Corrigendum: Clinical feasibility of miniaturized Lissajous scanning confocal laser endomicroscopy for indocyanine green-enhanced brain tumor diagnosis.

[This corrects the article DOI: 10.3389/fonc.2022.994054.].

Plasmon-enhanced optoacoustic transducer with Ecoflex thin film for broadband ultrasound generation using overdriven pulsed laser diode.

Significance: Ultrasonic transducers facilitate noninvasive biomedical imaging and therapeutic applications. Optoacoustic generation using nanoplasmonic structures provides a technical solution for highly efficient broadband ultrasonic transducer. However, bulky and high-cost nanosecond lasers as conventional excitation sources hinder a compact configuration of transducer. Aim: Here, we report a plasmon-enhanced optoacoustic transducer (PEAT) for broadband ultrasound generation, featuring an overdriven pulsed laser diode (LD) and an Ecoflex thin film. The PEAT module consists of an LD, a collimating lens, a focusing lens, and an Ecoflex-coated 3D nanoplasmonic substrate (NPS). Approach: The LD is overdriven above its nominal current and precisely modulated to achieve nanosecond pulsed beam with high optical peak power. The focused laser beam is injected on the NPS with high-density electromagnetic hotspots, which allows for the efficient plasmonic photothermal effect. The thermal expansion of Ecoflex finally generates broadband ultrasound. Results: The overdriven pulsed LD achieves a maximum optical peak power of 40 W, exceeding the average optical power of 3 W. The 22 µm thick Ecoflex-coated NPS exhibits an eightfold optoacoustic enhancement with a fractional -6 dB bandwidth higher than 160% and a peak frequency of 2.5 MHz. In addition, the optoacoustic amplitude is precisely controlled by the optical peak power or the laser pulse width. The PEAT-integrated microfluidic chip clearly demonstrates acoustic atomization by generating aerosol droplets at the air-liquid interface. Conclusions: Plasmon-enhanced optoacoustic generation using PEAT can provide an approach for compact and on-demand biomedical applications, such as ultrasound imaging and lab-on-a-chip technologies.

Fully Integrated Ultrathin Solid Immersion Grating Microspectrometer for Handheld Visible and Near-Infrared Spectroscopic Applications.

Despite advances in microfabrication, compact spectrometers still face challenges in shrinking their size without sacrificing optical performance. Here,  the solid immersion grating microspectrometer (SIG-µSPEC) for high spectral resolution in a broad operational wavelength range is reported. The spectroscopic module incorporates a silicon microslit, index-matched lens, plane mirrors, solid immersion grating (SIG), and a CMOS line sensor within a small form factor. The SIG facilitates high angular dispersion of light on a planar focal plane, resulting in an average spectral resolution of 5.8 nm, with over 76% maximum sensitivity from 400 to 800 nm. SIG-µSPEC measures the spectral reflectance of fruits at different ripening stages, clearly revealing changes in the chlorophyll absorption band. The measured spectrum is further utilized for the precise prediction of the soluble solid content (SSC) levels, achieving a high correlation (R2 = 0.91) and a ratio of prediction-to-deviation of 2.36. This compact microspectrometer holds the potential for precise and non-invasive spectral analysis across point-of-care fields.

Single-shot multi-channel plasmonic real-time polymerase chain reaction for multi-target point-of-care testing.

Plasmonic nucleic acid amplification tests demand high-throughput and multi-target detection of infectious diseases as well as short turnaround time and small size for point-of-care molecular diagnostics. Here, we report a multi-channel plasmonic real-time reverse-transcription polymerase chain reaction (mpRT-qPCR) assay for ultrafast and on-chip multi-target detection. The mpRT-qPCR system features two pairs of plasmonic thermocyclers for rapid nanostructure-driven amplification and microlens array fluorescence microscopes for in situ multi-color fluorescence quantification. Each channel shows a physical dimension of 32 mm, 75 mm, and 25 mm in width, length, and thickness. The ultrathin microscopes simultaneously capture four different fluorescence images from two PCR chambers of a single cartridge at a single shot exposure per PCR cycle of four different excitation light sources. The experimental results demonstrate a single assay result of high-throughput amplification and multi-target quantification for RNA-dependent RNA polymerase, nucleocapsid, and human ribonuclease P genes in SARS-CoV-2 RNA detection. The mpRT-PCR increases the number of tests four times over the single RT-PCR and exhibits a short detection time of 15 min for the four RT-PCR reactions. This point-of-care molecular diagnostic platform can reduce false negative results in clinical applications of virus detection and decentralize healthcare facilities with limited infrastructure.

