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Coherent Raman Scattering Fiber Endoscopy
Optics Letters
Abstract
Coherent Raman scattering methods allow for label-free imaging of tissue with chemical contrast and high spatial and temporal resolution. However, their imaging depth in scattering tissue is limited to less than 1 mm, requiring the development of endoscopes to obtain images deep inside the body. Here, we describe a coherent Raman endoscope that provides stimulated Raman scattering images at seven frames per second using a miniaturized fiber scanner, a custom-designed objective lens, and an optimized scheme for collection of scattered light from the tissue. We characterize the system and demonstrate chemical selectivity in mouse tissue images.
... Subsequently, endoscopic systems capable of generating coherent Raman signals were developed, fitting within a housing with an outer diameter of less than 1 mm (Deladurantaye et al. 2014;D. T. DePaoli et al. 2018;Saar et al. 2011). Other flexible nonlinear endoscopic systems utilize photonic crystal fibers to deliver femtosecond pulses, allowing the highly efficient generation of CARS, TPEF, and SHG in biological tissues (Ducourthial et al. 2015;Lombardini et al. 2018). ...
... Furthermore, endoscopic THG imaging has been achieved using GRIN lenses (Kuzmin et al. 2016), expanding the range of imaging modalities. The feasibility of an SRS endoscope has been demonstrated in principle (Saar et al. 2011), but further testing on biological tissue is still required. ...
Neurological disorders, including spinal cord injury, peripheral nerve injury, traumatic brain injury, and neurodegenerative diseases, pose significant challenges in terms of diagnosis, treatment, and understanding the underlying pathophysiological processes. Label-free multiphoton microscopy techniques, such as coherent Raman scattering, two-photon excited autofluo-rescence, and second and third harmonic generation microscopy, have emerged as powerful tools for visualizing nervous tissue with high resolution and without the need for exogenous labels. Coherent Raman scattering processes as well as third harmonic generation enable label-free visualization of myelin sheaths, while their combination with two-photon excited autofluorescence and second harmonic generation allows for a more comprehensive tissue visualization. They have shown promise in assessing the efficacy of therapeutic interventions and may have future applications in clinical diagnostics. In addition to multiphoton microscopy, vibrational spectroscopy methods such as infrared and Raman spectroscopy offer insights into the molecular signatures of injured nervous tissues and hold potential as diagnostic markers. This review summarizes the application of these label-free optical techniques in preclinical models and illustrates their potential in the diagnosis and treatment of neurological disorders with a special focus on injury, degeneration, and regeneration. Furthermore, it addresses current advancements and challenges for bridging the gap between research findings and their practical applications in a clinical setting.
... For image generation, the scanning can be performed at the proximal end of either a rigid endoscope or a flexible one, utilizing an imaging fiber bundle, where the spatial constraints are relaxed 6,7 . To produce small flexible scanning endoscopes, a compact scanner can be implemented at the distal end, using microelectro-mechanical systems (MEMS) as steerable mirrors or resonant fiber scanners [8][9][10][11] . The smallest CARS endoscope uses multimode gradient index fibers directly for CARS microscopy on the front surface, and scanning at the input site is achieved by using spatial light modulators 12 . ...
... The final key element of multimodal CARS endoscopes is highly corrected multi-element endomicroscopic objectives which have to be custom-designed for the particular laser source and scanner. Gradient index or GRIN lenses are frequently used because of their advantageous plane surfaces and the ability to correct aberrations with adapted index profiles 6,7,10,26,27 . ...
Multimodal non-linear microscopy combining coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excited fluorescence has proved to be a versatile and powerful tool enabling the label-free investigation of tissue structure, molecular composition, and correlation with function and disease status. For a routine medical application, the implementation of this approach into an in vivo imaging endoscope is required. However, this is a difficult task due to the requirements of a multicolour ultrashort laser delivery from a compact and robust laser source through a fiber with low losses and temporal synchronization, the efficient signal collection in epi-direction, the need for small-diameter but highly corrected endomicroobjectives of high numerical aperture and compact scanners. Here, we introduce an ultra-compact fiber-scanning endoscope platform for multimodal non-linear endomicroscopy in combination with a compact four-wave mixing based fiber laser. The heart of this fiber-scanning endoscope is an in-house custom-designed, single mode, double clad, double core pure silica fiber in combination with a 2.4 mm diameter NIR-dual-waveband corrected endomicroscopic objective of 0.55 numerical aperture and 180 µm field of view for non-linear imaging, allowing a background free, low-loss, high peak power laser delivery, and an efficient signal collection in backward direction. A linear diffractive optical grating overlays pump and Stokes laser foci across the full field of view, such that diffraction-limited performance is demonstrated for tissue imaging at one frame per second with sub-micron spatial resolution and at a high transmission of 65% from the laser to the specimen using a distal resonant fiber scanner.
... In CRS laser beams at two frequencies, defined as the pump, cO;,, and the Stokes, 0)5, are used to visualize the sample. When the difference frequency between the two laser beams is tuned to match an intrinsic molecular vibrational frequency in the sample cov/è, several non linear interactions occur: new light is generated at the anti-Stokes frequency, (Oas = 2cOp -(Os, by the CARS process, and some intensity is transferred from the pump to the Stokes beam by the SRS process (Saar et al., 2011). ...
... Both processes offer chemical selectivity and high spatial resolution, but SRS offers more desirable contrast and is free from image artifacts that plague CARS imaging (Saar et al., 2011). ...
Peptide and proteins are involved in a wide range of brain disease but they don't cross the blood brain barrier because of their hydrophilic nature and size. Nanofibrous systems are attracting increasing interest in the field of drug delivery and regenerative medicine. The aim of this work is to investigate the therapeutic applicability of peptide nanofibres as new drug delivery system to the Central Nervous System. Our working hypothesis was to choose a model hydrophilic peptide unable to enter the brain, make a lipophylic derivative from which monodomain nanofibres were constructed, in order to test them as a peptide carrier to the brain. Dalargin, a hexapeptide analogue of Leu-enkephalin, which is unable to cross the blood brain barrier was chosen as a model drug. On direct injection into the brain, Dalargin acts on brain opioid receptors, resulting in analgesia. An amphipatic derivative of dalargin, palmitoyl Dalargin (pDal) was synthesized resulting in a surfactant like peptide able to form high-axial-ratio nanostructures in aqueous environments. The self-assembly of the peptide amphilphile has been assessed experimentally and in silico. Intravenous injection of formulation of nanofibres resulted in analgesic response in mice. Brain peptide delivery was assessed with Raman microscopy as well as by measuring analgesia and peptide nanofibres pharmacokinetic profiles in biological matrices. While Dalargin was not detected in any of the tissue samples, palmitoyl Dalargin was measured in the brain tissue confirming the ability of peptide palmitoyl dalargin nanofibres to deliver the peptide across the blood brain barrier. Furthermore Raman microscopy revealed the presence of palmitoyl dalargin in the brain parenchyma. We conclude that peptide nanofibres offer a unique method for delivering hydrophilic peptides across the blood brain barrier.
... The back-emitted light from the tissue, is then captured with either the illumination fiber or using different fiber(s), and is directed to a photo detector element for image formation [4]. Such scanning endoscopic systems have been adapted to a plethora of optomedical imaging modalities, including but not limited to, optical coherence tomography (OCT), multi-photon endoscopy, confocal endoscopy and Raman spectroscopy [5][6][7][8]. Scanning fiber based devices have also been demonstrated as a display, through a modulated laser source to show the desired content to the viewer. However, previous scanning fiber displays utilize multiple optical components that makes the overall device bulky. ...
... where f x and f y are the drive frequencies for orthogonal directions and n x and n y are smallest integers satisfying Eq. (8). Lissajous scan pattern is created through driving the orthogonal electrodes of the piezoelectric actuator at different frequencies. ...
In this study, we propose a compact, lightweight scanning fiber microdisplay towards virtual and augmented reality applications. Our design that is tailored as a head-worn-display simply consists of a four-quadrant piezoelectric tube actuator through which a fiber optics cable is extended and actuated, and a reflective (or semi-reflective) ellipsoidal surface that relays the moving tip of the fiber onto the viewer’s retina. The proposed display, offers significant advantages in terms of architectural simplicity, form-factor, fabrication complexity and cost over other fiber scanner and MEMS mirror counterparts towards practical realization. We demonstrate the display of various patterns with ∼VGA resolution and further provide analytical formulas for mechanical and optical constraints to compare the performance of the proposed scanning fiber microdisplay with that of MEMS mirror-based microdisplays. Also we discuss the road steps towards improving the performance of the proposed scanning fiber microdisplay to high-definition video formats (such as HD1440), which is beyond what has been achieved by MEMS mirror based laser scanning displays.
