Fast Axial Scanning Research Articles

In-fiber illumination-detection-synchronized confocal microscopy for high-precision fast axial scanning

Confocal microscopy remains a major workhorse in the 3D surface topography measurement owing to its reliability and compatibility for workpieces with various reflectance, which, however, suffers from slow scanning speed and limited axial resolution. Commercial confocal microscopy primarily relies on the axial movement of a sample or objective lens to create a series of 2D image stacks, and then reconstruct 3D map. Nevertheless, most samples and objective lens are too bulky to scan rapidly, let alone that multiple scans are required to increase the signal-to-noise ratio (SNR). To address this issue, an original approach named illumination-detection-synchronized confocal microscopy was developed, which enabled high-precision fast axial scanning via a compact fiber-multiplexed port with the high-speed synchronous motion of point source and point detection. The approach can effectively improve the peak localization precision while not requiring a high-precision motion stage. Theoretically, via controlling the magnification of the optical system M > 1, the total peak localization precision of axial light intensity response curve can be improved by M3 times. Experiments show that the single-point repeat measurement precision by this approach is improved by 4 times, and the profile measurement precision is enhanced by 2.3 times. Moreover, this approach can effectively avoid collisions between the sample and objective lens, and has the potential to design a low-cost single-point confocal sensor or confocal profilometer.

Correction method for 3D non-linear drift distortions in atomic force microscopy raster measurements

A method to correct non-linear drift distortions in all three coordinate axes of atomic force microscope (AFM) images is presented. The method uses two measurements of the sample with two fast scan axes orthogonal to each other. Both AFM images are divided into segments and the shifts of the surface features of the segments of both images are determined. From these shifts subsequently the drift of both measurements is calculated. Depending on the segments used, significant non-linearities of the drift can be corrected. The two required measurements for this method do not have to be carried out in direct succession. With this method it is therefore possible to correct drift in an existing AFM image by measuring the sample again later. Although the method has been developed for AFM, it can also be used for other scanning probe microscopes.

System integration of hybrid assembled large aperture Micro Scanner Array for fast scanning LiDAR sensors

We present the design and system integration of a hybrid MEMS scanning mirror (MSM) array developed for real-time three-dimensional imaging with a panoramic optical field of view (FOV) of 360 deg × 60 deg (horizontal × vertical). The pulsed time-of-flight light detection and ranging (LiDAR) system targets a distance measurement range of 100 m with a video-like frame rate of 10 Hz. The fast vertical scan axis is realized by a synchronous scanning MSM array with large receiver aperture. It increases the scanning rate to 3200 Hz, which is four times faster in comparison with state-of-the-art fast macroscopic polygon scanning systems used in current LiDAR systems. A hybrid assembly of frequency selected scanner elements was chosen instead of a monolithic MEMS array to guaranty high yield of MEMS fabrication and a synchronous operation of all resonant MEMS elements at 1600 Hz with large FOV of 60 deg. The hybrid MSM array consists of a separate emitting mirror for laser scanning of the target and 22 reception elements resulting in a large reception aperture of Deff = 23 mm. All MSM are driven in parametric resonance to enable a fully synchronized operation of all individual MEMS scanner elements. Therefore, piezoresistive position sensors are integrated inside the MEMS chip, used for position feedback of the driving control. We focus on the MEMS system integration including the microassembly of multiple MEMS scanning elements using micromechanical self-alignment. We present technical details to meet the narrow tolerance budgets for (i) microassembly and (ii) synchronous driving of multiple MEMS scanner elements.

Extended range and aberration-free autofocusing via remote focusing and sequence-dependent learning.

Rapid autofocusing over long distances is critical for tracking 3D topological variations and sample motion in real time. Taking advantage of a deformable mirror and Shack-Hartmann wavefront sensor, remote focusing can permit fast axial scanning with simultaneous correction of system-induced aberrations. Here, we report an autofocusing technique that combines remote focusing with sequence-dependent learning via a bidirectional long short term memory network. A 120 µm autofocusing range was achieved in a compact reflectance confocal microscope both in air and in refractive-index-mismatched media, with similar performance under arbitrary-thickness liquid layers up to 1 mm. The technique was validated on sample types not used for network training, as well as for tracking of continuous axial motion. These results demonstrate that the proposed technique is suitable for real-time aberration-free autofocusing over a large axial range, and provides unique advantages for biomedical, holographic and other related applications.

