Ultrashort pulse Kagome hollow-core photonic crystal fiber delivery for nonlinear optical imaging.

We report ultrashort pulse delivery through a hypocycloid-core inhibited-coupling Kagome hollow-core photonic crystal fiber (HC-PCF). Undistorted 10 fs and 6.6 nJ pulses were launched through 1 m long fiber without fiber dispersion pre-compensation and 80% efficiency. The performance of this technology for biomedical imaging is demonstrated on a biological sample by incorporating the fiber into a two-photon excited fluorescence (TPEF) laser scanning microscope (LSM) achieving a pulse width of 15 fs at the sample location. To the best of our knowledge, this is the first report on undistorted TPEF imaging in a LSM with 15 fs pulses delivered through a 1 m long Kagome HC-PCF with high throughput.

Depth resolved label-free multimodal optical imaging platform to study morpho-molecular composition of tissue.

Multimodal imaging platforms offer a vast array of tissue information in a single image acquisition by combining complementary imaging techniques. By merging different systems, better tissue characterization can be achieved than is possible by the constituent imaging modalities alone. The combination of optical coherence tomography (OCT) with non-linear optical imaging (NLOI) techniques such as two-photon excited fluorescence (TPEF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) provides access to detailed information of tissue structure and molecular composition in a fast, label-free and non-invasive manner. We introduce a multimodal label-free approach for morpho-molecular imaging and spectroscopy and validate the system in mouse skin demonstrating the potential of the system for colocalized acquisition of OCT and NLOI signals.

Line Scan Raman Microspectroscopy for Label-Free Diagnosis of Human Pituitary Biopsies.

Pituitary adenomas are neoplasia of the anterior pituitary gland and can be subdivided into hormone-producing tumors (lactotroph, corticotroph, gonadotroph, somatotroph, thyreotroph or plurihormonal) and hormone-inactive tumors (silent or null cell adenomas) based on their hormonal status. We therefore developed a line scan Raman microspectroscopy (LSRM) system to detect, discriminate and hyperspectrally visualize pituitary gland from pituitary adenomas based on molecular differences. By applying principal component analysis followed by a k-nearest neighbor algorithm, specific hormone states were identified and a clear discrimination between pituitary gland and various adenoma subtypes was achieved. The classifier yielded an accuracy of 95% for gland tissue and 84-99% for adenoma subtypes. With an overall accuracy of 92%, our LSRM system has proven its potential to differentiate pituitary gland from pituitary adenomas. LSRM images based on the presence of specific Raman bands were created, and such images provided additional insight into the spatial distribution of particular molecular compounds. Pathological states could be molecularly differentiated and characterized with texture analysis evaluating Grey Level Cooccurrence Matrices for each LSRM image, as well as correlation coefficients between LSRM images.

In vivo tear film thickness measurement and tear film dynamics visualization using spectral domain optical coherence tomography.

Dry eye syndrome is a highly prevalent disease of the ocular surface characterized by an instability of the tear film. Traditional methods used for the evaluation of tear film stability are invasive or show limited repeatability. Here we propose a new non-invasive fully automated approach to measure tear film thickness based on spectral domain optical coherence tomography and on an efficient delay estimator. Silicon wafer phantom were used to validate the thickness measurement. The technique was applied in vivo in healthy subjects. Series of tear film thickness maps were generated, allowing for the visualization of tear film dynamics. Our results show that the in vivo central tear film thickness measurements are precise and repeatable with a coefficient of variation of about 0.65% and that repeatable tear film dynamics can be observed. The presented approach could be used in clinical setting to study patients with dry eye disease and monitor their treatments.

Single-pulse CARS based multimodal nonlinear optical microscope for bioimaging.