... CRS microscopy is a powerful method for label-free nerve imaging. Recently, several groups have investigated miniature and/or flexible fiber CRS imaging systems for clinical use [22][23][24][25][26][27][28]. On the other hand, for robot-assisted surgery, inserted part into the human body should be small diameter (typically smaller than 12 mm diameter) [29], but flexibility of the inserted part is not required. ...
... The features of the developed CARS rigid endoscope are 2.91 µm spatial resolution, 650 µm diameter field of view (450 µm × 450 µm square image), low distortion and high CARS intensity uniformity. The developed rigid endoscope is not so high resolution comparing with the previously reported CRS endoscopic systems [22][23][24][25][26][27][28]. However, our system provides the largest imaging field of view. ...
Label-free visualization of nerves and nervous plexuses will improve the preservation of neurological functions in nerve-sparing robot-assisted surgery. We have developed a coherent anti-Stokes Raman scattering (CARS) rigid endoscope to distinguish nerves from other tissues during surgery. The developed endoscope, which has a tube with a diameter of 12 mm and a length of 270 mm, achieved 0.91% image distortion and 8.6% non-uniformity of CARS intensity in the whole field of view (650 μm diameter). We demonstrated CARS imaging of a rat sciatic nerve and visualization of the fine structure of nerve fibers.
... Fibre bundle implementation of the PEM also offers a path towards full endoscopic implementation, as it permits static scanning, i.e., maintaining a static position at the distal end while the pump and probe beams are sequentially scanned through the bundle cores at the proximal end 20,35 . Alternate scanning configurations based on resonant vibration of single optical fibres have been used in optical and coherent Raman endoscopic imaging applications that could also be applied for endoscopically implementing PEM 36,37 . However, it is worth noting that future applications for our fibre technology are not limited to spatially resolved imaging as demonstrated with our proof-of-concept hypodermic needle-delivered Brillouin fibre spectrometer 38 . ...
This report presents an optical fibre-based endo-microscopic imaging tool that simultaneously measures the topographic profile and 3D viscoelastic properties of biological specimens through the phenomenon of time-resolved Brillouin scattering. This uses the intrinsic viscoelasticity of the specimen as a contrast mechanism without fluorescent tags or photoacoustic contrast mechanisms. We demonstrate 2 μm lateral resolution and 320 nm axial resolution for the 3D imaging of biological cells and Caenorhabditis elegans larvae. This has enabled the first ever 3D stiffness imaging and characterisation of the C. elegans larva cuticle in-situ. A label-free, subcellular resolution, and endoscopic compatible technique that reveals structural biologically-relevant material properties of tissue could pave the way toward in-vivo elasticity-based diagnostics down to the single cell level.
... inflammatory bowl disease 9 ), the development of endoscopic devices for non-linear spectroscopic imaging has been a subject of significant interest for many years. Different approaches have been presented: besides point scanning probes 10 , the most common ones are scanning fiber endoscopes [11][12][13][14][15][16][17][18] and using galvo scanning mirrors or microelectromechanical system (MEMS) scanners [19][20][21][22][23][24] . ...
Here, we report on the development and application of a compact multi-core fiber optical probe for multimodal non-linear imaging, combining the label-free modalities of Coherent Anti-Stokes Raman Scattering, Second Harmonic Generation, and Two-Photon Excited Fluorescence. Probes of this multi-core fiber design avoid moving and voltage-carrying parts at the distal end, thus providing promising improved compatibility with clinical requirements over competing implementations. The performance characteristics of the probe are established using thin cryo-sections and artificial targets before the applicability to clinically relevant samples is evaluated using ex vivo bulk human and porcine intestine tissues. After image reconstruction to counteract the data’s inherently pixelated nature, the recorded images show high image quality and morpho-chemical conformity on the tissue level compared to multimodal non-linear images obtained with a laser-scanning microscope using a standard microscope objective. Furthermore, a simple yet effective reconstruction procedure is presented and demonstrated to yield satisfactory results. Finally, a clear pathway for further developments to facilitate a translation of the multimodal fiber probe into real-world clinical evaluation and application is outlined.
... We determined the spatial resolution of the handheld DM-CARS by imaging DMSO and tting the rst derivative of the intensity pro le across the edge with a Gaussian distribution (Fig. 6c), which determined the spatial resolution to be about 2.4 µm, as good as the previously reported 40 . To evaluate the performance of the handheld DM-CARS in eliminating the background, we performed DM-CARS imaging of the PMMA beads-TiO 2 mixture and liver tumor tissues (Fig. 6e, f). ...
Coherent anti-Stokes Raman scattering (CARS) microscopy has been demonstrated to be a powerful platform for label-free, non-invasive, and chemically specific imaging of biological samples. Yet, the non-resonant background hinders its sensitive detection of weak Raman bands. Here, we demonstrate an approach to remove the non-resonant background in CARS based on delay modulation (DM), enabled by an acousto-optic modulator and spectral focusing. We show that the DM-CARS reduces the background by 10 times and achieves a detection sensitivity of 3ⅹ10 ⁵ molecules at a time constant of 7 μs, a 100 fold improvement over normal CARS. We demonstrated the potential clinical applications of DM-CARS in tracking heavy water metabolism in bacteria for antimicrobial susceptibility testing, which is challenging using normal CARS, and a fiber-delivered handheld DM-CARS device for liver tumor tissue imaging.
... This technique can be applied to a very broad range of investigations, from the mapping of the molecular mechanisms inside a cell to the characterization of tissues in living organisms, along with the alterations associated to different pathological conditions [14]. The use of this technique has been reported for mapping the neuronal activity in a label-free manner [15,16] and in its integration with approaches of micro-endoscopy [17,18]. Research in human biopsies or human-derived tissues is becoming more frequent and taking advantage of techniques, like SRS, for characterizing structural and molecular properties. ...
Stimulated Raman Scattering (SRS) microscopy is a light-based non-linear imaging method for visualizing a molecule based on its chemical properties, i.e., the vibrational energy states reflecting the molecule’s structure and its environment. This technique, relying on the specificity of the molecule’s spectral fingerprint, enables label-free, high-sensitivity, and high-resolution 3D reconstruction of the distribution and the properties of a molecule within a tissue. Despite its tremendous potentials, the application of SRS is still not frequent in the field of life science, where it could be applied over an extremely broad investigation range, from the study of the molecular interactions at subcellular level to the characterization of tissue alterations in clinical studies. Trying to fill this gap, here, after describing the general principles of SRS, we present the materials and the methods to integrate spectrally focused Stimulated Raman Spectroscopy (sf-SRS) on commercial multiphoton microscopes and highlight the critical aspects to consider.
... Az első CE-jelzésű multifoton rendszer a DermaInspect volt 2003-ban (JenLab GmbH, Germany), mely TPEF és SHG képalkotásra képes (71 (72). A száloptikás technika nagy előnye, hogy a korábban asztalhoz rögzített bonyolult tükör és lencserendszerek helyett hajlékony optikai szálakon keresztül is vezethető a lézernyaláb és a visszaérkező optikai információ, mely lehetővé teszi a betegágy melletti vizsgálatot, és akár az endoscopos jellegű felhasználást is (73). 2010-ben a JenLab megalkotta a flexibilis vizsgálófejjel rendelkező MPTflex multifoton tomográfiás eszközt, majd a közelmúltban bemutatásra került a CARS képalkotásra is alkalmas MPTflex-CARS rendszer (61). ...
As a result of advances in the development of medical imaging, the toolbox of dermatological imaging in the XXI. century expanded considerably. In addition to the histological examination that is currently the gold standard in diagnostics, a number of noninvasive imaging methods have emerged in recent decades that have become part of the clinical and preclinical practice. The characteristics of many dermatological diseases have been described in vivo studies with commercially available devices. The authors summarize the principles of terahertz imaging, photoacoustic imaging, reflectance and autofluorescence imaging, near-infrared spectroscopy, and nonlinear microscopy, as well as the in vivo dermatological results and clinical applications of these modalities.
