A prospective multicenter assessor blinded pilot study using confocal laser endomicroscopy for intraoperative brain tumor diagnosis.

In this multi-center, assessor-blinded pilot study, the diagnostic efficacy of cCeLL-Ex vivo, a second-generation confocal laser endomicroscopy (CLE), was compared against the gold standard frozen section analysis for intraoperative brain tumor diagnosis. The study was conducted across three tertiary medical institutions in the Republic of Korea. Biopsy samples from newly diagnosed brain tumor patients were categorized based on location and divided for permanent section analysis, frozen section analysis, and cCeLL-Ex vivo imaging. Of the 74 samples from 55 patients, the majority were from the tumor core (74.3%). cCeLL-Ex vivo exhibited a relatively higher diagnostic accuracy (89.2%) than frozen section analysis (86.5%), with both methods showing a sensitivity of 92.2%. cCeLL-Ex vivo also demonstrated higher specificity (70% vs. 50%), positive predictive value (PPV) (95.2% vs. 92.2%), and negative predictive value (NPV) (58.3% vs. 50%). Furthermore, the time from sample preparation to diagnosis was notably shorter with cCeLL-Ex vivo (13 min 17 s) compared to frozen section analysis (28 min 28 s) (p-value < 0.005). These findings underscore cCeLL-Ex vivo's potential as a supplementary tool for intraoperative brain tumor diagnosis, with future studies anticipated to further validate its clinical utility.

Real-time Histological Evaluation of Gastric Cancer Tissue by Using a Confocal Laser Endomicroscopic System.

BACKGROUND/AIM: The need for instant histological evaluation of fresh tissue, especially in cancer treatment, remains paramount. The conventional frozen section technique has inherent limitations, prompting the exploration of alternative methods. A recently developed confocal laser endomicroscopic system provides real-time imaging of the tissue without the need for glass slide preparation. Herein, we evaluated its applicability in the histologic evaluation of gastric cancer tissues. MATERIALS AND METHODS: A confocal laser endomicroscopic system (CLES) with a Lissajous pattern laser scanning, was developed. Fourteen fresh gastric cancer tissues and the same number of normal gastric tissues were obtained from advanced gastric cancer patients. Fluorescein sodium was used for staining. Five pathologists interpreted 100 endomicroscopic images and decided their histologic location and the presence of cancer. Following the review of matched hematoxylin and eosin (H&E) slides, their performance was evaluated with another 100 images. RESULTS: CLES images mirrored gastric tissue histology. Pathologists were able to detect the histologic location of the images with 65.7% accuracy and differentiate cancer tissue from normal with 74.7% accuracy. The sensitivity and specificity of cancer detection were 71.9% and 76.1%. Following the review of matched H&E images, the accuracy of identifying the histologic location was increased to 92.8% (p<0.0001), and that of detecting cancer tissue was also increased to 90.9% (p<0.001). The sensitivity and specificity of cancer detection were enhanced to 89.1% and 93.2% (p<0.0001). CONCLUSION: High-quality histological images were immediately acquired by the CLES. The operator training enabled the accurate detection of cancer and histologic location raising its potential applicability as a real-time tissue imaging modality.

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.].

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.

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.

Handheld endomicroscope using a fiber-optic harmonograph enables real-time and in vivo confocal imaging of living cell morphology and capillary perfusion.

Confocal laser endomicroscopy provides high potential for noninvasive and in vivo optical biopsy at the cellular level. Here, we report a fully packaged handheld confocal endomicroscopic system for real-time, high-resolution, and in vivo cellular imaging using a Lissajous scanning fiber-optic harmonograph. The endomicroscopic system features an endomicroscopic probe with a fiber-optic harmonograph, a confocal microscope unit, and an image signal processor. The fiber-optic harmonograph contains a single mode fiber coupled with a quadrupole piezoelectric tube, which resonantly scans both axes at ~ 1 kHz to obtain a Lissajous pattern. The fiber-optic harmonograph was fully packaged into an endomicroscopic probe with an objective lens. The endomicroscopic probe was hygienically packaged for waterproofing and disinfection of medical instruments within a 2.6-mm outer diameter stainless tube capable of being inserted through the working channel of a clinical endoscope. The probe was further combined with the confocal microscope unit for indocyanine green imaging and the image signal processor for high frame rate and high density Lissajous scanning. The signal processing unit delivers driving signals for probe actuation and reconstructs confocal images using the auto phase matching process of Lissajous fiber scanners. The confocal endomicroscopic system was used to successfully obtain human in vitro fluorescent images and real-time ex vivo and in vivo fluorescent images of the living cell morphology and capillary perfusion inside a single mouse.