Microlens array camera with variable apertures for single-shot high dynamic range (HDR) imaging.

We report a microlens array camera with variable apertures (MACVA) for high dynamic range (HDR) imaging by using microlens arrays with various sizes of apertures. The MACVA comprises variable apertures, microlens arrays, gap spacers, and a CMOS image sensor. The microlenses with variable apertures capture low dynamic range (LDR) images with different f-stops under single-shot exposure. The reconstructed HDR images clearly exhibit expanded dynamic ranges surpassing LDR images as well as high resolution without motion artifacts, comparable to the maximum MTF50 value observed among the LDR images. This compact camera provides, what we believe to be, a new perspective for various machine vision or mobile devices applications.

Deep focus light-field camera for handheld 3D intraoral scanning using crosstalk-free solid immersion microlens arrays.

3D in vivo imaging techniques facilitate disease tracking and treatment, but bulky configurations and motion artifacts limit practical clinical applications. Compact light-field cameras with microlens arrays offer a feasible option for rapid volumetric imaging, yet their utilization in clinical practice necessitates an increased depth-of-field for handheld operation. Here, we report deep focus light-field camera (DF-LFC) with crosstalk-free solid immersion microlens arrays (siMLAs), allowing large depth-of-field and high-resolution imaging for handheld 3D intraoral scanning. The siMLAs consist of thin PDMS-coated microlens arrays and a metal-insulator-metal absorber to extend the focal length with low optical crosstalk and specular reflection. The experimental results show that the immersion of MLAs in PDMS increases the focal length by a factor of 2.7 and the transmittance by 5.6%-27%. Unlike conventional MLAs, the siMLAs exhibit exceptionally high f-numbers up to f/6, resulting in a large depth-of-field for light-field imaging. The siMLAs were fully integrated into an intraoral scanner to reconstruct a 3D dental phantom with a distance measurement error of 82 ± 41 µm during handheld operation. The DF-LFC offers a new direction not only for digital dental impressions with high accuracy, simplified workflow, reduced waste, and digital compatibility but also for assorted clinical endoscopy and microscopy.

Highly Efficient On-Chip Photothermal Cell Lysis for Nucleic Acid Extraction Using Localized Plasmonic Heating of Strongly Absorbing Au Nanoislands.

Cell lysis serves as an essential role in the sample preparation for intracellular material extraction in lab-on-a-chip applications. However, recent microfluidic-based cell lysis chips still face several technical challenges such as reagent removal, complex design, and high fabrication cost. Here, we report highly efficient on-chip photothermal cell lysis for nucleic acid extraction using strongly absorbed plasmonic Au nanoislands (SAP-AuNIs). The highly efficient photothermal cell lysis chip (HEPCL chip) consists of a PDMS microfluidic chamber and densely distributed SAP-AuNIs with large diameters and small nanogaps, allowing for broad-spectrum light absorption. The SAP-AuNIs induce photothermal heat, resulting in a uniform temperature distribution within the chamber and rapidly reaching the target temperature for cell lysis within 30 s. Furthermore, the localized plasmonic heating of SAP-AuNIs expeditiously triggers phase transition and photoporation in the directly contacted lipid bilayer of the cell membrane, resulting in rapid and highly efficient cell lysis. The HEPCL chip successfully lysed 93% of PC9 cells at 90 °C for 90 s without nucleic acid degradation. This on-chip cell lysis offers a new sample preparation platform for integrated point-of-care molecular diagnostics.

Visible to near-infrared single pixel microspectrometer using electrothermal MEMS grating.

Compact spectrometers facilitate non-destructive and point-of-care spectral analysis. Here we report a single-pixel microspectrometer (SPM) for visible to near-infrared (VIS-NIR) spectroscopy using MEMS diffraction grating. The SPM consists of slits, electrothermally rotating diffraction grating, spherical mirror, and photodiode. The spherical mirror collimates an incident beam and focuses the beam on the exit slit. The photodiode detects spectral signals dispersed by electrothermally rotating diffraction grating. The SPM was fully packaged within 1.7 cm3 and provides a spectral response range of 405 nm to 810 nm with an average 2.2 nm spectral resolution. This optical module provides an opportunity for diverse mobile spectroscopic applications such as healthcare monitoring, product screening, or non-destructive inspection.