... Of course, an additional hurdle for true PK quantification of API concentrations using the S 4 RS system is the need for calibration methods to convert relative or semi-quantitative measurements into absolute quantitative measurements which could be benchmarked against other methodologies. Additionally, this technology is not limited to only cutaneous pharmacokinetic investigations but may also be extended to the study of local target-site concentrations through the use of endoscopic procedures [56]. ...
Stimulated Raman scattering (SRS) is a nondestructive and rapid technique for imaging of biological and clinical specimens with label-free chemical specificity. SRS spectral imaging is typically carried out either via broadband methods, or by tuning narrowband ultrafast light sources over narrow spectral ranges thus specifically targeting vibrational frequencies. We demonstrate a multi-window sparse spectral sampling SRS (S⁴RS) approach where a rapidly-tunable dual-output all-fiber optical parametric oscillator is tuned into specific vibrational modes across more than 1400 cm⁻¹ during imaging. This approach is capable of collecting SRS hyperspectral images either by scanning a full spectrum or by rapidly tuning into select target frequencies, hands-free and automatically, across the fingerprint, silent, and high wavenumber windows of the Raman spectrum. We further apply computational techniques for spectral decomposition and feature selection to identify a sparse subset of Raman frequencies capable of sample discrimination. Here we have applied this novel method to monitor spatiotemporal dynamic changes of active pharmaceutical ingredients in skin, which has particular relevance to topical drug product delivery.
... Endomicroscopy using coherent Raman scattering (CRS), such as coherent anti-Stokes Raman scattering (CARS) [11,12] and stimulated Raman scattering (SRS) [13,14], has been reported [15][16][17][18][19][20][21][22][23]. CARS and SRS provide 10 7 times higher efficiency than spontaneous Raman scattering [24] and can achieve imaging at video frame rates [25][26][27][28]. ...
We propose and demonstrate a method of suppressing four-wave mixing (FWM) in an optical fiber bundle to realize coherent anti-Stokes Raman scattering (CARS) endomicroscopy, which is the leading candidate for a definitive diagnosis of gastrointestinal cancer. Two excitation laser beams with different wavelengths are delivered via different cores to suppress FWM and are then combined with a polarization prism and a dual-wavelength wave plate and are focused to a spot. The background emission from the optical fiber bundle was suppressed to 1/3289, and we demonstrated CARS imaging of a polystyrene bead using the proposed method.
... These findings suggest that SRS microscopy can generate high-quality histological images for clinical diagnosis without the need for tissue fixation, sectioning, or staining. Noteworthy, with the recent advances of CARS and SRS endoscopy [112][113][114], further improvements to these Raman-based systems would open up exciting possibilities for in vivo, label-free, and non-invasive histopathological imaging and clinical diagnostics in the near future. ...
Simple Summary
Raman spectroscopy and imaging are label-free, non-destructive techniques to study cellular metabolism with subcellular spatial resolution. This review focuses on applications of Raman-based methods in a combination of stable isotope probing on cancer metabolism and cancer imaging.
Abstract
Metabolic reprogramming is a common hallmark in cancer. The high complexity and heterogeneity in cancer render it challenging for scientists to study cancer metabolism. Despite the recent advances in single-cell metabolomics based on mass spectrometry, the analysis of metabolites is still a destructive process, thus limiting in vivo investigations. Being label-free and nonperturbative, Raman spectroscopy offers intrinsic information for elucidating active biochemical processes at subcellular level. This review summarizes recent applications of Raman-based techniques, including spontaneous Raman spectroscopy and imaging, coherent Raman imaging, and Raman-stable isotope probing, in contribution to the molecular understanding of the complex biological processes in the disease. In addition, this review discusses possible future directions of Raman-based technologies in cancer research.
... Using devices similar to the coherent Raman scattering endoscopes 57 , we envision that DO-SRS, with its ability to image D-labeled macromolecules in living animals ( Figure 1D), could be applied to visualize metabolic patterns of internal organs, cortical metabolism for brain activities, and tumor metabolism for cancer progression through optical biopsy. Importantly, the sensitivity of this method is high enough to operate in the range of low D2O enrichment that is safe for humans. ...
Direct visualization of metabolic dynamics in living tissues with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines d euterium o xide (D 2 O) probing with s timulated R aman s cattering microscopy (DO-SRS) to image in situ metabolic activities. Enzymatic incorporation of D 2 O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ . Within the broad vibrational spectra of C-D bonds, we discovered lipid-, protein-, and DNA-specific Raman shifts and developed spectral unmixing methods to obtain C-D signals with macromolecular selectivity. DO-SRS enabled us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.
... However, the detection scheme in the report used a 10 × 10 mm photodiode with a hole drilled in the center, through which the excitation lasers were focused. A year later Saar et al. 103 presented a scanning-fiberendoscope version of the system using ∼130 mW total power for excitation and the same detection apparatus; in vivo work using the device has not been presented since. In 2018, building on earlier work in delay-line tuning, Liao et al. 88,104 presented a handheld hyperspectral SRS microscope capable of HWN spectroscopic images (15 cm −1 spectral resolution) on the order of 3 s [ Fig. 3(b)]. ...
Significance:
Although the clinical potential for Raman spectroscopy (RS) has been anticipated for decades, it has only recently been used in neurosurgery. Still, few devices have succeeded in making their way into the operating room. With recent technological advancements, however, vibrational sensing is poised to be a revolutionary tool for neurosurgeons.
Aim:
We give a summary of neurosurgical workflows and key translational milestones of RS in clinical use and provide the optics and data science background required to implement such devices.
Approach:
We performed an extensive review of the literature, with a specific emphasis on research that aims to build Raman systems suited for a neurosurgical setting.
Results:
The main translatable interest in Raman sensing rests in its capacity to yield label-free molecular information from tissue intraoperatively. Systems that have proven usable in the clinical setting are ergonomic, have a short integration time, and can acquire high-quality signal even in suboptimal conditions. Moreover, because of the complex microenvironment of brain tissue, data analysis is now recognized as a critical step in achieving high performance Raman-based sensing.
Conclusions:
The next generation of Raman-based devices are making their way into operating rooms and their clinical translation requires close collaboration between physicians, engineers, and data scientists.
... The endoscope is used to help diagnose the symptom in the stomach and intestines [1]. Researchers and doctors are always trying to find more effective and less invasive techniques to benefit patients for the endoscope [2][3][4]. Therefore, the large field of view (FOV), high resolution and complete pictures will save time of the doctor's examination process and improve the accuracy of judgment [5][6]. Endoscopic imaging system have been reported in previous literature [7][8][9][10], the common feature of which was the single-view function. ...
... Commercial CE-certified medical CARS imaging systems are already available 46 . Furthermore, CARS endoscopic solutions are currently beeing developed [47][48][49] . Inherently, any optical based bioanalytical imaging tool is limited to the field of view and penetration depth in tissue is small. ...
Human peripheral nerves hold the potential to regenerate after injuries; however, whether a successful axonal regrowth was achieved can be elucidated only months after injury by assessing function. The axolotl salamander is a regenerative model where nerves always regenerate quickly and fully after all types of injury. Here, de- and regeneration of the axolotl sciatic nerve were investigated in a single and double injury model by label-free multiphoton imaging in comparison to functional recovery. We used coherent anti-Stokes Raman scattering to visualize myelin fragmentation and axonal regeneration. The presence of axons at the lesion site corresponded to onset of functional recovery in both lesion models. In addition, we detected axonal regrowth later in the double injury model in agreement with a higher severity of injury. Moreover, endogenous two-photon excited fluorescence visualized macrophages and revealed a similar timecourse of inflammation in both injury models, which did not correlate with functional recovery. Finally, using the same techniques, axonal structure and status of myelin were visualized in vivo after sciatic nerve injury. Label-free imaging is a new experimental approach that provides mechanistic insights in animal models, with the potential to be used in the future for investigation of regeneration after nerve injuries in humans.
... 11 Scanning single fiber endoscopes have also been designed, incorporating the fiber inside a piezoelectric tube that expands and contracts at the resonant frequency, creating an expanding circular motion. [12][13][14] For this preliminary scientific study, however, the former approach (e.g., solenoid and magnetic bead on fiber) was adapted on a smaller scale to design, construct, and test a vibrating fiber for potential use in stone dusting during laser lithotripsy. ...
