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.
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.
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 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.
We present a wavelength coded volume holographic gratings (WC-VHGs) based dual wavelength fluorescence imaging system to simultaneously obtain two color fluorescence images of biological samples in one shot. The system utilizes wavelength coded recording technique based on Bragg degeneracy to generate the PQ-PMMA volume holographic gratings, which has high selectivity in both wavelength and angle. In order to improve the optical sectioning ability of the proposed system, we use HiLo image processing to suppress the out of focus signal. Experimental results demonstrate the imaging ability of the proposed system to observe multi-wavelength fluorescence images and HiLo images.
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.
Confocal microscopy has been widely used to acquire optical sectioning fluorescent image. However, traditional confocal technique requires point-by-point scanning which is time consuming. Alternative techniques to confocal microscopy, such as structured illumination, exist for fast sectioning images, but they require multiple axial planes to be imaged individually. Here, a non-axial line-scanning multifocal confocal microscopy is presented. The proposed system incorporates multiplex volume holographic grating (MVHG) in illumination and combination of multifocal image system. The detailed explanation for resolution on depth axial and simulation results are compared. Also both XY resolution is verified through resolution target. The ability of the proposed system to optical sectioning and multi-depth resolve image of fluorescently labeled microsphere and cornea is experimentally demonstrated.
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.
Under broadband illumination, a multiplane microscopy incorporating volume holographic gratings (VHGs) to observe three-dimensional structures of biological samples with different depth simultaneously is presented. VHGs formed in thick recording materials, including PQ-PMMA, provide strong angular and wavelength transmittance filtering properties, which enable acquiring spatial–spectral images of fine structures within biological samples. Here, we experimentally demonstrate this microscopic imaging capability to obtain multiple depth-resolved mixed pollen grains images of fine structures from eight depths in one shot.
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