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

Highly Adsorptive Au-TiO2 Nanocomposites for the SERS Face Mask Allow the Machine-Learning-Based Quantitative Assay of SARS-CoV-2 in Artificial Breath Aerosols.

Human respiratory aerosols contain diverse potential biomarkers for early disease diagnosis. Here, we report the direct and label-free detection of SARS-CoV-2 in respiratory aerosols using a highly adsorptive Au-TiO2 nanocomposite SERS face mask and an ablation-assisted autoencoder. The Au-TiO2 SERS face mask continuously preconcentrates and efficiently captures the oronasal aerosols, which substantially enhances the SERS signal intensities by 47% compared to simple Au nanoislands. The ultrasensitive Au-TiO2 nanocomposites also demonstrate the successful detection of SARS-CoV-2 spike proteins in artificial respiratory aerosols at a 100 pM concentration level. The deep learning-based autoencoder, followed by the partial ablation of nondiscriminant SERS features of spike proteins, allows a quantitative assay of the 101-104 pfu/mL SARS-CoV-2 lysates (comparable to 19-29 PCR cyclic threshold from COVID-19 patients) in aerosols with an accuracy of over 98%. The Au-TiO2 SERS face mask provides a platform for breath biopsy for the detection of various biomarkers in respiratory aerosols.

Handheld laser scanning microscope catheter for real-time and in vivo confocal microscopy using a high definition high frame rate Lissajous MEMS mirror.

A handheld confocal microscope using a rapid MEMS scanning mirror facilitates real-time optical biopsy for simple cancer diagnosis. Here we report a handheld confocal microscope catheter using high definition and high frame rate (HDHF) Lissajous scanning MEMS mirror. The broad resonant frequency region of the fast axis on the MEMS mirror with a low Q-factor facilitates the flexible selection of scanning frequencies. HDHF Lissajous scanning was achieved by selecting the scanning frequencies with high greatest common divisor (GCD) and high total lobe number. The MEMS mirror was fully packaged into a handheld configuration, which was coupled to a home-built confocal imaging system. The confocal microscope catheter allows fluorescence imaging of in vivo and ex vivo mouse tissues with 30 Hz frame rate and 95.4% fill factor at 256 × 256 pixels image, where the lateral resolution is 4.35 µm and the field-of-view (FOV) is 330 µm × 330 µm. This compact confocal microscope can provide diverse handheld microscopic applications for real-time, on-demand, and in vivo optical biopsy.

Clinical feasibility of miniaturized Lissajous scanning confocal laser endomicroscopy for indocyanine green-enhanced brain tumor diagnosis.

Background: Intraoperative real-time confocal laser endomicroscopy (CLE) is an alternative modality for frozen tissue histology that enables visualization of the cytoarchitecture of living tissues with spatial resolution at the cellular level. We developed a new CLE with a "Lissajous scanning pattern" and conducted a study to identify its feasibility for fluorescence-guided brain tumor diagnosis. Materials and methods: Conventional hematoxylin and eosin (H&E) histological images were compared with indocyanine green (ICG)-enhanced CLE images in two settings (1): experimental study with in vitro tumor cells and ex vivo glial tumors of mice, and (2) clinical evaluation with surgically resected human brain tumors. First, CLE images were obtained from cultured U87 and GL261 glioma cells. Then, U87 and GL261 tumor cells were implanted into the mouse brain, and H&E staining was compared with CLE images of normal and tumor tissues ex vivo. To determine the invasion of the normal brain, two types of patient-derived glioma cells (CSC2 and X01) were used for orthotopic intracranial tumor formation and compared using two methods (CLE vs. H&E staining). Second, in human brain tumors, tissue specimens from 69 patients were prospectively obtained after elective surgical resection and were also compared using two methods, namely, CLE and H&E staining. The comparison was performed by an experienced neuropathologist. Results: When ICG was incubated in vitro, U87 and GL261 cell morphologies were well-defined in the CLE images and depended on dimethyl sulfoxide. Ex vivo examination of xenograft glioma tissues revealed dense and heterogeneous glioma cell cores and peritumoral necrosis using both methods. CLE images also detected invasive tumor cell clusters in the normal brain of the patient-derived glioma xenograft model, which corresponded to H&E staining. In human tissue specimens, CLE images effectively visualized the cytoarchitecture of the normal brain and tumors. In addition, pathognomonic microstructures according to tumor subtype were also clearly observed. Interestingly, in gliomas, the cellularity of the tumor and the density of streak-like patterns were significantly associated with tumor grade in the CLE images. Finally, panoramic view reconstruction was successfully conducted for visualizing a gross tissue morphology. Conclusion: In conclusion, the newly developed CLE with Lissajous laser scanning can be a helpful intraoperative device for the diagnosis, detection of tumor-free margins, and maximal safe resection of brain tumors.