Our preliminary study investigates an automated, vibrating fiber optic tip for dusting of kidney stones during thulium fiber laser (TFL) lithotripsy. A (0.75-mm diameter and 5-mm length) magnetic bead was attached to the fiber jacket, centered 2 cm from distal fiber tip. A solenoid was placed parallel to the fiber with a 0.5-mm gap between solenoid and magnetic bead on fiber. The solenoid was used to create a magnetic force on the bead, inducing fiber vibration. Calibration tests for fiber motion in both air and water were performed. The ablation crater characteristics (surface area, volume, depth, and major/minor axis) of uric acid stones were measured using optical coherence tomography, after delivery of 1500 TFL pulses at 1908 nm, 33 mJ, 500 μs, and up to 300 Hz, through 50-, 100-, and 150-μm-core fibers. The resonant frequency was dependent on fiber diameter and rigidity, with a cutoff pivot point for optimum vibration amplitude at 4 cm. Maximum fiber displacement is about 1 mm in water and 4 mm in air. For 50-, 100-, and 150-μm-core fibers, ablated surface area averaged 1.7, 1.7, and 2.8 times greater with vibrating fiber than fixed fiber, respectively. For these fibers, ablation volume averaged 1.1, 1.5, and 1.1 times greater with vibrating fiber than fixed fiber, given a fixed energy per pulse, respectively. Our preliminary study demonstrates the functionality of an automated, vibrating fiber system for stone "dusting," with significantly larger surface area but similar ablation volumes as a fixed fiber. Future studies will focus on optimization of fiber parameters (especially displacement) and miniaturization of system components to facilitate integration into ureteroscopes. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
... A major challenge for in vivo metabolic imaging is the accessibility of tissues deep inside the body. Using devices similar to the coherent Raman scattering endoscopes 58 , we envision that DO-SRS microscopy could be applied to visualize metabolic patterns of internal organs and to study tumor metabolism through optical biopsy. The recent development of high-speed, volumetric stimulated Raman projection microscopy and tomography 59 also offers promise in deep-tissue, large-volume, in vivo imaging (e.g., imaging cortical metabolism). ...
... Brustlein et al. 10 showed efficient pulse delivery and coherent Raman signal collection from organic crystals using a single double-clad PCF in combination with a microscope after the fiber, to create an "endoscope-like" system with minimal FWM. Saar et al. 11 carried out coherent Raman imaging using 1 m of polarization-maintaining singlemode silica fibers for pulse delivery in combination with their in-house scanning fiber endoscope (SFE) probe. However, the detection scheme in the backward direction was not ideal, using a 10 mm × 10 mm photodiode with a hole cut in the middle for probe insertion. ...
Coherent Raman fiber probes have not yet found their way into the clinic despite their immense potential for label-free sensing and imaging. This is mainly due to the traditional bulky laser systems required to create the high peak power laser pulses needed for coherent Raman, as well as the complications that arise from the propagation of this type of energy through silica. Specifically, a coherent anti-Stokes Raman scattering (CARS) probe that could select its integration volume at high resolution, away from the tip of the fiber, is particularly interesting in the case of electrode implantation neurosurgeries, wherein it is possible to place optical fibers on-board the chronic electrode and provide optical guidance during its implantation, through the semi-transparent tip. To this clinical end, we have created an all fiber CARS system, consisting of small, rapidly tunable, turn-key fiber-lasers, capable of creating high wavenumber CARS spectra on the order of tens-of-milliseconds. The use of traditional silica fibers is made possible by the use of the laser's long pulse-widths (25 ps). The probe itself has an outer diameter of
250
μ
m
allowing it to fit within commercially available metal tubes that can replace deep brain stimulation (DBS) stylets. Using this system, we identified brain tissue types in intact nonhuman primates' brains and showed the ability to delineate white and gray matters with high resolution. Its advantages over spontaneous Raman stem from the orders of magnitude improvement in spatial resolution, its inherent translatability to three-dimensional (3-D) imaging, as well as the theoretical ability to remove parasitic Raman signal from probe encasements, such as a DBS electrode. The system is planned to have clinical implications in neurosurgical guidance as well as diseased tissue detection.
... The first rigid coherent Raman endoscope was published by Saar and co-workers. 30 In this case, a piezo scanner was used for scanning the proximal side of a focusing GRIN lens. In this manner, spatial resolutions of roughly 2 µm were achieved with an 80 µm diameter FoV. ...
Nonlinear optical endoscopy is an attractive technique for biomedical imaging since it promises to give access to high resolution imaging in vivo. Among the various techniques used for endoscopic contrast generation, coherent anti-Stokes Raman scattering (CARS) is especially interesting. CARS endoscopy allows molecule specific imaging of unlabeled samples. In this contribution, we describe the design, implementation, and experimental characterization of a rigid, compact CARS endoscope with a spatial resolution of 750 nm over a field of view of roughly 250 μm. Omission of the relay optics and use of a gradient index lens specifically designed for this application allow one to realize these specifications in an endoscopic unit which is 2.2 mm wide over a length of 187 mm, making clinical applications during surgical interventions possible. Multimodal use of the endoscope is demonstrated with images of samples with neurosurgical relevance.
... A major challenge for in vivo metabolic imaging is the accessibility of tissues deep inside the body. Using devices similar to the coherent Raman scattering endoscopes 58 , we envision that DO-SRS microscopy could be applied to visualize metabolic patterns of internal organs and to study tumor metabolism through optical biopsy. The recent development of high-speed, volumetric stimulated Raman projection microscopy and tomography 59 also offers promise in deep-tissue, large-volume, in vivo imaging (e.g., imaging cortical metabolism). ...
Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D2O-derived deuterium into macromolecules generates carbon-deuterium (C-D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C-D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C-D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.
... Therefore, the detector is preferred to be placed close to the sample for signal detection in an SRS endoscope. 157 Notably, SRS imaging can operate under ambient light, which is compatible with clinical applications. ...
Chemical imaging offers critical information to understand the fundamentals in biology and to assist clinical diagnostics. Label-free chemical imaging piques a general interest since it avoids the use of bio-perturbing molecular labels and holds promises to characterize human tissue in vivo. Coherent Raman scattering (CRS), which utilizes lasers to excite the vibrations of molecules, renders new modalities to map chemicals in living samples without the need of labeling and provides significantly improved speed, resolution, and sensitivity compared to spontaneous Raman scattering. Although microscopy systems based on CRS have seen rapid development in the past two decades, remaining challenges, which emerge in diverse aspects, start to impede the continuous advancement of the field. In this perspective, we review the history of CRS microscopy, scrutinize the pros and cons of different modalities, and discuss the current challenges and possible future directions of the field. Infiltration of conceptual and technological ideals from other fields will promote CRS microscopy towards a versatile tool for basic science and medical research.
... Performing CARS and/or SRS imaging via a fiber endoscope system is a crucial step towards imaging deep inside tissues and providing chemical imaging with a spatial resolution that is inaccessible using current fibered Raman probes 7,8 . Despite significant efforts over the last decade 9,10,11,12,13,14,15 , major technical challenges for pulsed laser delivery and signal collection have hindered the development of coherent Raman endoscopy. CARS/SRS endoscopy is more demanding than two-photon excited fluorescence (TPEF) or second harmonic generation (SHG) 16,17,18 as two spatially and temporally overlapping excitation beams are required. ...
A powerful technique called coherent Raman scattering microscopy could be used to visualize and study tissue inside the body thanks to a new light-transmitting fiber technology. Coherent Raman scattering generates images based on the interaction of light with molecules. It opens a window into cells and tissues that reveals information unavailable with simple illumination by light. Applying the technique deep within the body has proved difficult due to technical problems in delivering the necessary ultra-short pulses of light, and collecting the signals that are scattered back. Researchers in France and Germany, led by Hervé Rigneault at the Fresnel Institute in France, combined several innovations in fiber technology to bring high-resolution coherent Raman imaging into previously hidden locations. Their flexible endoscope literally shines new light to assist the diagnosis and surgical treatment of cancer and other diseases.
... 22 This nonspecific background overwhelms the relatively weak CRS signals and degrades the chemical selectivity of CRS imaging tremendously. 23 There has been a great effort in suppressing the background signals for CRS handheld microscopes and endoscopes. ...
