resolution optical images. With label-free and real-time imaging, optical microlens microscopy shows great potential in medicine and biology.
Теги: bionanoscopy microlens microscopy microsphere physics of living systems scanning probe microscopy бионаноскопия микролинзовая микроскопия микросферы сканирующая зондовая микроскопия физика живых систем
INTRODUCTION
In microlens optical microscopy, a transparent dielectric microsphere is placed in close proximity to the studied sample and it operates as an additional magnifier between the sample and the optical microscope objective. Efficiency and simplicity make it possible to use microspheres from different materials and with different geometric parameters in air and liquid media, as well as to integrate microspheres into other highly informative research methods in high-resolution microscopy [1].
This imaging technique was firstly introduced in 2011 [2]. Barium titanate microspheres with the diameter of 2-220 μm and a high refractive index (n ∼ 1.9-2.1) can be used to visualise nanostructures up to 100 nm in liquid [3]. Since then, different ways of using microlenses have emerged: it has been proposed to immerse spheres half in ethanol [4], in the sugar solution and immersion oil [5, 6], in elastomers [7-9], and microlenses themselves have been formed from nanoparticle solutions [10], from bacteria [11] and even from spider web [12].
Positioning the microlenses over the sample
One of the main challenges in the broad application of microlens microscopy is ensuring accurate positioning of the microspheres (Fig.1). Chemical, physical and mechanical methods of microsphere positioning can be used to scan the sample surface with microspheres. When chemical and physical methods are used, the region of interest is scanned by local catalytic reactions [13], optical tweezers, and acoustic fluids [14]. These methods do not have a limited field of view and do not fix the microsphere.
Scanning with a mechanical manipulator can be implemented in an atomic force microscope using a probe or capillary (Fig.2) and a three-dimensional moving table [15-17]. In this case positioning accuracy can reach several nanometres but the field of view is limited by the field of the piezo manipulator. A large installation space is also required. Microlens microscopy allows much better resolution when scanning two-dimensional samples, but it's inevitable to damage the sample or microlens when imaging a three-dimensional surface due to the lack of feedback of the microsphere position over the surface or the interaction strength between the probe and the sample. There are various methods for positioning microspheres more precisely in space: microlenses have been attached directly to the lens surface [18,19], bonded to a glass micropipette [20], attached to an AFM cantilever [21], and solid immersion lenses arrays were created [22].
Special microlens arrays are being developed, increasing the fields of view and somewhat simplifying the task of positioning the array over the sample surface. Thanks to a special holder connected to a piezoceramic stage, an image of the surface of a 900 µm2 Blu-ray disc, sequentially stitched from 210 images, was obtained using such an array [23].
In [24], researchers managed to attach a silicon microlens to a cantilever and grow a diamond probe to simultaneously scan topography and obtain an optical image of the sample surface. This allowed them to observe a microcircuit sample with an area of 90 × 90 μm2 and structures up to 60 nm in size.
Label-free microscopy
Microlens microscopy allows biological samples to be examined without labeling. Using an array of barium titanate microlenses, it was possible to visualise adenovirus particles with resolution of up to 100 nm [25]. Using sodium-lime-glass microspheres with a diameter of 25 µm, structures of mouse brain cells down to 100 nm have been studied [26]. Imaging lenses made of densely packed 15 nm titanium oxide spheres made it possible to visualise biological objects: tumour cells in air, spiral bacteria, Streptococcus thermophilus, mouse echinocytes in an aqueous medium [27]. Using barium titanate microlenses, erythrocyte transition into an echinocyte without fixation was demonstrated in real time (Fig.3) [28].
Combination with other methods
Advantage of microlens technology is in effective combination with other microscopy techniques.
Microspheres can be applied in combination with confocal [29], Raman [30], interferometric [31], holographic microscopy [32, 33], Mirau interferometry [34], Mueller matrix microscopy [35] and second optical harmonic microscopy.
Microspheres have been used to improve resolution in Raman microscopy for imaging graphene nanotubes [36]. Combination of microlenses and a copper vapour laser not only improves resolution but also avoids undesirable phenomena such as photodamage in the study of biological objects due to the use of low light intensity [37].
Microspheres can be combined with fluorescence microscopy to study biological objects such as glioblastoma cells [38], mouse hepatocytes [39], adenoviruses [40]. In [41], atomic force microscopy and fluorescence microscopy were combined to image the surface of live mouse myoblast cells (C2C12) and human breast cancer cells (MCF-7) using constant height mode.
A collagen-containing lung fibrous tissue specimen was imaged with up to 125 nm resolution through a 14 µm barium titanate sphere by combining microlenses and Second Harmonic Generation (SHG) microscopy [42].
Microspheres have improved lateral resolution in digital holographic microscopy in the study of erythrocytes from patients with thalassemia, which is characterized by a decrease in hemoglobin production [43].
In addition to commercially available microspheres, there is research on the use of biological lenses – bacteria, yeast, stem cells, lymphocytes and monocytes (44). Using a cell as a microlens and optical tweezers to position it, it has been possible to resolve the fibrous cytoskeleton within an epithelial cell and the bilayer structures on the cell membrane, which were indistinguishable in a conventional microscope (45).
