Multiplane differential saturated excitation microscopy using varifocal lenses.

Saturated excitation microscopy, which collects nonlinear fluorescence signals generated by saturation, has been proposed to improve three-dimensional spatial resolution. Differential saturated excitation (dSAX) microscopy can further improve the detection efficiency of a nonlinear fluorescence signal. By comparing signals obtained at different saturation levels, high spatial resolution can be achieved in a simple and efficient manner. High-resolution multiplane microscopy is perquisite for volumetric imaging of thick samples. To the best of our knowledge, no reports of multiplane dSAX have been made. Our aim is to obtain multiplane high-resolution optically sectioned images by adapting differential saturated excitation in confocal laser scanning fluorescence microscopy. To perform multiplane dSAX microscopy, a variable focus lens is employed in a telecentric design to achieve focus tunability with constant magnification and contrast throughout the axial scanning range. Multiplane fluorescence imaging of two different types of pollen grains shows improved resolution and contrast. Our system's imaging performance is evaluated using standard targets, and the results are compared with standard confocal microscopy. Using a simple and efficient method, we demonstrate multiplane high-resolution fluorescence imaging. We anticipate that high-spatial resolution combined with high-speed focus tunability with invariant contrast and magnification will be useful in performing 3D imaging of thick biological samples.

Volume holographic illuminator for Airy light-sheet microscopy.

Airy light sheets combined with the deconvolution approach can provide multiple benefits, including large field of view (FOV), thin optical sectioning, and high axial resolution. The efficient design of an Airy light-sheet fluorescence microscope requires a compact illumination system. Here, we show that an Airy light sheet can be conveniently implemented in microscopy using a volume holographic grating (VHG). To verify the FOV and the axial resolution of the proposed VHG-based Airy light-sheet fluorescence microscope, ex-vivo fluorescently labeled Caenorhabditis elegans (C. elegans) embryos were imaged, and the Richardson-Lucy deconvolution method was used to improve the image contrast. Optimized parameters for deconvolution were compared with different methods. The experimental results show that the FOV and the axial resolution were 196 µm and 3 µm, respectively. The proposed method of using a compact VHG to replace the common spatial light modulator provides a direct solution to construct a compact light-sheet fluorescence microscope.

Low-cost, open-source XYZ nanopositioner for high-precision analytical applications.

Nanoscale positioning has numerous applications in both academia and industry. A growing number of applications require devices with long working distances and nanoscale resolutions. Friction-inertia piezoelectric positioners, which are based on the stick-slip mechanism, achieve both nanometer resolution and centimeter-scale travel. However, the requirements of complex preload mechanism, precision machining, and precise assembly increase the cost of conventional friction-inertia nanopositioners. Herein we present the design of an open-source XYZ-axis nanopositioning system. Utilizing a magnet-based stick-slip driving mechanism, the proposed XYZ nanopositioner provides several advantages, including sub-nanometer resolution, a payload capacity of up to 12 kg (horizontal), compact size, low cost, and easy assembly; furthermore, the system is adjustment-free. The performance tests validate the precision of the system in both scanning and stepping operation modes. Moreover, the resonant spectra affirm the rigidity and dynamic response of the mechanism. In addition, we demonstrate the practical applications of this nanopositioner in various measurement techniques, including scanning electron microscopy, vibrometry, and atomic force microscopy. Furthermore, we present 11 variations of the nanopositioner designs that are either compatible with ultra-high-vacuum systems and other existing systems, 3D printable, or hacking commercial linear slides.

Telecentric design for digital-scanning-based HiLo optical sectioning endomicroscopy with an electrically tunable lens.

Confocal endoscopy has been widely used to obtain fine optically sectioned images. However, confocal endomicroscopic images are formed by point-by-point scanning in both lateral and axial directions, which results in long image acquisition time. Here, an endomicroscope with telecentric configuration is presented to achieve nonmechanical and rapid axial scanning for volumetric fluorescence imaging. In our system, optical sectioning in wide-field fashion is obtained through HiLo imaging with a digital micromirror device. Axial scanning, without mechanical moving parts, is conducted by digital focus adjustment using an electrically tunable lens, offering constant magnification and contrast. We demonstrate imaging performance of our system with optically sectioned images using fluorescently labeled beads, as well as ex vivo mice cardiac tissue samples. Our system provides multiple advantages, in terms of improved scanning range, and reduced image acquisition time, which shows great potentials for three-dimensional biopsies of volumetric biological samples.

Quantitative differential phase contrast imaging at high resolution with radially asymmetric illumination.

Half-circle illumination-based differential phase contrast (DPC) microscopy has been utilized to recover phase images through a pair of images along multiple axes. Recently, the half-circle based DPC using 12-axis measurements significantly provides a circularly symmetric phase transfer function to improve accuracy for more stable phase recovery. Instead of using half-circle-based DPC, we propose a new scheme of DPC under radially asymmetric illumination to achieve circularly symmetric phase transfer function and enhance the accuracy of phase recovery in a more stable and efficient fashion. We present the design, implementation, and experimental image data demonstrating the ability of our method to obtain quantitative phase images of microspheres, as well as live fibroblast cell samples.

High-sensitivity imaging with lateral resonance mode atomic force microscopy.