Spectroscopic stimulated Raman scattering (SRS) microscopy is a label-free technique that generates chemical maps of live cells or tissues. A handheld SRS imaging system using an optical fiber for laser delivery will further enable in situ and in vivo compositional analysis for applications such as medical diagnosis and surgical guidance. In fiber-delivered SRS, the interaction of two ultrashort pulses in the confined mode area creates a significant background that overwhelms the stimulated Raman signal from a sample. Here, we report the first background-free fiber-delivered handheld SRS microscope for in situ chemical imaging. By temporally separating the two ultrafast pulses propagating in the fiber and then overlapping them on a sample through a highly dispersive material, we detected a stimulated Raman signal that is 200 times weaker than the background induced by fiber. Broad applications of the handheld SRS microscope were demonstrated through in situ ambient-light chemical mapping of pesticide on a spinach leaf, cancerous tissue versus healthy brain tissue in a canine model, and cosmetic distribution on live human skin. A lab-built objective lens further reduced the size of the pen-shaped microscope to about one centimeter in diameter.
... In the past decades, increasing attention has addressed developing minimized optical fiber-based CARS imaging probes for direct in vivo applications at clinical settings. [67][68][69][70] Our group has been studying the mechanisms of optical fiber delivered CARS, [71][72][73][74] and recently reported a fiber-based miniaturized endomicroscope probe design that incorporates a small lens part with microelectro-mechanical systems. 75 This device has opened up the possibility of applying CARS in vivo to provide reliable architectural and cellular information without the need for invasive and sometimes repetitive tissue removal. ...
Lung cancer is the most prevalent type of cancer and the leading cause of cancer-related deaths worldwide. Coherent anti-Stokes Raman scattering (CARS) is capable of providing cellular-level images and resolving pathologically related features on human lung tissues. However, conventional means of analyzing CARS images requires extensive image processing, feature engineering, and human intervention. This study demonstrates the feasibility of applying a deep learning algorithm to automatically differentiate normal and cancerous lung tissue images acquired by CARS. We leverage the features learned by pretrained deep neural networks and retrain the model using CARS images as the input. We achieve 89.2% accuracy in classifying normal, small-cell carcinoma, adenocarcinoma, and squamous cell carcinoma lung images. This computational method is a step toward on-the-spot diagnosis of lung cancer and can be further strengthened by the efforts aimed at miniaturizing the CARS technique for fiber-based microendoscopic imaging. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
... 97 Coherent Raman methods could easily provide sufficient signal levels for this application. Narrowband CRI approaches have been implemented in fiber, [231][232][233] so could be adapted to needle biopsy, but are probably not suited for this application since disease detection would be based strictly on spectra, and thus benefit greatly from sensitivity to multiple peaks in the fingerprint spectral region. Current broadband spectroscopic CRI approaches based on impulsive excitation or spectral focusing will be difficult to implement in a fiber delivery, but other approaches may be possible in the future. ...
Histopathology plays a central role in diagnosis of many diseases including solid cancers. Efforts are underway to transform this subjective art form to an objective and quantitative science. Coherent Raman imaging (CRI), a label-free imaging modality with sub-cellular spatial resolution and molecule-specific contrast possesses characteristics which could support the qualitative-to-quantitative transition of histopathology. In this work we briefly survey major themes related to modernization of histopathology, review applications of CRI to histopathology and, finally, discuss potential roles for CRI in the transformation of histopathology that is already underway.
... Additionally, Raman spectrum by photo-excitation in the near-infrared region has little spectral interference from water, which provides excellent advantages for its endoscopic application ( Figure 3). For example, CARS endoscopy utilizes an NIR laser-line, such as 1064 nm of picosecond pulses, which enables quantitative chemical analysis of major cellular components with Raman spectra arising from water, lipid and proteins (27)(28)(29). Most continuous wave-based Raman endoscopies utilize a relatively shorter NIR laserline (e.g. ...
In this review, we assessed endoscopic imaging using surface-enhanced Raman scattering (SERS). As white-light endoscopy, the current standard for gastrointestinal endoscopy, is limited to morphology, Raman endoscopy using surface-enhanced Raman scattering nanoparticles (SERS endoscopy) was introduced as one of the novel functional modalities. SERS endoscopy has multiplex capability and high sensitivity with low autofluorescence and photobleaching. As a result, multiple molecular characteristics of the lesion can be accurately evaluated in real time while performing endoscopy using SERS probes and appropriate instrumentation. Especially, recently developed dual modality of fluorescence and SERS endoscopy offers easy localization with identification of multiple target molecules. For clinical use of SERS endoscopy in the future, problems of limited field of view and cytotoxicity should be addressed by fusion imaging, topical administration, and non-toxic coating of nanoparticles. We expect SERS endoscopic imaging would be an essential endoscopic technique for diagnosis of cancerous lesions, assessment of resection margins and evaluation of therapeutic responses.
... Performing CARS and/or SRS imaging in a fiber endoscope system is a crucial step to image deep inside tissues and provide information that is inaccessible with current microscopy tools. Despite significant efforts over the last decade 7,8,9,10,11,12,13 , major technical challenges in the pulsed laser delivery and signal collection have hindered the development of coherent Raman endoscopy. CARS/SRS endoscopy is more demanding than 2-photon or second harmonic generation (SHG) 14,15 as two spatially and temporally overlapping excitation beams are required. ...
Coherent Raman scattering microscopy is a fast, label-free and chemically specific imaging technique that shows high potential for future in-vivo optical histology. However, the imaging depth in tissues is limited to the sub-millimeter range because of absorption and scattering. Realization of coherent Raman imaging using a fiber endoscope system is a crucial step towards imaging deep inside living tissues and providing information that is inaccessible with current microscopy tools. Until now, the development of coherent Raman endoscopy has been hampered by several issues, mainly related to the fiber delivery of the excitation pulses and signal collection. Here, we present a flexible, compact, coherent Raman and multimodal nonlinear endoscope (4.2 mm outer diameter, 71 mm rigid length) based on a resonantly scanned hollow-core Kagomé-lattice double-clad fiber. The fiber design enables distortion-less, background-free delivery of femtosecond excitation pulses and back-collection of nonlinear signals through the same fiber. Sub-micrometer spatial resolution over a large field of view is obtained by combination of a miniature objective lens with a silica microsphere lens inserted into the fiber core. We demonstrate high-resolution, high-contrast coherent anti-Stokes Raman scattering and second harmonic generation endoscopic imaging of biological tissues over a field of view of 320 µm at a rate of 0.8 frames per second. These results pave the way for intra-operative label-free imaging applied to real-time histopathology diagnosis and surgery guidance.
... Electrostatic [6][7][8][9], electrothermal [10,11], electromagnetic [12] and many other kinds of actuation mechanisms are utilized to drive a mirror plate to perform tipping, tilting or piston motion. For forward-view imaging, the dominating method should be piezo microactuators [13][14][15][16][17][18] vibrating the free end of an tiny optical fiber to project a range of patterns, such as Lissajour, spiral or raster, on the imaging target. However most of endoscopic optical probes aforementioned have needle shapes indicating their long rigid parts as long as 40 mm. ...
In this article we present a miniaturized two dimensional optical scanner for endoscopic imaging applications. The optical scanner is based on a hybrid actuation, which is composed of a planar waveguide (PWG)beam deflector for the fast-axis and an electrothermally actuated flexible printed circuit beam deflector for the slow-axis. The PWG beam deflector utilizing a microheater prism array to rapidly modulate the refractive index of three layers of the PWG is based on the thermo-optic effect. Maximum optical deflection angles of 8.6° for the PWG and 12.4° for the FPC beam deflectors are experimentally achieved. 1.2 Hz two dimensional imaging rate is tested. By assembling two beam deflectors together as well as two rod lenses, an optical scanner highlights small outline dimensions and low drive voltages, which makes its feasibility for endoscopic imaging purposes.
Coherent anti‐Stokes Raman scattering (CARS) microscopy has demonstrated a powerful platform for label‐free, non‐invasive, and chemically specific imaging of biological samples. However, the non‐resonant background limits its ability to detect weak Raman signals. Here, an approach to eliminate the non‐resonant background in CARS using delay modulation (DM) is presented, which is enabled by an acousto‐optic modulator and spectral focusing. The results demonstrate that DM‐CARS reduces the non‐resonant background by 10 times and improves detection sensitivity by 100‐fold over normal CARS. It also shows that DM‐CARS has potential applications in tracking heavy water metabolism in bacteria for antimicrobial susceptibility testing and imaging liver tumor tissues. Furthermore, a handheld DM‐CARS device has also demonstrated for in vivo imaging, which can be beneficial in various applications.