Noninvasive label-free imaging of biological systems raises demand not only for high-speed three-dimensional prescreening of morphology over a wide-field of view but also it seeks to extract the microscopic functional and molecular details within. Capitalizing on the unique advantages brought out by different nonlinear optical effects, a multimodal nonlinear optical microscope can be a powerful tool for bioimaging. Bringing together the intensity-dependent contrast mechanisms via second harmonic generation, third harmonic generation and four-wave mixing for structural-sensitive imaging, and single-beam/single-pulse coherent anti-Stokes Raman scattering technique for chemical sensitive imaging in the finger-print region, we have developed a simple and nearly alignment-free multimodal nonlinear optical microscope that is based on a single wide-band Ti:Sapphire femtosecond pulse laser source. Successful imaging tests have been realized on two exemplary biological samples, a canine femur bone and collagen fibrils harvested from a rat tail. Since the ultra-broad band-width femtosecond laser is a suitable source for performing high-resolution optical coherence tomography, a wide-field optical coherence tomography arm can be easily incorporated into the presented multimodal microscope making it a versatile optical imaging tool for noninvasive label-free bioimaging.

Hybrid single-source online Fourier transform coherent anti-Stokes Raman scattering/optical coherence tomography.

We demonstrate a multimodal optical coherence tomography (OCT) and online Fourier transform coherent anti-Stokes Raman scattering (FTCARS) platform using a single sub-12 femtosecond (fs) Ti:sapphire laser enabling simultaneous extraction of structural and chemical ("morphomolecular") information of biological samples. Spectral domain OCT prescreens the specimen providing a fast ultrahigh (4×12 µm axial and transverse) resolution wide field morphologic overview. Additional complementary intrinsic molecular information is obtained by zooming into regions of interest for fast label-free chemical mapping with online FTCARS spectroscopy. Background-free CARS is based on a Michelson interferometer in combination with a highly linear piezo stage, which allows for quick point-to-point extraction of CARS spectra in the fingerprint region in less than 125 ms with a resolution better than 4 cm(-1) without the need for averaging. OCT morphology and CARS spectral maps indicating phosphate and carbonate bond vibrations from human bone samples are extracted to demonstrate the performance of this hybrid imaging platform.

Flavin fluorescence lifetime and autofluorescence optical redox ratio for improved visualization and classification of brain tumors.

Purpose: Modern techniques for improved tumor visualization have the aim to maximize the extent of resection during brain tumor surgery and thus improve patient prognosis. Optical imaging of autofluorescence is a powerful and non-invasive tool to monitor metabolic changes and transformation in brain tumors. Cellular redox ratios can be retrieved from fluorescence emitted by the coenzymes reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). Recent studies point out that the influence of flavin mononucleotide (FMN) has been underestimated. Experimental design: Fluorescence lifetime imaging and fluorescence spectroscopy were performed through a modified surgical microscope. We acquired 361 flavin fluorescence lifetime (500-580 nm) and fluorescence spectra (430-740 nm) data points on freshly excised different brain tumors: low-grade gliomas (N=17), high-grade gliomas (N=42), meningiomas (N=23), metastases (N=26) and specimens from the non-tumorous brain (N=3). Results: Protein-bound FMN fluorescence in brain tumors did increase with a shift toward a more glycolytic metabolism (R=-0.87). This increased the average flavin fluorescence lifetime in tumor entities with respect to the non-tumorous brain. Further, these metrics were characteristic for the different tumor entities and showed promise for machine learning based brain tumor classification. Conclusions: Our results shed light on FMN fluorescence in metabolic imaging and outline the potential for supporting the neurosurgeon in visualizing and classifying brain tumor tissue during surgery.

Precise thickness measurements of Bowman's layer, epithelium, and tear film.

