In confocal microscopy, multifocal illumination can reduce image capture time compared to single point scanning. However, due to increased system complexity, attaining uniform multifocal illumination across the field of view with traditional approaches is difficult. For multifocal confocal imaging in lateral as well as in axial directions, we propose two methods using a volume holographic lenslet array illuminator (VHLAI) and a volume holographic axial multi-plane illuminator (VHAMI) respectively. A super Gaussian (SG) beam shaping was incorporated with VHLAI with a 43% efficiency and utilized in a confocal microscope to provide uniform array illumination. Multiplexed holographic gratings are used to achieve simultaneous multi-plane illumination. Multiple photo detector arrangements have been done to carry out simultaneous multi-plane imaging without mechanical or electro-optic axial scanning. The design methodologies for photopolymer-based volume holographic beam shapers are discussed, as well as their benefits. we demonstrate the ability of proposed approaches by comparing optically sectioned microscopic images of fluorescence beads, florescence pollen grains, and biological specimens to wide-field images. The proposed systems can greatly reduce image acquisition time while maintaining image quality. When compared to former spatial light modulating devices, the illumination approach employed in our confocal systems are more direct and compact. Multifocal illumination may considerably shorten scanning time without requiring a change in scanning devices, while also providing uniform illumination across the field of view. These multifocal confocal systems construct images more quickly while requiring no modifications to scanning hardware. The approaches shown here might be useful in high-speed multifocal microscopy platforms.
Optical microscopic techniques are the most commonly used methods in biology and medical research. In recent years, structured light plays vital roles to enhance resolution. In addition, metasurfaces, the emerging optical techniques, with unique optical capabilities to manipulate the basic characteristics of light have received a significant amount of interest in the optical microscopic field. This talk will introduce the latest studies on the biomedical use of structured light, as well as metasurface. The following are the topics that will be covered: quantitative cell imaging, optical sectioning microscopy; light sheet microscopy; and optical tweezers. This talk will also discuss the technological challenges presently encountered with metasurface from the point of view of preclinical and clinical systems.
Conventional optical imaging systems have played a crucial role in various biomedical applications, including diagnosis, drug development, and tissue engineering. However, they usually suffer from the requirements of bulky optical components, particularly when operating in environments with limited space. Metasurfaces, composed of artificially thin nanostructures that manipulate light at the nanoscale level enabling precise control of the phase, amplitude, and polarization of light, offer a promising solution to the limitation. These technologies can be used in a wide range of bio-imaging applications, including light sheet microscopy, endoscopy, confocal microscopy, and more. In this talk, we will explore the use of metasurfaces in various bio-imaging applications and discuss their potential for revolutionizing diagnosis and treatment in the biomedical field.
Microscopy is an essential tool for biomedical research and clinical diagnosis. Nevertheless, conventional wide-field imaging system lacks optical sectioning ability, restricting applications in thick tissues. Recently, HiLo microscopy improved sectioning efficiency but still requires axial movement. Here, we propose the Moiré metalens to develop the varifocal metalens based fluorescence optical sectioning microscopy. It leveraging metasurfaces capabilities to control optical properties. Our system enables optical sectioning and 3D imaging capability. We demonstrate multiplane HiLo optically sectioned images of fluorescent microspheres and ex-vivo mouse brain tissues. This technique opens new possibilities for high-contrast microscopy in biomedical research and clinical diagnosis.
We present the use of Moiré metalens in fluorescence microscopy system combining with telecentric system and HiLo algorithm for bio-imaging. The Moiré metalens is composed of paired phase metasurfaces. The system is capable of tuning the focal length from 10 mm to 125 mm by tuning the relative angle between the paired metasurfaces without axial displacement. In addition, the speckle illumination HiLo microscopy is used to mitigate the out-of-focus light scattering, the background noise. The labeled beads and ex vivo mouse intestinal tissue samples are imaged to show the optical sectioning capacity of our system. It is anticipated that the proposed design of varifocal metalens would have significant applications in fluorescence microscopy and endoscopy.
We have successfully designed, fabricated, and demonstrated the use of a planar dielectric metasurface to generate AAF beams for biomedical applications. Our results demonstrate the potential of using metasurface for precise control of the local phase of light and the generation of unique optical properties. The high-intensity contrast between the initial and focal plane was used for fluorescence-guided light treatment of microspheres and mouse cardiac slices. Our ultrathin (800 nm) Nano-photonics metasurface allows for designing miniature integrated optical devices for biomedical instruments. The AAF beam& #39;s high-energy-density hotspot could be useful in Nano-surgery, high-resolution imaging, and photo-dynamic therapy.