Multi-label invivo STED microscopy by parallelized switching of reversibly switchable fluorescent proteins.

Despite the tremendous success of super-resolution microscopy, multi-color invivo applications are still rare. Here we present live-cell multi-label STED microscopy invivo and invitro by combining spectrally separated excitation and detection with temporal sequential imaging of reversibly switchable fluorescent proteins (RSFPs). Triple-label STED microscopy resolves pre- and postsynaptic nano-organizations invivo in mouse visual cortex employing EGFP, Citrine, and the RSFP rsEGP2. Combining the positive and negative switching RSFPs Padron and Dronpa-M159T enables dual-label STED microscopy. All labels are recorded quasi-simultaneously by parallelized on- and off-switching of the RSFPs within the fast-scanning axis. Depletion is performed by a single STED beam so that all channels automatically co-align. Such an addition of a second or third marker merely requires a switching laser, minimizing setup complexity. Our technique enhances invivo STED microscopy, making it a powerful tool for studying multiple synaptic nano-organizations or the tripartite synapse invivo.

Precompensation of 3D field distortions in remote focus two-photon microscopy.

Remote focusing is widely used in 3D two-photon microscopy and 3D photostimulation because it enables fast axial scanning without moving the objective lens or specimen. However, due to the design constraints of microscope optics, remote focus units are often located in non-telecentric positions in the optical path, leading to significant depth-dependent 3D field distortions in the imaging volume. To address this limitation, we characterized 3D field distortions arising from non-telecentric remote focusing and present a method for distortion precompensation. We demonstrate its applicability for a 3D two-photon microscope that uses an acousto-optic lens (AOL) for remote focusing and scanning. We show that the distortion precompensation method improves the pointing precision of the AOL microscope to < 0.5 µm throughout the 400 × 400 × 400 µm imaging volume.

Multi-photon 3D imaging with an electrothermal actuator with low thermal and inertial mass

Design and testing of a low-mass electrothermal vertical scanning mirror for fast axial scanning during multi-photon microscopy is presented. The mirror makes use of a novel honeycomb mechanical support structure to substantially reduce both thermal and inertial mass relative to mirror size. At the same time, undesirable mirror curvature due to residual thermal stress is controlled without adversely affecting the image quality. In addition, the support structure makes the complete mirror area usable which otherwise is not possible in devices that rely on isotropic silicon etching. A high actuator efficiency with static displacement per unit power of 2.08 μm/mW was achieved due to the reduced thermal capacitance at the actuator legs, when using a 700 nm thin structural layer of SiO2. The resulting scanning mirror achieves open loop time constant as small as 3.42 ms with both thermal and mechanical bandwidths exceeding 100 Hz. Mirror surface non-idealities related to the mirror structure and their influence on image quality are discussed. Sample 3D multi-photon images captured by performing remote scanning in both lateral and axial direction on a benchtop testbed are presented. The efficacy of remote axial scan is ascertained by comparisons with images obtained by moving specimens relative to the objective.

Simulation and analysis of variable numerical aperture wide-field microscopy for telecentricity with constant resolution

In this work, we propose simulation and analysis of a novel combination of a variable numerical aperture wide-field microscope objective with an Electrically Tunable Lens (ETL) for axial scanning with a telecentric image space. This work resolves the challenges of the conventional ETL based wide-field microscopic systems viz. variable magnification, field-of-view (FOV), an inconsistent lateral and axial resolution. The proposed combination performs the tuning of the object plane by changing ETL current and effectively modifies the object space numerical aperture using an aperture controller to provide a stationary telecentric image plane. Moreover, with the proposed combination, the lateral resolution of the system is preserved over an axial range of 7.586mm with a magnification error of 2.55 %. The proposed work witnesses the advantages of providing fast axial scanning, extended axial scanning range, autofocusing, invariant magnification, constant FOV, and constant resolution along with a compact optical setup.

Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

Axial resolution improvement of two-photon microscopy by multi-frame reconstruction and adaptive optics.