Baicalin improves the in vitro developmental capacity of pig embryos by inhibiting apoptosis, regulating mitochondrial activity and activating sonic hedgehog signaling.

Baicalin, a traditional Chinese medicinal monomer whose chemical structure is known, can be used to treat female infertility. However, the effect of baicalin on embryonic development is unknown. This study investigated the effects of baicalin on in vitro development of parthenogenetically activated (PA) and in vitro fertilized (IVF) pig embryos and the underlying mechanisms involved. Treatment with 0.1 µg/ml baicalin significantly improved (P < 0.05) the in vitro developmental capacity of PA pig embryos by reducing the reactive oxygen species (ROS) levels and apoptosis and increasing the mitochondrial membrane potential (ΔΨm) and ATP level. mRNA and protein expression of sonic hedgehog (SHH) and GLI1, which are related to the SHH signaling pathway, in PA pig embryos at the 2-cell stage, were significantly higher in the baicalin-treated group than in the control group. To confirm that the SHH signaling pathway is involved in the mechanism by which baicalin improves embryonic development, we treated embryos with baicalin in the absence or presence of cyclopamine (Cy), an inhibitor of this pathway. Cy abolished the effects of baicalin on in vitro embryonic development. In conclusion, baicalin improves the in vitro developmental capacity of PA and IVF pig embryos by inhibiting ROS production and apoptosis, regulating mitochondrial activity and activating SHH signaling.

Lissajous Scanning Two-photon Endomicroscope for In vivo Tissue Imaging.

An endomicroscope opens new frontiers of non-invasive biopsy for in vivo imaging applications. Here we report two-photon laser scanning endomicroscope for in vivo cellular and tissue imaging using a Lissajous fiber scanner. The fiber scanner consists of a piezoelectric (PZT) tube, a single double-clad fiber (DCF) with high fluorescence collection, and a micro-tethered-silicon-oscillator (MTSO) for the separation of biaxial resonant scanning frequencies. The endomicroscopic imaging exhibits 5 frames/s with 99% in scanning density by using the selection rule of scanning frequencies. The endomicroscopic scanner was compactly packaged within a stainless tube of 2.6 mm in diameter with a high NA gradient-index (GRIN) lens, which can be easily inserted into the working channel of a conventional laparoscope. The lateral and axial resolutions of the endomicroscope are 0.70 µm and 7.6 µm, respectively. Two-photon fluorescence images of a stained kidney section and miscellaneous ex vivo and in vivo organs from wild type and green fluorescent protein transgenic (GFP-TG) mice were successfully obtained by using the endomicroscope. The endomicroscope also obtained label free images including autofluorescence and second-harmonic generation of an ear tissue of Thy1-GCaMP6 (GP5.17) mouse. The Lissajous scanning two-photon endomicroscope can provide a compact handheld platform for in vivo tissue imaging or optical biopsy applications.

Scanning MEMS Mirror for High Definition and High Frame Rate Lissajous Patterns.

Scanning MEMS (micro-electro-mechanical system) mirrors are attractive given their potential use in a diverse array of laser scanning display and imaging applications. Here we report on an electrostatic MEMS mirror for high definition and high frame rate (HDHF) Lissajous scanning. The MEMS mirror comprised a low Q-factor inner mirror and frame mirror, which provided two-dimensional scanning at two similar resonant scanning frequencies with high mechanical stability. The low Q inner mirror enabled a broad frequency selection range. The high definition and high frame rate (HDHF) Lissajous scanning of the MEMS mirror was achieved by selecting a set of scanning frequencies near its resonance with a high greatest common divisor (GCD) and a high total lobe number. The MEMS mirror had resonant scanning frequencies at 5402 Hz and 6702 Hz in x and y directions, respectively. The selected pseudo-resonant frequencies of 5450 Hz and 6700 Hz for HDHF scanning provided 50 frames per second with 94% fill factor in 256 × 256 pixels. This Lissajous MEMS mirror could be utilized for assorted HDHF laser scanning imaging and display applications.