Fluorescence microscopy is a common method to observe the structural features of biological samples. Conventional tunable lenses are usually used to axially scan the volumetric samples for obtaining fluorescence optical sectioning images. However, they suffer from spherical aberration and distortions. In this work, we propose a compact imaging system with optical sectioning capability based on Moiré metalens which consists of two complementary phase metasurfaces to perform fluorescence bio-imaging applications in visible regions. The focal length of system can be tuned from 10 mm to 125 mm at the wavelength of 532 nm by changing mutual angles between two metasurfaces. In addition, the speckle illumination HiLo microscope is used to reduce the effect of out-of-focus light scattering. To demonstrate the optical sectioning capability of our system, the labeled beads as well as ex vivo mice intestine tissue samples are imaged. The presented design of varifocal metalens is anticipated to realize important applications in fluorescence microscopy and endoscopy.
Based on the Moiré effect, a pairwise ultrathin flat Moiré metalens is designed and fabricated. The diameter of the metalens is 1.6 mm. According to the mutual angles between two metasurfaces, the focal length tuning range of Moiré metalens is ~115 mm with ~40 % transmission efficiency at 532 nm. In addition, the Moiré metalens is implemented to a telecentric design to form the long axial scanning range imaging system with constant magnification. The scanning range of the telecentric imaging system is around 75 μm. The long tuning range with constant magnification is demonstrated by the imaging resolution chart that shows the lateral resolution of the system is around 2 μm. The proposed telecentric imaging system combines with structure illumination-based HiLo imaging principle to obtain the fine optical sectioning fluorescence images with invariant image contrast in the scanning range. The experiment results of the fluorescence beads show the optical sectioning capability of the system is around 7 μm. The ex-vivo fluorescence image results of the mice intestine tissue indicate that the system has the ability to obtain three different depths sectioning images. With the help of the HiLo imaging process, the defocus background noise can be suppressed, and the in focus villi detailed structure can be captured with high signal-to-noise ratio. The proposed varifocal ultrathin size of Moiré metalens has great potentials to replace the conventional bulky varifocal lens for compact system design of optical systems.
Quantitative differential phase contrast (qDPC) microscopy is utilized to observe label-free specimens by asymmetric illumination and intensity measurements. To provide uniform phase contrast images with minimum acquisition time, dual-color linear gradient pupils are applied to generate structured light. It is shown that the proposed pupils in qDPC can outperform half-circle and vortex pupils, and isotropic phase transfer function can be achieved with only 2-axis measurements. With the implantation of digital pupils in our system, the limitation of the time-consuming multi-axis measurement and reconstruction artifacts caused by missing frequencies in a half-circle pupil can be overcome. The required frame of dual-color coded microscopy reduces to two due to the color encoding method used in pupil design so that one-axis information can be obtained within a single shot. The improvement in imaging speed can help in monitoring fast developmental cell processes for various biological applications. Standard micro-lens-array and rat astrocyte cells were used to evaluate the performance of our microscopy system. We demonstrated time-lapse phase imaging of living cells and observed detailed morphology and dynamics changes. Present studies show the potential of the dual-color coded qDPC system for quantitative biomedical imaging for cell research. Due to our label-free approach, the natural contents of the cells remain intact and the dynamic processes through the samples can readily be observed. With our method, the nature of the structural changes in the samples can be evaluated in terms of quantitative phase changes inside acquired images.
Significance: Quantitative differential phase contrast (qDPC) microscopy enhances phase contrast by asymmetric illumination using partially coherent light and multiple intensity measurements. However, for live cell imaging, motion artifacts and image acquisition time are important issues. For live cell imaging, a large number of intensity measurements can limit the imaging quality and speed. The minimum number of intensity measurements in qDPC can greatly enhance performance for live imaging.
Aim: To obtain high-contrast, isotropic qDPC images with two intensity measurements and perform time-lapse imaging of biological samples.
