We have developed a microscopic adaptive optics (AO) system that corrects wavefront phase errors induced by complex structures of biological samples. The technique of correlation-based Shack-Hartmann (SH) sensing used in the AO system enables wavefront measurement using complex structures in a target as the reference. However, sub-images in the SH sensor become deformed dependently on the positions of sub-apertures as the NA of the microscopic objective is higher. This often deteriorates the accuracy of wavefront sensing. To mitigate the undesirable effect, we here propose a differential wavefront sensing technique with a mathematical formula, which is expected to measure wavefront at a better precision. Because differences in image shapes are less significant between nearby SAs, correlations between adjacent SAs are measured in the proposed method. We confirmed that the AO system worked as designed by experiments.
<p>A stable multimodal system is developed by combining two common-path digital holographic microscopes (DHMs): coherent and incoherent, for simultaneous recording and retrieval of three-dimensional (3-D) phase and 3-D fluorescence imaging (FI), respectively, of a biological specimen. The 3-D FI is realized by a single-shot common-path off-axis fluorescent DHM developed recently by our group. In addition, we accomplish, the phase imaging by another single-shot, highly stable common-path off-axis DHM based on a beam splitter. In this DHM configuration, a beam splitter is used to divide the incoming object beam into two beams. One beam serves as the object beam carrying the useful information of the object under study, whereas another beam is spatially filtered at its Fourier plane by using a pinhole and it serves as a reference beam. This DHM setup, owing to a common-path geometry, is less vibration-sensitive and compact, having a similar field of view but with high temporal phase stability in comparison to a two-beam Mach–Zehnder-type DHM. The performance of the proposed common-path DHM and the multimodal system is verified by conducting various experiments on fluorescent microspheres and fluorescent protein-labeled living cells of the moss <italic>Physcomitrella patens</italic>. Moreover, the potential capability of the proposed multimodal system for 3-D live fluorescence and phase imaging of the fluorescent beads is also demonstrated. The obtained experimental results corroborate the feasibility of the proposed multimodal system and indicate its potential applications for the analysis of functional and structural behaviors of a biological specimen and enhancement of the understanding of physiological mechanisms and various biological diseases.</p>
Adaptive optics (AO) is a promising technique for correcting wavefront errors induced by complex structures of biological samples which significantly causes image degradation. We develop a microscopic AO system with a Shack-Hartmann wavefront sensor based on image correlation. The correlation-based wavefront sensing is feasible using an extended object under bright-field illumination, as well as spot fluorescence. To make the correlation-based sensing more reliable, we newly introduce a technique of excluding sub-images with insufficient quality. We show experimental results under a variety of conditions for objects, light sources, and wavefront error sources. In any cases, we confirmed that the AO system effectively worked so as to improve image qualities.
Mobile phone technology has led to implementation of portable and inexpensive microscopes. Light-emitting diode (LED) array microscopes support various multicontrast imaging by flexible illumination patterns of the LED array that can be achieved without changing the optical components of the microscope. Here, we demonstrate a mobile-phone-based LED array microscope to realize multimodal imaging with bright-field, dark-field, differential phase-contrast, and Rheinberg illuminations using as few as 37 LED bulbs. Using this microscope, we obtained high-contrast images of living cells. Furthermore, by changing the color combinations of Rheinberg illumination, we were able to obtain images of living chromatic structures with enhanced or diminished contrast. This technique is expected to be a foundation for high-contrast microscopy used in modern field studies.
This paper presents lateral spatial resolution improvement by scanning a focused spot array pattern. To enhance the effective point spread function (PSF), we employed a phase-only computer generated hologram (CGH) that can reduce the spot size comparing to the single diffraction limited spot. A CGH was designed based on the Gerchberg-Saxton algorithm with a specific constraint to control the dispersion of light energy and the phase of generated spots. As a design example, we obtained a CGH for generating 3x3 optical spots whose sizes were reduced to 78.5% of the single diffraction limited spot. We also confirmed by simulation that the effective PSF was improved from 208 nm to 183 nm when using the subdiffraction limit spots for excitation.
This letter proposes a method of configuring a testing target to evaluate the performance of adaptive optics microscopes. In this method, a testing slide with fluorescent beads is used to simultaneously determine the point spread function and the field of view. The point spread function is reproduced to simulate actual biological samples by etching a microstructure on the cover glass. The fabrication process is simplified to facilitate an onsite preparation. The artificial tissue consists of solid materials and silicone oil and is stable for use in repetitive experiments.
Adaptive optics is useful not only for the suppression of the blur of image, but also for the reduction of the aberration on the transmitted light. Recent years, methods for optical manipulation of biological tissue under the microscope is becoming available, whereas the live tissue often causes the considerable amount of optical aberration that prevents the clear convergence of the laser beam onto the target cell. This research shows the basic experiments to improve the convergence of the laser beam focused on the tissue in microscope by correcting the optical aberration using adaptive optics.
Live-cell imaging using fluorescent molecules is now essential for biological researches. However, images of living cells are accompanied with blur, which becomes stronger according to the depth inside the cells and tissues. This image blur is caused by the disturbance on light that goes through optically inhomogeneous living cells and tissues. Here, we show adaptive optics (AO) imaging of living plant cells. AO has been developed in astronomy to correct the disturbance on light caused by atmospheric turbulence. We developed AO microscope effective for the observation of living plant cells with strong disturbance by chloroplasts, and successfully obtained clear images inside plant cells.
The improvement of the optical devices in this decade, such as the MEMS-SLM ( Micro Electro Mechanical Systems- Spatial Light Modulator ) and wave front sensor with micro lens device, is making adaptive optics commonly available. It also gives the new basis of the design of adaptive optics with the improved accuracy and the compactness. We have developed an adaptive optics bench from such a point of view, and the application to the optical microscope has attained effective results in the observation of the live cell samples. In this presentation, our recent results will be shown. The result includes analysis of blur by the fine structures in biological sample and result of the image correction by the adaptive optics.