1.65 mm diameter forward-viewing confocal endomicroscopic catheter using a flip-chip bonded electrothermal MEMS fiber scanner.

We report a 1.65 mm diameter forward-viewing confocal endomicroscopic catheter using a flip-chip bonded electrothermal MEMS fiber scanner. Lissajous scanning was implemented by the electrothermal MEMS fiber scanner. The Lissajous scanned MEMS fiber scanner was precisely fabricated to facilitate flip-chip connection, and bonded with a printed circuit board. The scanner was successfully combined with a fiber-based confocal imaging system. A two-dimensional reflectance image of the metal pattern 'OPTICS' was successfully obtained with the scanner. The flip-chip bonded scanner minimizes electrical packaging dimensions. The inner diameter of the flip-chip bonded MEMS fiber scanner is 1.3 mm. The flip-chip bonded MEMS fiber scanner is fully packaged with a 1.65 mm diameter housing tube, 1 mm diameter GRIN lens, and a single mode optical fiber. The packaged confocal endomicroscopic catheter can provide a new breakthrough for diverse in-vivo endomicroscopic applications.

Frequency selection rule for high definition and high frame rate Lissajous scanning.

Lissajous microscanners are very attractive in compact laser scanning applications such as endomicroscopy or pro-projection display owing to high mechanical stability and low operating voltages. The scanning frequency serves as a critical factor for determining the scanning imaging quality. Here we report the selection rule of scanning frequencies that can realize high definition and high frame-rate (HDHF) full-repeated Lissajous scanning imaging. The fill factor (FF) monotonically increases with the total lobe number of a Lissajous curve, i.e., the sum of scanning frequencies divided by the great common divisor (GCD) of bi-axial scanning frequencies. The frames per second (FPS), called the pattern repeated rate or the frame rate, linearly increases with GCD. HDHF Lissajous scanning is achieved at the bi-axial scanning frequencies, where the GCD has the maximum value among various sets of the scanning frequencies satisfying the total lobe number for a target FF. Based on this selection rule, the experimental results clearly demonstrate that conventional Lissajous scanners substantially increase both FF and FPS by slightly modulating the scanning frequencies at near the resonance within the resonance bandwidth of a Lissajous scanner. This selection rule provides a new guideline for HDHF Lissajous scanning in compact laser scanning systems.

Electrothermal MEMS fiber scanner for optical endomicroscopy.

We report a novel MEMS fiber scanner with an electrothermal silicon microactuator and a directly mounted optical fiber. The microactuator comprises double hot arm and cold arm structures with a linking bridge and an optical fiber is aligned along a silicon fiber groove. The unique feature induces separation of resonant scanning frequencies of a single optical fiber in lateral and vertical directions, which realizes Lissajous scanning during the resonant motion. The footprint dimension of microactuator is 1.28 x 7 x 0.44 mm3. The resonant scanning frequencies of a 20 mm long optical fiber are 239.4 Hz and 218.4 Hz in lateral and vertical directions, respectively. The full scanned area indicates 451 µm x 558 µm under a 16 Vpp pulse train. This novel laser scanner can provide many opportunities for laser scanning endomicroscopic applications.

Micromachined tethered silicon oscillator for an endomicroscopic Lissajous fiber scanner.

This work reports micromachined tethered silicon oscillators (MTSOs) for endoscopic Lissajous fiber scanners. An MTSO comprises an offset silicon spring for stiffness modulation of a scanning fiber and additional mass for modulation of resonant scanning frequency in one body. MTSOs were assembled with a resonant fiber scanner and enhanced scanning reliability of the scanner by eliminating mechanical cross coupling. The fiber scanner with MTSOs was fully packaged as an endomicroscopic catheter and coupled with a conventional laparoscope and spectral domain OCT system. The endomicroscope was maneuvered with the integrated laparoscope and in vivo swine tissue OCT imaging was successfully demonstrated during open surgery. This new component serves as an important element inside an endoscopic Lissajous fiber scanner for early cancer detection or on-demand minimum lesional margin decision during noninvasive endoscopic biopsy.