Two-photon microscopy (TPM) has been widely used in biological imaging owing to its intrinsic optical sectioning and deep penetration abilities. However, the conventional TPM suffers from poor axial resolution, which makes it difficult to recognize some three-dimensional fine features. We present multi-frame reconstruction two-photon microscopy (MR-TPM) using a liquid lens as a fast axial scanning engine. A sensorless adaptive optics (AO) approach is adopted to correct the aberrations caused by both the liquid lens and the optical system. By overcoming the effect of optical aberrations, inadequate sampling, and poor focusing capability of a conventional TPM, the axial resolution can be improved by a factor of 3 with a high signal-to-noise ratio. The proposed technology is compatible with the conventional TPM and requires no optical post-processing. We demonstrate the proposed method by imaging fluorescent beads, in vitro imaging of the neural circuit of mouse brain slice, and in vivo time-lapse imaging of the morphological changes of microglial cells in septic mice model. The results suggest that the axon of the neural circuit and the process of microglia along the axial direction, which cannot be resolved using conventional TPM, become distinguishable using the proposed AO MR-TPM.

A stainless-steel micro-scanner for rapid 3D confocal imaging

This paper summarizes the design, fabrication, and characterization of a magnetically actuated stainless-steel based micro-scanner. The out-of-plane deflection of the proposed device is calculated by using a custom depth scan setup. The main advantage of laser cutting technology, which is utilized in manufacturing the proposed steel scanner, is its rapid fabrication capability at low cost, while still offering high frequency scan for imaging and/or ablation with high frame-rates. In the lateral plane, the scanner delivers 5 degrees of total optical scan angle for a current drive of 60 mA for both slow scan and fast scan axes at 998 Hz and 2795 Hz, respectively. Furthermore, the device provides an out-of-plane pumping mode at 1723 Hz that could be utilized for axial scanning to create focal shift at the target. Fabricated scanner is integrated into a confocal microscopy setup and tested with a resolution target and a Convallaria rhizome sample, accomplishing a 240 m 240 m field of view with 2.8 m resolution. The device offers 218 m depth of field (in tissue) and based on acquired resonance frequencies, we estimate rapid scanning of a three-dimensional block of tissue (240 m 240 m 218 m size) with approximately 3 block per second with 50% fill rate and total coverage of 87% for 1 s scan. Finally, a custom setup is proposed for 3D imaging and validity of the 3D beam steering of the micro-scanner is tested.

High-Speed AFM Imaging of Nanopositioning Stages Using H$_{\infty }$ and Iterative Learning Control

This paper presents a method that combines a robust controller (H $_{\infty }$ ) and an iterative learning controller (ILC) to control a low mechanical bandwidth nanopositioning stage for high-speed atomic force microscopy imaging. In conventional scanning configurations, the imaging speed of a low-resonance frequency scanner is limited to a few Hz. However, the images obtained using the proposed method have no obvious anamorphosis with a scan speed of up to 80 Hz. This method uses a sinusoidal scanning mode in the fast-scan axis, which effectively reduces the mechanical vibration of the XY -scanner and improves the imaging bandwidth. In addition, a compact high-bandwidth Z -scanner configured with a symmetrical dual-actuator was developed to replace the Z -axis of the nanopositioning stage for high-speed tracking of the sample topography. To further improve the imaging performance, an ILC is designed to suppress the nonlinear behavior of piezoelectric and reduce the tracking error. In addition, a model-based H $_{\infty }$ is designed to reduce the measurement error and enhance the image quality. All algorithms and real-time control are implemented with a field-programmable gate array platform. The experimental results demonstrated that these configurations exhibit significant performance improvements by comparison with conventional scanning modes.

Hybrid assembled MEMS scanner array with large aperture for fast scanning LIDAR systems

Abstract This article presents a large aperture micro scanning mirror (MSM) array especially developed for the panoramic 3D-ToF camera Fovea-3D. The Fovea-3D system uses a fiber amplified pulsed laser ToF technique at λ = 1550 nm \lambda =1550\hspace{0.1667em}\text{nm} with 1 MVoxel distance measuring rate. It targets for real time 3D imaging with a panoramic optical field of view (FOV) of 360 ° × 60 ° 360^\circ \times 60^\circ (horizontal × vertical) combined with a large distance measurement range of 100 m and a video-like frame rate of 10 Hz. For fast vertical scan axis a MEMS scanner module with large receiver aperture was especially developed. It increases the scanning rate to 3200 Hz which is four times faster in comparison to state-of-the-art fast macroscopic polygon scanning systems used for LIDAR systems. To guarantee at the same time a large reception aperture of D eff = 23 mm {D_{\mathit{eff}}}=23\hspace{0.1667em}\text{mm} , large FOV of 60 ° 60^\circ and high vertical scanning rate of 3200 Hz, a hybrid assembled MSM array was developed consisting of 22 reception mirrors and a separate emitting mirror for laser scanning of the target. For Fovea3D hybrid assembly of frequency selected scanner elements was chosen instead of a monolithic MEMS scanner array to guaranty a high yield of MEMS fabrication. All MSM are driven in parametric resonance to enable a fully synchronized operation of all individual MEMS scanner elements. Therefore, piezo-resistive position sensors are integrated on each MEMS chip for position feedback of driving control. The paper discusses details of the MEMS system integration including the synchronized operation of multiple MEMS scanning elements.