Large-Area and Ultrathin MEMS Mirror Using Silicon Micro Rim.

A large-area and ultrathin MEMS (microelectromechanical system) mirror can provide efficient light-coupling, a large scanning area, and high energy efficiency for actuation. However, the ultrathin mirror is significantly vulnerable to diverse film deformation due to residual thin film stresses, so that high flatness of the mirror is hardly achieved. Here, we report a MEMS mirror of large-area and ultrathin membrane with high flatness by using the silicon rim microstructure (SRM). The ultrathin MEMS mirror with SRM (SRM-mirror) consists of aluminum (Al) deposited silicon nitride membrane, bimorph actuator, and the SRM. The SRM is simply fabricated underneath the silicon nitride membrane, and thus effectively inhibits the tensile stress relaxation of the membrane. As a result, the membrane has high flatness of 10.6 m-1 film curvature at minimum without any deformation. The electrothermal actuation of the SRM-mirror shows large tilting angles from 15° to -45° depending on the applied DC voltage of 0~4 VDC, preserving high flatness of the tilting membrane. This stable and statically actuated SRM-mirror spurs diverse micro-optic applications such as optical sensing, beam alignment, or optical switching.

Extraordinary sensitivity enhancement of Ag-Au alloy nanohole arrays for label-free detection of Escherichia Coli.

Alloy nanostructures unveil extraordinary plasmonic phenomena that supersede the mono-metallic counterparts. Here we report silver-gold (Ag-Au) alloy nanohole arrays (α-NHA) for ultra-sensitive plasmonic label-free detection of Escherichia Coli (E. coli). Large-area α-NHA were fabricated by using nanoimprint lithography and concurrent thermal evaporation of Ag and Au. The completely miscible Ag-Au alloy exhibits an entirely different dielectric function in the near infra-red wavelength range compared to mono-metallic Ag or Au. The α-NHA demonstrate substantially enhanced refractive index sensitivity of 387 nm/RIU, surpassing those of Ag or Au mono-metallic nanohole arrays by approximately 40%. Moreover, the α-NHA provide highly durable material stability to corrosion and oxidation during over one-month observation. The ultra-sensitive α-NHA allow the label-free detection of E. coli in various concentration levels ranging from 103 to 108 cfu/ml with a calculated limit of detection of 59 cfu/ml. This novel alloy plasmonic material provides a new outlook for widely applicable biosensing and bio-medical applications.

Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.

Ultrathin arrayed camera for high-contrast near-infrared imaging.

We report an ultrathin arrayed camera (UAC) for high-contrast near infrared (NIR) imaging by using microlens arrays with a multilayered light absorber. The UAC consists of a multilayered composite light absorber, inverted microlenses, gap-alumina spacers and a planar CMOS image sensor. The multilayered light absorber was fabricated through lift-off and repeated photolithography processes. The experimental results demonstrate that the image contrast is increased by 4.48 times and the MTF 50 is increased by 2.03 times by eliminating optical noise between microlenses through the light absorber. The NIR imaging of UAC successfully allows distinguishing the security strip of authentic bill and the blood vessel of finger. The ultrathin camera offers a new route for diverse applications in biometric, surveillance, and biomedical imaging.

Spread spectrum SERS allows label-free detection of attomolar neurotransmitters.