Approach: Based on the color-coded design, a dual-color linear-gradient pupil is proposed to achieve isotropic phase contrast response with two intensity measurements. In our method, the purpose of designing a dual-color coded pupil is twofold: first, to obtain a linear amplitude gradient for asymmetric illumination, which is required to get a circular symmetry of transfer function, and second, to reduce the required number of frames for phase retrieval.
Results: To demonstrate the imaging performance of our system, standard microlens arrays were used as samples. We performed time-lapse quantitative phase imaging of rat astrocytes under a low-oxygen environment. Detailed morphology and dynamic changes such as the apoptosis process and migration of cells were observed.
Conclusions: It is shown that dual-color linear-gradient pupils in qDPC can outperform half-circle and vortex pupils, and isotropic phase transfer function can be achieved with only two-axis measurements. The reduced number of frames helps in achieving faster imaging speed as compared to the typical qDPC system. The imaging performance of our system is evaluated by time-lapse imaging of rat astrocytes. Different morphological changes in cells during their life cycle were observed in terms of quantitative phase change values.
A volume holographic optical element is designed and fabricated using photopolymer (PQ-PMMA) for super
Gaussian beam. A Lee hologram and Digital Micro Mirror Device (DMD) are used for modulating the laser beam to a
uniform one all over its beam diameter. The efficiency and uniformity of super Gaussian beams generated through
volume holographic optical component is experimentally demonstrated with optimized parameters. Super Gaussian beam
generated by volume holographic grating with unique diffractive properties are advantageous for variety of laser
applications.
For acquiring optically sectioned images of volumetric samples from multiple depths without axial scanning, a
confocal system which is able to obtain multi-plane images with a Digital Micro-Mirror Device (DMD) has been
proposed. The focal points of the laser beam can be arbitrarily positioned to any multiple planes in the three dimensional
object space by switching multiplexed binary hologram encoded in the DMD by Lee hologram. The radius of the
spherical wavefronts generated directly determines the location of the focal points and DMD can function either as a
convex or concave mirror. The optical sectioning capabilities of system are verified and which can be applicable in
various biomedical imaging applications.
Understanding the propagating of light in biological tissues have long been an important issue. For this purpose,
modeling scattering of light from laser radiation into turbid media has been studied. One of the common modeling tools is
Monte Carlo simulation, and Mueller matrix method is a commonly used technique to study birefringence and
depolarization properties of biological samples. Based on electric field Monte Carlo simulation in this paper, we present
backscattering Muller matrix calculations for different size of particles and performed comparative studies. Our results
may find important applications in laser tissue interaction.
We present a telecentric design to obtain constant magnification with long axial scanning range in an optical sectioning
microscope. A Moiré lens is included and utilized for this purpose. The design principles of our system are explained
through mathematical expression and simulation results are provided for different conditions, which include the case of
variation displacement of the focal plane with different Moiré lens focal length, and the scanning range in terms of the
magnification value. Furthermore, we also use the Zemax ray-tracing simulation method to show the telecentric examples for
different magnification value with corresponding Moiré lens focal length.
The feature of rapid divergence of conventional Gaussian beam restricts the field of view in light sheet microscopy.
Comparison with Gaussian beam, the Airy beams provides many advantages in light sheet microscopy such as larger field
of view (FOV), longer penetration path, and self-reconstruction. C. elegans is a model organism for developmental
biological studies. Here, we design Airy light sheet microscope to perform in-vivo imaging of C. elegans. A spatial light
modulator is used to generate 1D Airy beam for illumination purpose. Imaging performance is experimentally evaluated
for our system. Present results may find important applications for studying internal structures in-vivo imaging of C.
elegans.
The optical sectioning images of volumetric biological sample was obtained by varifocal metalens with Moiré effect in fluorescence microscopy system in conjunction with telecentricity and HiLo image processing method. The varifocal metalens is capable of changing the focal depth ranging from 10 mm to 125 mm by tuning the relative angle between its paired metasurfaces. The standard resolution target and fluorescent mircrosphere are imaged and analyzed; its lateral resolution and optical sectioning capability are 2.46 μm and 7.5 μm, respectively. Our study offers a demonstration, a solid foundation, for developing more compact optical microscopy systems based on metasurface optics.
Significance: Differential phase contrast (DPC) is a well-known imaging technique for phase imaging. However, simultaneously acquiring multidepth DPC images is a non-trivial task. We propose simultaneous multiplane DPC imaging using volume holographic microscopy (VHM).