Analysis of axial scanning range and magnification variation in wide‐field microscope for measurement using an electrically tunable lens

Inserting an electrically tunable lens (ETL), such as liquid lens or tunable acoustic gradient lens, into a microscope can enable fast axial scanning, autofocusing, and extended depth of field. However, placing the ETL at different positions has different influences on image quality. Specially, in a wide-field microscope for measurement, the magnification has to be constant when introducing an ETL, otherwise it will affect measurement accuracy. To determine the best position of ETL, axial scanning range and magnification variation are quantitatively analyzed and discussed in finite and infinite microscopes through theoretical analysis, optical simulation, and experiment for four configurations: when ETL is placed at the back focal plane of objective, at the conjugate plane of objective's back focal plane between two relay lenses, or behind two relay lenses, and at imaging detector plane. The obtained results are as follows. When ETL is placed at the back focal plane, the system has a large scanning range, but the magnification varies because the back focal plane is inside the objective. When ETL is placed between two relay lenses, the magnification stays constant, but the scanning range is small. When ETL is placed behind two relay lenses, the magnification keeps invariant and the scanning range is large, but ETL and two relay lenses are inside the microscope and the system has to be customized. Finally, when ETL is placed at imaging detector plane, the magnification stays constant, but the scanning range is 0, which means the system has no axial scanning capability. RESEARCH HIGHLIGHTS: An electrically tunable lens (ETL) is introduced into a wide-field microscope for measurement. Axial scanning range and magnification variation are analyzed and discussed. Theoretical analysis, ZEMAX optical simulation and experiments are performed.

Dual-mode phase and fluorescence imaging with a confocal laser scanning microscope

We present dual-mode phase and fluorescence imaging in a confocal laser scanning microscopy (CLSM) system. For phase imaging, the depth of field of the CLSM system is extended by fast axial scanning with a tunable acoustic gradient index of refraction lens. Under transillumination, intensity images of the sample are recorded at a few different defocusing distances. The phase image is reconstructed from these intensity images by using the transport-of-intensity equation. The 3D fluorescence image is obtained by confocal scanning. The dual-mode images with pixel-to-pixel correspondence yield complementary quantitative structural and functional information. Combination of the two imaging modalities enables standalone determination of the refractive index of live cells.

Laser scanners with oscillatory elements: Design and optimization of 1D and 2D scanning functions

• Multi-parameter analysis (with optical, mechanical, and electrical aspects) of laser scanners with oscillatory elements. • Analysis of common scanning functions (sinusoidal, sawtooth, and triangular) from the point of view of their duty cycle. • Development of 1D optimal scanning functions with the maximum possible duty cycle (and minimum distortions). • Trade-off between the duty cycle and the voltage peaks of the command functions of scanners with oscillatory elements. • Development and optimization of 2D scanning functions of galvanometer scanners or Micro-Electro-Mechanical Systems (MEMS). Optomechatronic laser scanners with oscillatory mirrors, built as galvanometer scanners (GSs) or as Micro-Electro-Mechanical Systems (MEMS) are approached from the point of view of their scanning functions. The non-linearity of the 1D scanning functions which are the output signals of such scanners (i.e., the current angular position of their oscillatory mirrors) are first discussed for the three most common input signals (triangular, sawtooth, and sinusoidal), for which the effective duty cycle/time efficiency (of the output) was modeled using Optical Coherence Tomography (OCT) imaging, in contrast to the theoretical duty cycle (of the input). Second, optimal linear plus non-linear 1D scanning functions are designed, with specially introduced parabolic portions, in order to maximize the duty cycle of the scanning process and to provide the most distortion-free scanning, for example for OCT. A trade-off is also discussed between this duty cycle and the peaks of the voltage that has to be applied to the motor of the scanner, in order to minimize these peaks and thus to protect the system from an electrical point of view. Finally, using all the results above, both angular and linear 2D scanning functions are designed and analyzed, for their possible variants. For the fast scan axis the triangular 1D functions obtained above (with or without non-linear portions introduced between the constant speed, useful scan) are utilized. For the slow scan axis the two most utilized scanning algorithms are employed: with a step-by-step or with a continuous angular movement. The characteristic parameters of their different variants are analyzed in order to optimize these scanning functions. We demonstrate that the step-by-step angular movement is the best one, in order to avoid producing acceleration (therefore inertia torque and voltage) spikes. Equations that avoid such issues are proposed. These developments are placed in the context of the requirements of high-end applications like OCT, both for biomedical imaging and for Non-Destructive Testing (NDT) in industry, including for hot topics, such as handheld or endoscope scanning probes.