PURPOSE: To visualize corneal microstructure such as tear film, epithelium, and Bowman's layer in three dimensions with spectral domain optical coherence tomography (SDOCT) exhibiting 1.3 µm axial resolution at 100,000 A-scans/s. This enables measurement of epithelial and Bowman layer thickness across an area of 8.4 mm × 8.4 mm and measuring the tear film thickness at the central cornea. METHODS: We designed a high-performance SDOCT system, which uses a broad bandwidth TiSapph Laser and a high-speed complementary metal-oxide-semiconductor detector technology, providing a resolution in tissue of 1.3 µm and an acquisition speed of 100,000 A-scans/s. Such speed and resolution is a prerequisite if precise anatomy is to be determined. The high resolution gives access to corneal microstructure such as the epithelium layer as well as the boundaries of Bowman's layer and stroma. Even more interestingly, the tear film can be distinguished on the surface of the cornea. The Bowman's layer and epithelial thickness for both eyes of nine subjects have been measured out of which two subjects underwent photorefractive keratectomy treatment. RESULTS: Three-dimensional volumes of the human cornea have been recorded in vivo at an A-scan rate of 100,000 scans/s. Epithelial thickness was measured to be 55.8 ± 3.3 µm and Bowman's layer thickness 18.7 ± 2.5 µm in normal eyes. Epithelial thickness in the eyes after refractive surgery was measured to be 68.2 ± 5.0 µm. The Bowman layer was degenerated in these eyes. The average tear film thickness of four eyes was 5.1 ± 0.5 µm. CONCLUSIONS: Using a high-performance SDOCT system with high-imaging speed and ultrahigh resolution, we produced precise thickness maps of the epithelium and for the first time of the Bowman's layer. Such a system will give insight into high-fidelity three-dimensional corneal microstructure helping to precisely plan refractive surgery. It may furthermore yield new perspectives on studying and understanding tear film dynamics.

Frequency-doubled DBR-tapered diode laser for direct pumping of Ti:sapphire lasers generating sub-20 fs pulses.

For the first time a single-pass frequency doubled DBR-tapered diode laser suitable for pumping Ti:sapphire lasers generating ultrashort pulses is demonstrated. The maximum output powers achieved when pumping the Ti:sapphire laser are 110 mW (CW) and 82 mW (mode-locked) respectively at 1.2 W of pump power. This corresponds to a reduction in optical conversion efficiencies to 75% of the values achieved with a commercial diode pumped solid-state laser. However, the superior electro-optical efficiency of the diode laser improves the overall efficiency of the Ti:sapphire laser by a factor > 2. The optical spectrum emitted by the Ti:sapphire laser when pumped with our diode laser shows a spectral width of 112 nm (FWHM). Based on autocorrelation measurements, pulse widths of less than 20 fs can therefore be expected.

Enhanced medical diagnosis for dOCTors: a perspective of optical coherence tomography.

SIGNIFICANCE: After three decades, more than 75,000 publications, tens of companies being involved in its commercialization, and a global market perspective of about USD 1.5 billion in 2023, optical coherence tomography (OCT) has become one of the fastest successfully translated imaging techniques with substantial clinical and economic impacts and acceptance. AIM: Our perspective focuses on disruptive forward-looking innovations and key technologies to further boost OCT performance and therefore enable significantly enhanced medical diagnosis. APPROACH: A comprehensive review of state-of-the-art accomplishments in OCT has been performed. RESULTS: The most disruptive future OCT innovations include imaging resolution and speed (single-beam raster scanning versus parallelization) improvement, new implementations for dual modality or even multimodality systems, and using endogenous or exogenous contrast in these hybrid OCT systems targeting molecular and metabolic imaging. Aside from OCT angiography, no other functional or contrast enhancing OCT extension has accomplished comparable clinical and commercial impacts. Some more recently developed extensions, e.g., optical coherence elastography, dynamic contrast OCT, optoretinography, and artificial intelligence enhanced OCT are also considered with high potential for the future. In addition, OCT miniaturization for portable, compact, handheld, and/or cost-effective capsule-based OCT applications, home-OCT, and self-OCT systems based on micro-optic assemblies or photonic integrated circuits will revolutionize new applications and availability in the near future. Finally, clinical translation of OCT including medical device regulatory challenges will continue to be absolutely essential. CONCLUSIONS: With its exquisite non-invasive, micrometer resolution depth sectioning capability, OCT has especially revolutionized ophthalmic diagnosis and hence is the fastest adopted imaging technology in the history of ophthalmology. Nonetheless, OCT has not been completely exploited and has substantial growth potential-in academics as well as in industry. This applies not only to the ophthalmic application field, but also especially to the original motivation of OCT to enable optical biopsy, i.e., the in situ imaging of tissue microstructure with a resolution approaching that of histology but without the need for tissue excision.