Aim: To design and implement a new configuration of DPC-VHM for multiplane imaging.
Approach: The angularly multiplexed volume holographic gratings (AMVHGs) and the wavelength-coded volume holographic gratings (WC-VHGs) are used for this purpose. To obtain asymmetric illumination for DPC images, a dynamic illumination system is designed by modifying the regular Köhler illumination using a thin film transistor panel (TFT-panel).
Results: Multidepth DPC images of standard resolution chart and biosamples were used to compare imaging performance with the corresponding bright-field images. An average contrast enhancement of around three times is observed for target resolution chart by DPC-VHM. Imaging performance of our system is studied by modulation transfer function analysis, which suggests that DPC-VHM not only suppresses the DC component but also enhances high-frequency information.
Conclusions: Proposed DPC-VHM can acquire multidepth-resolved DPC images without axial scanning. The illumination part of the system is adjustable so that the system can be adapted to bright-field mode, phase contrast mode, and DPC mode by controlling the pattern on the TFT-panel.
Significance: Two-photon (2P) fluorescence imaging can provide background-free high-contrast images from the scattering tissues. However, obtaining a multiplane image is not straightforward. We present a two-photon volume holographic imaging (2P-VHI) system for multiplane imaging.
Aim: Our goal was to design and implement a 2P-VHI system that can provide the high-contrast optically sectioned images at multiple planes.
Approach: A 2P-VHI system is presented that incorporates angularly multiplexed volume holographic gratings and a femtosecond laser source for fluorescence excitation for multiplane imaging. A volume hologram with multiplexed gratings provides multifocal observation, whereas nonlinear excitation using a femtosecond laser helps in significantly enhancing both depth resolution and contrast of images.
Results: Standard fluorescent beads are used to demonstrate the imaging performance of the 2P-VHI system. Two-depth resolved optical-sectioning images of fluorescently labeled thick mice intestine samples were obtained. In addition, the optical sectioning capability of our system is measured and compared with that of a conventional VHI system.
Conclusions: Results demonstrated that 2P excitation in VHI systems provided the optical sectioning ability that helps in reducing background noise in the images. Integration of nonlinear fluorescence excitation in the VHI provides some unique advantages to the system and has potential to design multidepth optical sectioned spatial–spectral imaging systems.
We propose a Light-sheet fluorescent microscopy (LSFM) system with a flat metalens, which is used to replace the bulky illuminating components. The metalens is a diffractive optics elements (DOEs) composed of various gallium nitride (GaN) nanopillar for light-sheet generation, and it can be readily integrated in existing LSFM systems. In contrast to the traditional LSFM system, the metalens-based system is more compact and flexible, and its sectioning capability is comparable to the conventional illumination arm in LSFMs. The live fluorescent-labeled transgenic Caenorhabditis elegans (C. elegans) are exploited to verify the performance of this system. The intensity profile of oocytes in C. elegans with cellular resolution level was obtained.
Utilizing three unique defining properties of volume holograms, namely, wavelength degeneracy, angular selectivity, and multiplexing capability, here we show the recording and simultaneous reconstruction of the Airy and Dual Airy beam from multiplexed volume holographic gratings (MVHGs). Each grating acts independently and creates its own diffraction pattern corresponding to the shape of the grating. Multicolor reconstruction of MVHGs are shown. Experimental results demonstrate that volume holograms are capable of reproducing optical wavefront with high precision without affecting the structural properties of beams at any optical wavelengths. These MGHGs acts as wavelength-independent mode shaper and can be used to make compact optical systems. The volume hologram based beam shaping technique is simple and cost-effective and has potential for the mass production.
We report on the implementation of spiral phase contrast imaging at multiple planes using forked-shaped defocus grating. The dual function of grating helps in simultaneous generation of multiple edge enhanced images corresponding to different depths. Present method is simple, direct and is applicable to coherent and incoherent imaging system.
In the unfolding process a uniformly polarized optical vortex evolves into a complex polarization distribution within a birefringent crystal. In this paper we introduce the “unfolding region” to characterize this process. This is the real space region over which the polarization state travels half way around the Poincar´e sphere relative to that observed at the center of the beam. Ideally the region is a circle, but it can be distorted into an ellipse by a small tilt between the ordinary and extraordinary rays. We have applied the above analysis to an actual unfolding process observed with a birefringent interferometer.
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