The erythrocyte can swell from a disk volume of 90 fl to a sphere volume of 150 fl, changing the focal distance from negative to positive values, so it is also used as a biolens [46]. Due to cell membranes elasticity, the shape of the erythrocyte can be easily transformed by optical forces changing the focal distance from 3.3 to 6.5 μm. Polystyrene particles of 500 nm have been imaged using erythrocyte microlenses [47].
In [48], microrelief was studied using Hela cells and it was shown that astigmatism caused by the cell nucleus, especially during cell division, can lead to aberrations up to hundreds of nanometers and inaccurate observations in high-resolution microscopy.
In our works for measurements in liquid we use barium titanate microlenses BTGMS-4.25, diameter 30-100 µm, density 4.22 g/cm3, refractive index 1.9; for measurements in air we use polymethylacrylate (PMA) microspheres 9 µm in diameter with an intrinsic index of refraction 1.49 [49].
Two optical microscopes are used, a Zeiss Model AxioSkop-40 straight microscope in reflected light mode and a Nikon Ti-U Eclipse inverted microscope in transmitted light mode. Lenses used: Nikon Plan Fluor 10x/0.30 and Nikon S Plan Fluor 40x/0.60.
By using an inverted microscope it is possible to study biological objects dynamically in liquid by placing microlenses on the bottom of a Petri dish and placing the sample on microspheres. Also for measurements in liquid it is possible to place microlenses and a sample in liquid between two glasses. In this case it is possible to capture the objects movement in volume as the sample dries.
CONCLUSIONS
Despite the rapid development of the use of microlenses in resolution of static objects, calibration gratings, microarrays, and optical disks, not many works are devoted to dynamic phenomena in biology. This is partly due to the lack of a standard approach to integrating them into existing optical instruments, the need to constantly calibrate the setup to experimental conditions. A microsphere collects a evanescent wave in the near field and converts it into a propagating wave. This means that the distance between the microsphere and the sample must be smaller than the wavelength, which is why it is so important to precisely control the position of the microsphere with feedback. In addition, the field of view of individual microspheres is quite small – comparable to the diameter of the microspheres. Image stitching technology during scanning increases the field of view, but makes it difficult to process the results.
In improving microlenses working methods there are still many gaps, but the method has great potential to encrease optical microscopy resolution quickly and relatively easily, and we are actively working to optimise this technology for biological applications.
ACKNOWLEDGMENTS
The study of A.I.Akhmetova was supported by the Russian Science Foundation (project No. 23-74-30003), and the work of S.A.Senotrusova was supported by the Theoretical Physics and Mathematics Advancement Foundation "BASIS" (contract No. 22-2-9-19-1).
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.
In microlens optical microscopy, a transparent dielectric microsphere is placed in close proximity to the studied sample and it operates as an additional magnifier between the sample and the optical microscope objective. Efficiency and simplicity make it possible to use microspheres from different materials and with different geometric parameters in air and liquid media, as well as to integrate microspheres into other highly informative research methods in high-resolution microscopy [1].
This imaging technique was firstly introduced in 2011 [2]. Barium titanate microspheres with the diameter of 2-220 μm and a high refractive index (n ∼ 1.9-2.1) can be used to visualise nanostructures up to 100 nm in liquid [3]. Since then, different ways of using microlenses have emerged: it has been proposed to immerse spheres half in ethanol [4], in the sugar solution and immersion oil [5, 6], in elastomers [7-9], and microlenses themselves have been formed from nanoparticle solutions [10], from bacteria [11] and even from spider web [12].
Positioning the microlenses over the sample
One of the main challenges in the broad application of microlens microscopy is ensuring accurate positioning of the microspheres (Fig.1). Chemical, physical and mechanical methods of microsphere positioning can be used to scan the sample surface with microspheres. When chemical and physical methods are used, the region of interest is scanned by local catalytic reactions [13], optical tweezers, and acoustic fluids [14]. These methods do not have a limited field of view and do not fix the microsphere.
Scanning with a mechanical manipulator can be implemented in an atomic force microscope using a probe or capillary (Fig.2) and a three-dimensional moving table [15-17]. In this case positioning accuracy can reach several nanometres but the field of view is limited by the field of the piezo manipulator. A large installation space is also required. Microlens microscopy allows much better resolution when scanning two-dimensional samples, but it's inevitable to damage the sample or microlens when imaging a three-dimensional surface due to the lack of feedback of the microsphere position over the surface or the interaction strength between the probe and the sample. There are various methods for positioning microspheres more precisely in space: microlenses have been attached directly to the lens surface [18,19], bonded to a glass micropipette [20], attached to an AFM cantilever [21], and solid immersion lenses arrays were created [22].
Special microlens arrays are being developed, increasing the fields of view and somewhat simplifying the task of positioning the array over the sample surface. Thanks to a special holder connected to a piezoceramic stage, an image of the surface of a 900 µm2 Blu-ray disc, sequentially stitched from 210 images, was obtained using such an array [23].