In the operation of a dynamic mode atomic force microscope, a micro-fabricated rectangular cantilever is typically oscillated at or near its mechanical resonance frequency. Lateral bending resonances of cantilevers are rarely used because the resonances are not expected to be detected by the beam-deflection method. In this work, we found that micro-cantilevers with a large tip produced an out-of-plane displacement in lateral resonance (LR), which could be detected with the beam-deflection method. Finite-element analysis indicated that the presence of a large tip is the major source of the out-of-plane coupling for the LR. We also imaged a heterogeneous sample by operating a cantilever in LR, torsional resonance, and tapping modes. LR mode yielded a small deformation and noise level in the height maps as well as a high contrast and small noise level in the phase maps. LR mode also had a resonance frequency that was orders of magnitude higher than that of tapping mode. Operation with LR mode may have the benefits of high-speed scanning, high-sensitivity imaging, and mapping of in-plane mechanical properties of the sample surface. In general, LR mode may become a powerful new atomic force microscopy technique for characterizing sample materials.

Optical imaging module for astigmatic detection system.

In this paper, an optical imaging module design for an astigmatic detection system (ADS) is presented. The module is based on a commercial optical pickup unit (OPU) and it contains a coaxial illuminant for illuminating a specimen. Furthermore, the imaging module facilitates viewing the specimen and the detection laser spot of the ADS with a lateral resolution of approximately 1 µm without requiring the removal of an element of the OPU. Two polarizers and one infrared filter are used to eliminate stray laser light in the OPU and stray light produced by the illuminant. Imaging modules designed for digital versatile disks (DVDs) and Blu-ray DVDs were demonstrated. Furthermore, the module can be used for imaging a small cantilever with approximate dimensions of 2 µm (width) × 5 µm (length), and therefore, it has the potential to be used in high-speed atomic force microscopy.

Low-voltage and high-performance buzzer-scanner based streamlined atomic force microscope system.

In this paper we present a novel scanner design in a quad-rod actuation structure, actuated by piezoelectric disk buzzers, and a new type of atomic force microscope (AFM), which uses this buzzer-scanner and a compact disk/digital-versatile-disk astigmatic optical pickup unit (OPU) for the detection of cantilever movements. Commercially available piezoelectric disk buzzers have a low capacitance and can be driven by low-voltage signal sources, such as analog outputs from a data acquisition card, without additional voltage or current amplifiers. Various scanning ranges can be realized through changing the dimensions of the actuation structure and/or the choice of disk buzzer. We constructed a buzzer-scanner and evaluated its performance. The scanner had a scanning range of 15 µm in the X and Y directions and an actuation range of 3.5 µm on the Z axis, with nonlinearity of 2.11%, 2.73%, and 2.19% for the X,Y and Z axes, respectively. The scanner had a resonance frequency of approximately 360 Hz on the X and Y axes, and 4.12 kHz on the Z axis. An OPU-AFM with this buzzer-scanner can resolve single atomic steps of a graphite substrate with a noise level of 0.06 nm. The obtained topographic images exhibit much less distortion than those obtained with an AFM using a piezoelectric tube scanner.

Torsional resonance mode atomic force microscopy in liquid with Lorentz force actuation.

In this work, we present a design based on Lorentz force induction to excite pure torsional resonances of different types of cantilevers in air as well as in water. To demonstrate the atomic force microscopy imaging capability, the phase-modulation torsional resonance mode is employed to resolve fine features of purple membranes in a buffer solution. Most importantly, force-versus-distance curves using a relatively stiff cantilever can clearly detect the characteristic oscillatory profiles of hydration layers at a water-mica interface, indicating the high force sensitivity of the torsional mode. The high resonance frequencies and high quality-factors for the torsional mode may be of great potential for high-speed and high-sensitivity imaging in aqueous environment.

Motility measurement of a mouse sperm by atomic force microscopy.

Eukaryotic flagella are responsible for the motile organelles that cause the migration of mammalian sperms. The lashing force and torque of the sperm flagellum contain critical information regarding the sperm health, as important evaluation factors for sperm screening. The objective of the study was to investigate the lashing force and torque of a sperm under physiological conditions using atomic force microscopy (AFM). At a distance of about 18.5 µm from its head-tail junction, a lashing force of 0.96 ± 0.20 nN was measured. Its corresponding lashing torque was 1.77(± 0.37)× 10(-14) N·m. The torque increases in proportion to the square of the head-tail junction distance. Our results reasonably conclude that the axonemal motility is linear dependent on the flagellum length of the sperm. Our developed measurement system can consistently determine the lashing force and torque of a sperm, which can contribute to further studies concerning the mechanism of sperm transport and fertilization.

Real-time detection of linear and angular displacements with a modified DVD optical head.

Here we demonstrate that an astigmatic detection system (ADS), constructed with a modified digital-versatile-disk (DVD) optical head, can achieve real-time measurement of a linear displacement and two-dimensional (2D) tilt angles with a high sensitivity. An atomic force microscope (AFM), using our detection system to sense the deflection of microfabricated cantilevers, can resolve single atomic steps on graphite surfaces with a noise level less than 0.04 nm in topographic images. This astigmatic detection system can even detect mechanical resonances due to thermal vibrations of microfabricated cantilevers. The high sensitivity, small detecting size and high bandwidth of this detection system is suitable for dynamic characterization of elements in micromachined components. Further optimization of the system will promise many other applications in diverse technological fields.