The quantitative label-free detection of neurotransmitters provides critical clues in understanding neurological functions or disorders. However, the identification of neurotransmitters remains challenging for surface-enhanced Raman spectroscopy (SERS) due to the presence of noise. Here, we report spread spectrum SERS (ss-SERS) detection for the rapid quantification of neurotransmitters at the attomolar level by encoding excited light and decoding SERS signals with peak autocorrelation and near-zero cross-correlation. Compared to conventional SERS measurements, the experimental result of ss-SERS shows an exceptional improvement in the signal-to-noise ratio of more than three orders of magnitude, thus achieving a high temporal resolution of over one hundred times. The ss-SERS measurement further allows the attomolar SERS detection of dopamine, serotonin, acetylcholine, γ-aminobutyric acid, and glutamate without Raman reporters. This approach opens up opportunities not only for investigating the early diagnostics of neurological disorders or highly sensitive biomedical SERS applications but also for developing low-cost spectroscopic biosensing applications.

Lissajous scanning structured illumination microscopy.

High-resolution fluorescent microscopic imaging techniques are in high demand to observe detailed structures or dynamic mechanisms of biological samples. Structured illumination microscopy (SIM) has grabbed much attention in super-resolution imaging due to simple configuration, high compatibility with common fluorescent molecules, and fast image acquisition. Here, we report Lissajous scanning SIM (LS-SIM) by using a high fill-factor Lissajous scanning micromirror and laser beam modulation. The LS-SIM was realized by a Lissajous scanned structured illumination module, relay optics, and a conventional fluorescent microscope. The micromirror comprises an inner mirror and an outer frame, which are scanned at pseudo-resonance with electrostatic actuation. The biaxial scanning frequencies are selected by the frequency selection rule for high fill-factor (> 80%) Lissajous scanning. Structured illumination (SI) was then realized by modulating the intensity of a laser beam at the least common multiple (LCM) of the scanning frequencies. A compact Lissajous scanned SI module containing a fiber-optic collimator and Lissajous micromirror has been fully packaged and coupled with relay optics and a fiber-based diode pumped solid state (DPSS) laser including acousto-optic-modulator (AOM). Various structured images were obtained by shifting the phase and orientation of the illumination patterns and finally mounted with a conventional fluorescent microscope. The LS-SIM has experimentally demonstrated high-resolution fluorescent microscopic imaging of reference targets and human lung cancer cell PC-9 cells. The LS-SIM exhibits the observable region in spatial frequency space over 2x, the line-edge sharpness over 1.5x, and the peak-to-valley (P-V) ratio over 2x, compared to widefield fluorescent microscopy. This method can provide a new route for advanced high-resolution fluorescent microscopic imaging.

Lissajous scanned variable structured illumination for dynamic stereo depth map.

Structured illumination plays an important role in advanced photographic and microscopic imaging applications. Here we report variable structured illumination (VSI) using Lissajous scanning techniques. The variable structured illumination module comprises Lissajous scanning micromirror and fiber-based diode pumped solid state (DPSS) laser with intensity modulation, combined with a stereo camera for dynamic stereo depth map. The micromirror projects static and discrete patterns by modulating the intensity of a laser beam at the least common multiple (LCM) of two scanning frequencies. The pattern density is increased by either decreasing the greatest common divisor (GCD) of scanning frequencies or decreasing the duty rate of the laser modulation. The scanning amplitude also controls the field-of-view (FOV) for the exact illumination of a target object for dynamic stereo depth map. The variable structured illumination module provides a new route for advanced imaging applications such as high-quality depth map, super-resolution, or motion recognition.

Multifocal microlens arrays using multilayer photolithography.

We report a new microfabrication method of multifocal microlens arrays (MF-MLAs) for extended depth-of-field (DoF) using multilayer photolithography and thermal reflow. Microlenses of different focal lengths were simultaneously fabricated on a single glass wafer by using repeated photolithography with multiple photomasks to define microposts of different thicknesses and concurrent thermal reflow of multi-stacked microposts. The diverse lens curvatures of MF-MLAs are precisely controlled by the thickness of the micropost. Hexagonally packaged MF-MLAs clearly show three different focal lengths of 249 µm, 310 µm, and 460 µm for 200 µm in lens diameter and result in multifocal images on a single image sensor. This method provides a new route for developing various three-dimensional (3D) imaging applications such as light-field cameras or 3D medical endoscopes.