Toward Quantitative in vivo Label-Free Tracking of Lipid Distribution in a Zebrafish Cancer Model.

Cancer cells often adapt their lipid metabolism to accommodate the increased fatty acid demand for membrane biogenesis and energy production. Upregulation of fatty acid uptake from the environment of cancer cells has also been reported as an alternative mechanism. To investigate the role of lipids in tumor onset and progression and to identify potential diagnostic biomarkers, lipids are ideally imaged directly within the intact tumor tissue in a label-free way. In this study, we investigated lipid accumulation and distribution in living zebrafish larvae developing a tumor by means of coherent anti-Stokes Raman scattering microscopy. Quantitative textural features based on radiomics revealed higher lipid accumulation in oncogene-expressing larvae compared to healthy ones. This high lipid accumulation could reflect an altered lipid metabolism in the hyperproliferating oncogene-expressing cells.

Morpho-Molecular Metabolic Analysis and Classification of Human Pituitary Gland and Adenoma Biopsies Based on Multimodal Optical Imaging.

Pituitary adenomas count among the most common intracranial tumors. During pituitary oncogenesis structural, textural, metabolic and molecular changes occur which can be revealed with our integrated ultrahigh-resolution multimodal imaging approach including optical coherence tomography (OCT), multiphoton microscopy (MPM) and line scan Raman microspectroscopy (LSRM) on an unprecedented cellular level in a label-free manner. We investigated 5 pituitary gland and 25 adenoma biopsies, including lactotroph, null cell, gonadotroph, somatotroph and mammosomatotroph as well as corticotroph. First-level binary classification for discrimination of pituitary gland and adenomas was performed by feature extraction via radiomic analysis on OCT and MPM images and achieved an accuracy of 88%. Second-level multi-class classification was performed based on molecular analysis of the specimen via LSRM to discriminate pituitary adenomas subtypes with accuracies of up to 99%. Chemical compounds such as lipids, proteins, collagen, DNA and carotenoids and their relation could be identified as relevant biomarkers, and their spatial distribution visualized to provide deeper insight into the chemical properties of pituitary adenomas. Thereby, the aim of the current work was to assess a unique label-free and non-invasive multimodal optical imaging platform for pituitary tissue imaging and to perform a multiparametric morpho-molecular metabolic analysis and classification.

Diagnosis of Pituitary Adenoma Biopsies by Ultrahigh Resolution Optical Coherence Tomography Using Neuronal Networks.

Objective: Despite advancements of intraoperative visualization, the difficulty to visually distinguish adenoma from adjacent pituitary gland due to textural similarities may lead to incomplete adenoma resection or impairment of pituitary function. The aim of this study was to investigate optical coherence tomography (OCT) imaging in combination with a convolutional neural network (CNN) for objectively identify pituitary adenoma tissue in an ex vivo setting. Methods: A prospective study was conducted to train and test a CNN algorithm to identify pituitary adenoma tissue in OCT images of adenoma and adjacent pituitary gland samples. From each sample, 500 slices of adjacent cross-sectional OCT images were used for CNN classification. Results: OCT data acquisition was feasible in 19/20 (95%) patients. The 16.000 OCT slices of 16/19 of cases were employed for creating a trained CNN algorithm (70% for training, 15% for validating the classifier). Thereafter, the classifier was tested on the paired samples of three patients (3.000 slices). The CNN correctly predicted adenoma in the 3 adenoma samples (98%, 100% and 84% respectively), and correctly predicted gland and transition zone in the 3 samples from the adjacent pituitary gland. Conclusion: Trained convolutional neural network computing has the potential for fast and objective identification of pituitary adenoma tissue in OCT images with high sensitivity ex vivo. However, further investigation with larger number of samples is required.