Hematoxylin and eosin (H&E) staining, the century-old technique, has been the gold standard tool for pathologists to detect anomalies in tissues and diseases such as cancer. H&E staining is a cumbersome, time-consuming process that delays and wastes precious minutes during an intraoperative diagnosis. However, even in the modern era, real-time label-free imaging techniques such as simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy have delivered several more layers of information to characterize a tissue with high precision. Still, they have yet to translate to the clinic. The slow translation rate can be attributed to the lack of direct comparisons between the old and new techniques. Our approach to solving this problem is to: 1) reduce dimensions by pre-sectioning the tissue in 500 μm slices, and 2) produce fiducial laser markings which appear in both SLAM and histological imaging. High peak-power femtosecond laser pulses enable ablation in a controlled and contained manner. We perform laser marking on a grid of points encompassing the SLAM region of interest. We optimize laser power, numerical aperture, and timing to produce axially extended marking, hence multilayered fiducial markers, with minimal damage to the surrounding tissues. We performed this co-registration over an area of 3x3 mm2 of freshly excised mouse kidney and intestine, followed by standard H&E staining. Reduced dimensionality and the use of laser markings provided a comparison of the old and new techniques, giving a wealth of correlative information and elevating the potential of translating nonlinear microscopy to the clinic for rapid pathological assessment.
Deuterium labeling has been widely used for SRS metabolic imaging. It is the minimum label that causes little perturbation to the biochemical property of target molecules. In this chapter, we introduce the applications of deuterium-probed SRS metabolic imaging in living organisms. We first review recent developments of two SRS imaging techniques that enable simultaneous visualization of the metabolic dynamics of a variety of biomolecules (lipids, protein, and DNA) in living organisms. One novel technique uses D2O as the deuterium source and combines it with SRS (DO-SRS) microscopy for metabolic imaging. The other uses deuterated glucose for visualizing various newly synthesized biomolecules by spectral tracing of deuterium (STRIDE) in the living organism. Next, we overview a volumetric tissue clearing-enhanced SRS imaging technique that increases imaging depth by 10-fold. At last, we review the applications of SRS for imaging protein metabolic dynamics in mouse tissues and organs, by in vivo intracarotid injection of the deuterated amino acid (D-AA).
Medical imaging plays an indispensable role not only in diagnostics but also in guiding and monitoring treatments. Currently deployed techniques in this context are either inapplicable or insufficient across all patient cohorts and medical conditions. While significant advances in optical imaging have been made academically, they have also primarily remained confined to academic institutions. Through this chapter, we aim to encourage clinical transition of novel spectroscopic imaging methods, particularly label-free nonlinear techniques, which could address several challenges in the field. An overview of the currently applied medical imaging techniques is presented, followed by a focused description of optical spectroscopic imaging modalities that are likely to migrate to clinical intraoperative use in the short term to midterm.
Clinical medicine continues to seek novel rapid non-invasive tools capable of providing greater insight into disease progression and management. Raman scattering based technologies constitute a set of tools under continuing development to address outstanding challenges spanning diagnostic medicine, surgical guidance, therapeutic monitoring, and histopathology. Here we review the mechanisms and clinical applications of Raman scattering, specifically focusing on high-speed imaging methods that can provide spatial context for translational biomedical applications.
Coherent Raman scattering (CRS) processes, including both the coherent anti-Stokes Raman scattering and stimulated Raman scattering, have been utilized in state-of-the-art microscopy platforms for chemical imaging of biological samples. The key advantage of CRS microscopy over fluorescence microscopy is label-free, which is an attractive characteristic for modern biological and medical sciences. Besides, CRS has other advantages such as higher selectivity to metabolites, no photobleaching, and narrow peak width. These features have brought fast-growing attention to CRS microscopy in biological research. In this review article, we will first briefly introduce the history of CRS microscopy, and then explain the theoretical background of the CRS processes in detail using the classical approach. Next, we will cover major instrumentation techniques of CRS microscopy. Finally, we will enumerate examples of recent applications of CRS imaging in biological and medical sciences.
Background
Label-free multiphoton microscopy has been suggested for intraoperative recognition and delineation of brain tumors. For any future clinical application, appropriate approaches for image acquisition and analysis have to be developed. Moreover, an evaluation of the reliability of the approach, taking into account inter- and intrapatient variability, is needed.
Methods
Coherent anti Stokes Raman scattering (CARS), two photon excited fluorescence (TPEF) and second harmonic generation (SHG) were acquired on cryosections of brain tumors of 382 patients and 28 human non-tumor brain samples. Texture parameters of those images were calculated and used as input for linear discriminant analysis.
Results
The combined analysis of texture parameters of the CARS and TPEF signal proved to be most suited for the discrimination of non-tumor brain versus brain tumors (low and high grade astrocytoma, oligodendroglioma, glioblastoma, recurrent glioblastoma, brain metastases of lung, colon, renal and breast cancer and of malignant melanoma) leading to a correct rate of 96% (sensitivity: 96%, specificity: 100%). To approximate the clinical setting, the results were validated on 42 fresh, unfixed tumor biopsies. 82% of the tumors and, most important, all of the non-tumor samples were correctly recognized. An image resolution of 1 µm was sufficient to distinguish brain tumors and non-tumor brain. Moreover, the vast majority of single fields of view of each patient’s sample were correctly classified with high probabilities, which is important for clinical translation.
Conclusions
Label-free multiphoton imaging might allow fast and accurate intraoperative delineation of primary and secondary brain tumors in combination with endoscopic systems.
Optical microscopy is a standard tool to study biological objects. Diffraction limits the achievable lateral resolution of an optical microscope to around 200 nm restricting its applicability. However, recent advances in optical super-resolution techniques have shown that the diffraction does not impose a fundamental limit to resolution and can be circumvented. These super-resolution techniques can provide resolution approaching the nanometer scale and are enabling studies of biological objects with unprecedented capabilities. However, most of the super-resolution techniques are based on using fluorescent dyes, complicating their use in medical research and applications. Therefore, a need exists for label-free super-resolution techniques, which could be especially valuable for clinical applications. To answer this need, several such techniques have been recently developed. In this chapter, we introduce the reader to some of these techniques, discuss their applications, and of the possible future directions. In order to limit the scope of this vastly expanding topic, we focus on techniques based on structured illumination and intrinsic nonlinear responses of materials.
This book presents the advances in super-resolution microscopy in physics and biomedical optics for nanoscale imaging. In the last decade, super-resolved fluorescence imaging has opened new horizons in improving the resolution of optical microscopes far beyond the classical diffraction limit, leading to the Nobel Prize in Chemistry in 2014. This book represents the first comprehensive review of a different type of super-resolved microscopy, which does not rely on using fluorescent markers. Such label-free super-resolution microscopy enables potentially even broader applications in life sciences and nanoscale imaging, but is much more challenging and it is based on different physical concepts and approaches. A unique feature of this book is that it combines insights into mechanisms of label-free super-resolution with a vast range of applications from fast imaging of living cells to inorganic nanostructures. This book can be used by researchers in biological and medical physics. Due to its logically organizational structure, it can be also used as a teaching tool in graduate and upper-division undergraduate-level courses devoted to super-resolved microscopy, nanoscale imaging, microscopy instrumentation, and biomedical imaging.
We present a compact dual-view endoscope objective lens with a field of view (FOV) of ±80°and F/# of 3.4. The endoscope consists of two optical configurations for increasing FOV within the volume constraint. The front view configuration is a fisheye lens with a FOV of ±55°, and the side view configuration is a panoramic annular lens that covers the remaining FOV. The two configurations are combined by a hybrid lens that consists of center refractive portion and side catadioptric portion. Both the front and rear surfaces of the hybrid lens are aspherized with the use of annularly stitched Q-type aspheres. Thus, a compact endoscope is successfully implemented with fewer lenses, with a total length of 11.5 mm and a maximum diameter of 5.5 mm. The modulation transfer function at 167 lp/mm is above 0.4 over the entire FOV. The relative illumination is more than 0.65 and the optical distortion is within 10%. Moreover, the near telecentric condition is fulfilled and supports constant magnification focusing.