In [24], researchers managed to attach a silicon microlens to a cantilever and grow a diamond probe to simultaneously scan topography and obtain an optical image of the sample surface. This allowed them to observe a microcircuit sample with an area of 90 × 90 μm2 and structures up to 60 nm in size.
Label-free microscopy
Microlens microscopy allows biological samples to be examined without labeling. Using an array of barium titanate microlenses, it was possible to visualise adenovirus particles with resolution of up to 100 nm [25]. Using sodium-lime-glass microspheres with a diameter of 25 µm, structures of mouse brain cells down to 100 nm have been studied [26]. Imaging lenses made of densely packed 15 nm titanium oxide spheres made it possible to visualise biological objects: tumour cells in air, spiral bacteria, Streptococcus thermophilus, mouse echinocytes in an aqueous medium [27]. Using barium titanate microlenses, erythrocyte transition into an echinocyte without fixation was demonstrated in real time (Fig.3) [28].
Combination with other methods
Advantage of microlens technology is in effective combination with other microscopy techniques.
Microspheres can be applied in combination with confocal [29], Raman [30], interferometric [31], holographic microscopy [32, 33], Mirau interferometry [34], Mueller matrix microscopy [35] and second optical harmonic microscopy.
Microspheres have been used to improve resolution in Raman microscopy for imaging graphene nanotubes [36]. Combination of microlenses and a copper vapour laser not only improves resolution but also avoids undesirable phenomena such as photodamage in the study of biological objects due to the use of low light intensity [37].
Microspheres can be combined with fluorescence microscopy to study biological objects such as glioblastoma cells [38], mouse hepatocytes [39], adenoviruses [40]. In [41], atomic force microscopy and fluorescence microscopy were combined to image the surface of live mouse myoblast cells (C2C12) and human breast cancer cells (MCF-7) using constant height mode.
A collagen-containing lung fibrous tissue specimen was imaged with up to 125 nm resolution through a 14 µm barium titanate sphere by combining microlenses and Second Harmonic Generation (SHG) microscopy [42].
Microspheres have improved lateral resolution in digital holographic microscopy in the study of erythrocytes from patients with thalassemia, which is characterized by a decrease in hemoglobin production [43].
In addition to commercially available microspheres, there is research on the use of biological lenses – bacteria, yeast, stem cells, lymphocytes and monocytes (44). Using a cell as a microlens and optical tweezers to position it, it has been possible to resolve the fibrous cytoskeleton within an epithelial cell and the bilayer structures on the cell membrane, which were indistinguishable in a conventional microscope (45).
The erythrocyte can swell from a disk volume of 90 fl to a sphere volume of 150 fl, changing the focal distance from negative to positive values, so it is also used as a biolens [46]. Due to cell membranes elasticity, the shape of the erythrocyte can be easily transformed by optical forces changing the focal distance from 3.3 to 6.5 μm. Polystyrene particles of 500 nm have been imaged using erythrocyte microlenses [47].
In [48], microrelief was studied using Hela cells and it was shown that astigmatism caused by the cell nucleus, especially during cell division, can lead to aberrations up to hundreds of nanometers and inaccurate observations in high-resolution microscopy.
In our works for measurements in liquid we use barium titanate microlenses BTGMS-4.25, diameter 30-100 µm, density 4.22 g/cm3, refractive index 1.9; for measurements in air we use polymethylacrylate (PMA) microspheres 9 µm in diameter with an intrinsic index of refraction 1.49 [49].
Two optical microscopes are used, a Zeiss Model AxioSkop-40 straight microscope in reflected light mode and a Nikon Ti-U Eclipse inverted microscope in transmitted light mode. Lenses used: Nikon Plan Fluor 10x/0.30 and Nikon S Plan Fluor 40x/0.60.
By using an inverted microscope it is possible to study biological objects dynamically in liquid by placing microlenses on the bottom of a Petri dish and placing the sample on microspheres. Also for measurements in liquid it is possible to place microlenses and a sample in liquid between two glasses. In this case it is possible to capture the objects movement in volume as the sample dries.
CONCLUSIONS
Despite the rapid development of the use of microlenses in resolution of static objects, calibration gratings, microarrays, and optical disks, not many works are devoted to dynamic phenomena in biology. This is partly due to the lack of a standard approach to integrating them into existing optical instruments, the need to constantly calibrate the setup to experimental conditions. A microsphere collects a evanescent wave in the near field and converts it into a propagating wave. This means that the distance between the microsphere and the sample must be smaller than the wavelength, which is why it is so important to precisely control the position of the microsphere with feedback. In addition, the field of view of individual microspheres is quite small – comparable to the diameter of the microspheres. Image stitching technology during scanning increases the field of view, but makes it difficult to process the results.
In improving microlenses working methods there are still many gaps, but the method has great potential to encrease optical microscopy resolution quickly and relatively easily, and we are actively working to optimise this technology for biological applications.
ACKNOWLEDGMENTS
The study of A.I.Akhmetova was supported by the Russian Science Foundation (project No. 23-74-30003), and the work of S.A.Senotrusova was supported by the Theoretical Physics and Mathematics Advancement Foundation "BASIS" (contract No. 22-2-9-19-1).
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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