Fluorescence Lifetime Imaging and Spectroscopic Co-Validation for Protoporphyrin IX-Guided Tumor Visualization in Neurosurgery.

Maximal safe resection is a key strategy for improving patient prognosis in the management of brain tumors. Intraoperative fluorescence guidance has emerged as a standard in the surgery of high-grade gliomas. The administration of 5-aminolevulinic acid prior to surgery induces tumor-specific accumulation of protoporphyrin IX, which emits red fluorescence under blue-light illumination. The technology, however, is substantially limited for low-grade gliomas and weakly tumor-infiltrated brain, where low protoporphyrin IX concentrations are outweighed by tissue autofluorescence. In this context, fluorescence lifetime imaging has shown promise to distinguish spectrally overlapping fluorophores. We integrated frequency-domain fluorescence lifetime imaging in a surgical microscope and combined it with spatially registered fluorescence spectroscopy, which can be considered a research benchmark for sensitive protoporphyrin IX detection. Fluorescence lifetime maps and spectra were acquired for a representative set of fresh ex-vivo brain tumor specimens (low-grade gliomas n = 15, high-grade gliomas n = 80, meningiomas n = 41, and metastases n = 35). Combining the fluorescence lifetime with fluorescence spectra unveiled how weak protoporphyrin IX accumulations increased the lifetime respective to tissue autofluorescence. Infiltration zones (4.1ns ± 1.8ns, p = 0.017) and core tumor areas (4.8ns ± 1.3ns, p = 0.040) of low-grade gliomas were significantly distinguishable from non-pathologic tissue (1.6ns ± 0.5ns). Similarly, fluorescence lifetimes for infiltrated and reactive tissue as well as necrotic and core tumor areas were increased for high-grade gliomas and metastasis. Meningioma tumor specimens showed strongly increased lifetimes (12.2ns ± 2.5ns, p = 0.005). Our results emphasize the potential of fluorescence lifetime imaging to optimize maximal safe resection in brain tumors in future and highlight its potential toward clinical translation.

Fast dispersion encoded full range optical coherence tomography for retinal imaging at 800 nm and 1060 nm.

The dispersion mismatch between sample and reference arm in frequency-domain optical coherence tomography (OCT) can be used to iteratively suppress complex conjugate artifacts and thereby increase the imaging range. In this paper, we propose a fast dispersion encoded full range (DEFR) algorithm that detects multiple signal components per iteration. The influence of different dispersion levels on the reconstruction quality is analyzed experimentally using a multilayered scattering phantom and in vivo retinal tomograms at 800 nm. Best results have been achieved with 30 mm SF11, with neglectable resolution decrease due to finite resolution of the spectrometer. Our fast DEFR algorithm achieves an average suppression ratio of 55 dB and typically converges within 5 to 10 iterations. The processing time on non-dedicated hardware was 5 to 10 seconds for tomograms with 512 depth scans and 4096 sampling points per depth scan. Application of DEFR to the more challenging 1060 nm wavelength region is also demonstrated by introducing an additional optical fibre in the sample arm.

Towards ultrahigh resolution OCT based endoscopical pituitary gland and adenoma screening: a performance parameter evaluation.