Nonlinear multimodal microscopy offers a series of label‐free techniques with potential for intraoperative identification of tumor borders in situ using novel endoscopic devices. Here, we combined coherent anti‐Stokes Raman scattering (CARS), two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to analyze biopsies of different human brain tumors, with the aim to understand whether the morphological information carried by single field‐of‐view images, similar to what delivered by present endoscopic systems, is sufficient for tumor recognition. We imaged 40 human biopsies of high and low grade glioma, meningioma, as well as brain metastases of melanoma, breast, lung and renal carcinoma, in comparison with normal brain parenchyma. Furthermore, five biopsies of schwannoma were analyzed and compared with non‐pathological nerve tissue. Besides the high cellularity, the typical features of tumor, which were identified and quantified, are intracellular and extracellular lipid droplets, aberrant vessels, extracellular matrix collagen and diffuse TPEF. Each tumor type displayed a particular morphochemistry characterized by specific patterns of the above mentioned features. Nonlinear multimodal microscopy performed on fresh unprocessed biopsies confirmed that the technique has the ability to visualize tumor structures and discern normal from neoplastic tissue likewise in conditions close to in situ.
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Cells and tissues often display pronounced spatial and dynamical metabolic heterogeneity. Common glucose-imaging techniques report glucose uptake or catabolism activity, yet do not trace the functional utilization of glucose-derived anabolic products. Here we report a microscopy technique for the optical imaging, via the spectral tracing of deuterium (STRIDE), of diverse macromolecules derived from glucose. Based on stimulated Raman-scattering imaging, STRIDE visualizes the metabolic dynamics of newly synthesized macromolecules, such as DNA, protein, lipids and glycogen, via the enrichment and distinct spectra of carbon–deuterium bonds transferred from the deuterated glucose precursor. STRIDE can also use spectral differences derived from different glucose isotopologues to visualize temporally separated glucose populations using a pulse–chase protocol. We also show that STRIDE can be used to image glucose metabolism in many mouse tissues, including tumours, brain, intestine and liver, at a detection limit of 10 mM of carbon–deuterium bonds. STRIDE provides a high-resolution and chemically informative assessment of glucose anabolic utilization.
Multiphoton microscopy of cellular autofluorescence and second harmonic generation from collagen facilitates imaging of living cells and tissues without the need for additional fluorescent labels. Here, a compact multiphoton endomicroscope for label‐free in vivo imaging in small animals via side‐viewing needle objectives is presented. Minimal invasive imaging at cellular resolution is performed in colonoscopy of mice without surgical measures and without fluorescent dyes as a contrast agent. The colon mucosa is imaged repeatedly in the same animal in a mouse model of acute intestinal inflammation to study the process of inflammation at the tissue level within a time period of ten days, demonstrating the capabilities of label‐free endomicroscopy for longitudinal studies for the first time.
We present a simple technique that significantly enhances the interaction of pump pulses with a supercontinuum Stokes generated by a particular nonlinear fiber for time-gated experiments such as coherent anti-Stokes Raman scattering (CARS). The enhancement is achieved through a synchronized power-tuning/time delay scheme that we call spectral surfing. In this Letter, we introduce spectral surfing and demonstrate how its application to an economical CARS hypermicroscopy scheme increases the brightness, contrast, and spectral scanning range, while potentially reducing sample light exposure.
A stimulated Raman scattering microscope with near-infrared picosecond laser pulses at high repetition rates (76 MHz) and radio-frequency lock-in detection is accomplished. Based on stimulated Raman loss detection, we demonstrate noninvasive point-by-point vibrational mapping of chemical and biological samples with high sensitivity and without the requirement for labeling of the sample with natural or artificial fluorophores. We experimentally demonstrate a major benefit of this technique, which is the capability to respond exclusively to the linear Raman-resonance properties of the sample, thus allowing a direct quantitative interpretation of image contrast in terms of the number density of Raman-active modes.
Optical imaging in vivo with molecular specificity is important in biomedicine because of its high spatial resolution and
sensitivity compared with magnetic resonance imaging. Stimulated Raman scattering (SRS) microscopy allows highly sensitive
optical imaging based on vibrational spectroscopy without adding toxic or perturbative labels. However, SRS imaging in living
animals and humans has not been feasible because light cannot be collected through thick tissues, and motion-blur arises from
slow imaging based on backscattered light. In this work, we enable in vivo SRS imaging by substantially enhancing the collection
of the backscattered signal and increasing the imaging speed by three orders of magnitude to video rate. This approach allows
label-free in vivo imaging of water, lipid, and protein in skin and mapping of penetration pathways of topically applied drugs
in mice and humans.
Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique that is capable of real-time, nonperturbative examination of living cells and organisms based on molecular vibrational spectroscopy. Recent advances in detection schemes, understanding of contrast mechanisms, and developments of laser sources have enabled superb sensitivity and high time resolution. Emerging applications, such as metabolite and drug imaging and tumor identification, raise many exciting new possibilities for biology and medicine.
We demonstrate a fiber-based probe for maximum collection of the coherent anti-Stokes Raman scattering (CARS) signal in biological tissues. We discuss the design challenges including capturing the backscattered forward generated CARS signal in the sample and the effects of fiber nonlinearities on the propagating pulses. Three different single mode fibers (fused silica fiber, photonic crystal fiber and double-clad photonic crystal fiber) were tested for the probe design. We investigated self-phase modulation, stimulated Raman scattering (SRS) and four-wave-mixing (FWM) generation in the fiber: nonlinear processes expected to occur in a two-beam excitation based probe. While SPM and SRS induced spectral broadening was negligible, a strong non phase-matched FWM contribution was found to be present in all the tested fibers for excitation conditions relevant to CARS microscopy of tissues. To spectrally suppress this strong contribution, the pro design incorporates separate fibers for excitation light delivery and for signal detection, in combination with dichroic optics. CARS images of the samples were recorded by collecting the back-scattered forward generated CARS signal in the sample through a multi-mode fiber. Different biological tissues were imaged ex vivo in order to assess the performance of our fiber-delivered probe for CARS imaging, a tool which we consider an important advance towards label-free, in vivo probing of superficial tissues.
We demonstrate the use of coherent anti-Stokes Raman scattering (CARS) microscopy to image brain structure and pathology ex vivo. Although non-invasive clinical brain imaging with CT, MRI and PET has transformed the diagnosis of neurologic disease, definitive pre-operative distinction of neoplastic and benign pathologies remains elusive. Definitive diagnosis still requires brain biopsy in a significant number of cases. CARS microscopy, a nonlinear, vibrationally-sensitive technique, is capable of high-sensitivity chemically-selective three-dimensional imaging without exogenous labeling agents. Like MRI, CARS can be tuned to provide a wide variety of possible tissue contrasts, but with sub-cellular spatial resolution and near real time temporal resolution. These attributes make CARS an ideal technique for fast, minimally invasive, non-destructive, molecularly specific intraoperative optical diagnosis of brain lesions. This promises significant clinical benefit to neurosurgical patients by providing definitive diagnosis of neoplasia prior to tissue biopsy or resection. CARS imaging can augment the diagnostic accuracy of traditional frozen section histopathology in needle biopsy and dynamically define the margins of tumor resection during brain surgery. This report illustrates the feasibility of in vivo CARS vibrational histology as a clinical tool for neuropathological diagnosis by demonstrating the use of CARS microscopy in identifying normal brain structures and primary glioma in fresh unfixed and unstained ex vivo brain tissue.
We provide a proof-of-principle demonstration of CARS endoscopy. The design utilizes a single mode optical fiber with a focusing unit attached to the distal end. Picosecond pump and Stokes pulse trains in the near infrared are delivered through the fiber with nearly unaltered spectral and temporal characteristics at intensities needed for endoscopy. CARS endoscopic images are recorded by collecting the epi-CARS signal generated at the sample and raster scanning the sample with respect to the fiber. This CARS endoscope prototype represents an important step towards in situ chemically selective imaging for biomedical applications.
Spectrally encoded confocal microscopy (SECM) is a technique that allows confocal microscopy to be performed through the confines of a narrow diameter optical fiber probe. We present a novel scheme for performing SECM in which a rapid wavelength swept source is used. The system allows large field of view images to be acquired at rates up to 30 frames/second. Images of resolution targets and tissue specimens acquired ex vivo demonstrate high lateral (1.4 mum) and axial (6 mum) resolution. Imaging of human skin was performed in vivo at depths of up to 350 mum, allowing cellular and sub-cellular details to be visualized in real time.