Two-Photon Microscopy with a Double-Wavelength Metasurface Objective Lens.

Two-photon microscopy is a key imaging technique in life sciences due to its superior deep-tissue imaging capabilities. Light-weight and compact two-photon microscopes are of great interest because of their applications for in vivo deep brain imaging. Recently, dielectric metasurfaces have enabled a new category of small and lightweight optical elements, including objective lenses. Here we experimentally demonstrate two-photon microscopy using a double-wavelength metasurface lens. It is specifically designed to focus 820 and 605 nm light, corresponding to the excitation and emission wavelengths of the measured fluorophors, to the same focal distance. The captured two-photon images are qualitatively comparable to the ones taken by a conventional objective lens. Our metasurface lens can enable ultracompact two-photon microscopes with similar performance compared to current systems that are usually based on graded-index-lenses. In addition, further development of tunable metasurface lenses will enable fast axial scanning for volumetric imaging.

High-speed large area atomic force microscopy using a quartz resonator

A high-speed atomic force microscope for scanning large areas, utilizing a quartz bar driven close to resonance to provide the motion in the fast scan axis is presented. Images up to 170 × 170 μm2 have been obtained on a polydimethylsiloxane (PDMS) grating in 1 s. This is provided through an average tip-sample velocity of 28 cm s−1 at a line rate of 830 Hz. Scan areas up to 80 × 80 μm2 have been obtained in 0.42 s with a line rate of 1410 Hz. To demonstrate the capability of the scanner the spherulitic crystallization of a semicrystalline polymer was imaged in situ at high speed.

MEMS-tunable dielectric metasurface lens

Varifocal lenses, conventionally implemented by changing the axial distance between multiple optical elements, have a wide range of applications in imaging and optical beam scanning. The use of conventional bulky refractive elements makes these varifocal lenses large, slow, and limits their tunability. Metasurfaces, a new category of lithographically defined diffractive devices, enable thin and lightweight optical elements with precisely engineered phase profiles. Here we demonstrate tunable metasurface doublets, based on microelectromechanical systems (MEMS), with more than 60 diopters (about 4%) change in the optical power upon a 1-μm movement of one metasurface, and a scanning frequency that can potentially reach a few kHz. They can also be integrated with a third metasurface to make compact microscopes (~1 mm thick) with a large corrected field of view (~500 μm or 40 degrees) and fast axial scanning for 3D imaging. This paves the way towards MEMS-integrated metasurfaces as a platform for tunable and reconfigurable optics.

Multifocus optical-resolution photoacoustic microscope using ultrafast axial scanning of single laser pulse

A tunable acoustic gradient (TAG) lens has been introduced in the fast axial scanning in optical imaging. However, it still needs additional imaging time for depth scanning. In this study, we split a single laser pulse into three sub-pulses and introduce them into three fibers with different lengths. The sub-pulses out of the fibers were combined thereafter. We then obtained a pulse train with a time interval of 120 ns. By controlling the fire time of the pulse train and the driving signal of the TAG lens, we can receive three focal spots in one A line data acquisition using a single input laser pulse. The depth of focus (DoF) of the system was measured to be 360 μm, which is three times of that of previous systems without the sacrifice of time resolution. A mouse ear and mouse cerebral vasculature were imaged in-vivo to demonstrate the feasibility of the extended DoF of our system.