Ultrahigh resolution optical coherence tomography (UHR-OCT) for differentiating pituitary gland versus adenoma tissue has been investigated for the first time, indicating more than 80% accuracy. For biomarker identification, OCT images of paraffin embedded tissue are correlated to histopathological slices. The identified biomarkers are verified on fresh biopsies. Additionally, an approach, based on resolution modified UHR-OCT ex vivo data, investigating optical performance parameters for the realization in an in vivo endoscope is presented and evaluated. The identified morphological features-cell groups with reticulin framework-detectable with UHR-OCT showcase a promising differentiation ability, encouraging endoscopic OCT probe development for in vivo application.

Macroscopic fluorescence-lifetime imaging of NADH and protoporphyrin IX improves the detection and grading of 5-aminolevulinic acid-stained brain tumors.

Maximal safe tumor resection remains the key prognostic factor for improved prognosis in brain tumor patients. Despite 5-aminolevulinic acid-based fluorescence guidance the neurosurgeon is, however, not able to visualize most low-grade gliomas (LGG) and infiltration zone of high-grade gliomas (HGG). To overcome the need for a more sensitive visualization, we investigated the potential of macroscopic, wide-field fluorescence lifetime imaging of nicotinamide adenine dinucleotide (NADH) and protoporphyrin IX (PPIX) in selected human brain tumors. For future intraoperative use, the imaging system offered a square field of view of 11 mm at 250 mm free working distance. We performed imaging of tumor tissue ex vivo, including LGG and HGG as well as brain metastases obtained from 21 patients undergoing fluorescence-guided surgery. Half of all samples showed visible fluorescence during surgery, which was associated with significant increase in PPIX fluorescence lifetime. While the PPIX lifetime was significantly different between specific tumor tissue types, the NADH lifetimes did not differ significantly among them. However, mainly necrotic areas exhibited significantly lower NADH lifetimes compared to compact tumor in HGG. Our pilot study indicates that combined fluorescence lifetime imaging of NADH/PPIX represents a sensitive tool to visualize brain tumor tissue not detectable with conventional 5-ALA fluorescence.

Functional optical coherence tomography and photoacoustic microscopy imaging for zebrafish larvae.

We present a dual modality functional optical coherence tomography and photoacoustic microscopy (OCT-PAM) system. The photoacoustic modality employs an akinetic optical sensor with a large imaging window. This imaging window enables direct reflection mode operation, and a seamless integration of optical coherence tomography (OCT) as a second imaging modality. Functional extensions to the OCT-PAM system include Doppler OCT (DOCT) and spectroscopic PAM (sPAM). This functional and non-invasive imaging system is applied to image zebrafish larvae, demonstrating its capability to extract both morphological and hemodynamic parameters in vivo in small animals, which are essential and critical in preclinical imaging for physiological, pathophysiological and drug response studies.

Towards real-time wide-field fluorescence lifetime imaging of 5-ALA labeled brain tumors with multi-tap CMOS cameras.

Fluorescence guided neurosurgery based on 5-aminolevulinic acid (5-ALA) has significantly increased maximal safe resections. Fluorescence lifetime imaging (FLIM) of 5-ALA could further boost this development by its increased sensitivity. However, neurosurgeons require real-time visual feedback which was so far limited in dual-tap CMOS camera based FLIM. By optimizing the number of phase frames required for reconstruction, we here demonstrate real-time 5-ALA FLIM of human high- and low-grade glioma with up to 12 Hz imaging rate over a wide field of view (11.0 x 11.0 mm). Compared to conventional fluorescence imaging, real-time FLIM offers enhanced contrast of weakly fluorescent tissue.

Surgical microscope with integrated fluorescence lifetime imaging for 5-aminolevulinic acid fluorescence-guided neurosurgery.

SIGNIFICANCE: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed. AIM: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging. APPROACH: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology. RESULTS: Our imaging system enabled macroscopic FLIM of a 6.5 × 6.5 mm2 field of view at spatial resolutions <20 µm. A frame of 512 × 512 pixels with a lifetime accuracy <1 ns was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology. CONCLUSIONS: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.