We theoretically show that the shot-noise-limited sensitivity of stimulated Raman scattering (SRS) microscopy, which enables high-contrast vibrational imaging, is similar to that of coherent anti-Stokes Raman scattering microscopy. We experimentally confirm that the sensitivity of our SRS microscope is lower than the shot-noise limit only by <15 dB, which indicates that the high-sensitivity of SRS microscopy is readily available.
Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.
Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH2 stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.
• nonlinear microscopy
• vibrational imaging
• back scattering
The ability to conduct high-resolution fluorescence imaging in internal organs of small animal models in situ and over time can make a significant impact in biomedical research. Toward this goal, we developed a real-time confocal and multiphoton endoscopic imaging system. Using 1-mm-diameter endoscopes based on gradient index lenses, we demonstrate video-rate multicolor multimodal imaging with cellular resolution in live mice.
Combined two-photon fluorescence microscopy and femtosecond laser microsurgery has many potential biomedical applications as a powerful "seek-and-treat" tool. Towards developing such a tool, we demonstrate a miniaturized probe which combines these techniques in a compact housing. The device is 10 x 15 x 40 mm(3) in size and uses an aircore photonic crystal fiber to deliver femtosecond laser pulses at 80 MHz repetition rate for imaging and 1 kHz for microsurgery. A fast two-axis microelectromechanical system scanning mirror is driven at resonance to produce Lissajous beam scanning at 10 frames per second. Field of view is 310 microm in diameter and the lateral and axial resolutions are 1.64 microm and 16.4 microm, respectively. Combined imaging and microsurgery is demonstrated using live cancer cells.
Sarcomeres are the basic contractile units of striated muscle. Our knowledge about sarcomere dynamics has primarily come from in vitro studies of muscle fibres and analysis of optical diffraction patterns obtained from living muscles. Both approaches involve highly invasive procedures and neither allows examination of individual sarcomeres in live subjects. Here we report direct visualization of individual sarcomeres and their dynamical length variations using minimally invasive optical microendoscopy to observe second-harmonic frequencies of light generated in the muscle fibres of live mice and humans. Using microendoscopes as small as 350 microm in diameter, we imaged individual sarcomeres in both passive and activated muscle. Our measurements permit in vivo characterization of sarcomere length changes that occur with alterations in body posture and visualization of local variations in sarcomere length not apparent in aggregate length determinations. High-speed data acquisition enabled observation of sarcomere contractile dynamics with millisecond-scale resolution. These experiments point the way to in vivo imaging studies demonstrating how sarcomere performance varies with physical conditioning and physiological state, as well as imaging diagnostics revealing how neuromuscular diseases affect contractile dynamics.
A novel type of non-linear Raman microscopy, femtosecond stimulated Raman microscopy (FSRM), is introduced. It employs femtosecond white light pulses and intense picosecond pulses which are derived from a femtosecond laser/amplifier system. The pulses are coupled into a microscope set-up and induce a stimulated Raman process at the focus. The Raman interaction spectrally modulates the white light. These modulations are read-out in multi-channel fashion and allow recording of a complete Raman spectrum of the focal region. By raster-scanning the sample, complete Raman images can be obtained. Raman images of polystyrene beads in water demonstrate the feasibility of the approach.
A multiphoton microscopy based on coherent anti-Stokes Raman scattering is accomplished with near-infrared ultrashort laser pulses. We demonstrate vibrational imaging of chemical and biological samples with high sensitivity, high spatial resolution, noninvasiveness, and three-dimensional sectioning capability. {copyright} {ital 1999} {ital The American Physical Society}
An endoscope capable of Coherent Anti-Stokes Raman scattering (CARS) imaging would be of significant clinical value for improving early detection of endoluminal cancers. However, developing this technology is challenging for many reasons. First, nonlinear imaging techniques such as CARS are single point measurements thus requiring fast scanning in a small footprint if video rate is to be achieved. Moreover, the intrinsic nonlinearity of this modality imposes several technical constraints and limitations, mainly related to pulse and beam distortions that occur within the optical fiber and the focusing objective. Here, we describe the design and report modeling results of a new CARS endoscope. The miniature microscope objective design and its anticipated performance are presented, along with its compatibility with a new spiral scanningfiber imaging technology developed at the University of Washington. This technology has ideal attributes for clinical use, with its small footprint, adjustable field-of-view and high spatial-resolution. This compact hybrid fiber-based endoscopic CARS imaging design is anticipated to have a wide clinical applicability.
We demonstrate a novel miniaturized multimodal coherent anti-Stokes Raman scattering (CARS) microscope based on microelectromechanical systems (MEMS) scanning mirrors and custom miniature optics. A single Ti:sapphire femtosecond pulsed laser is used as the light source to produce the CARS, two photon excitation fluorescence (TPEF) and second harmonic generation (SHG) images using this miniaturized microscope. The high resolution and distortion-free images obtained from various samples such as a USAF target, fluorescent and polystyrene microspheres and biological tissue successfully demonstrate proof of concept, and pave the path towards future integration of parts into a handheld multimodal CARS probe for non- or minimally-invasive in vivo imaging.
In modern endoscopy, wide field of view and full color are considered necessary for navigating inside the body, inspecting tissue for disease and guiding interventions such as biopsy or surgery. Current flexible endoscope technologies suffer from reduced resolution when device diameter shrinks. Endoscopic procedures today, using coherent fiber-bundle technology on the scale of 1 mm, are performed with such poor image quality that the clinician's vision meets the criteria for legal blindness. Here, we review a new and versatile scanning fiber-imaging technology and describe its implementation for ultrathin and flexible endoscopy. This scanning fiber endoscope (SFE) or catheterscope enables high-quality, laser-based, video imaging for ultrathin clinical applications, while also providing new options for in vivo biological research of subsurface tissue and high resolution fluorescence imaging.
Remote optical imaging of human tissue in vivo has been the foundation for the growth of minimally invasive medicine. This article describes a new type of endoscopic imaging that has been developed and applied to the human esophagus, pig bile duct, and mouse colon. The technology is based on a single optical fiber that is scanned at the distal tip of an ultrathin and flexible shaft that projects red, green, and blue laser light onto tissue in a spiral pattern. The resulting images are high-quality color video that is expected to produce future endoscopes that are thinner, longer, more flexible, and able to directly integrate the many recent advances of laser diagnostics and therapies.
Advances in surgery have focused on minimizing the invasiveness of surgical procedures, such that a significant paradigm shift has occurred for some procedures in which surgeons no longer directly touch or see the structures on which they operate. Advancements in video imaging, endoscope technology, and instrumentation have made it possible to convert many procedures in many surgical specialties from open surgeries to endoscopic ones. The use of computers and robotics promises to facilitate complex endoscopic procedures by virtue of voice control over the networked operating room, enhancement of dexterity to facilitate microscale operations, and development of virtual simulator trainers to enhance the ability to learn new complex operations. Future research will focus on delivery of diagnostic and therapeutic modalities through natural orifices in which investigation is under remote control and navigation, so that truly "noninvasive" surgery will be a reality.
A confocal laser endoscopy system has recently been developed that may allow subsurface imaging of living cells in colonic tissue in vivo. The aim of the present study was to assess its potential for prediction of histology during screening colonoscopy for colorectal cancer.
Twenty-seven patients underwent colonoscopy with the confocal endoscope using acriflavine hydrochloride or fluorescein sodium with blue laser illumination. Furthermore, 42 patients underwent colonoscopy with this system using fluorescein sodium. Standardized locations and circumscript lesions were examined by confocal imaging before taking biopsy specimens. Confocal images were graded according to cellular and vascular changes and correlated with conventional histology in a prospective and blinded fashion.
Acriflavine hydrochloride and fluorescein sodium both yielded high-quality images. Whereas acriflavine hydrochloride strongly labeled the superficial epithelial cells, fluorescein sodium offered deeper imaging into the lamina propria. Fluorescein sodium was thus used for the prospective component of the study in which 13,020 confocal images from 390 different locations were compared with histologic data from 1038 biopsy specimens. Subsurface analysis during confocal laser endoscopy allowed detailed analysis of cellular structures. The presence of neoplastic changes could be predicted with high accuracy (sensitivity, 97.4%; specificity, 99.4%; accuracy, 99.2%).
Confocal laser endoscopy is a novel diagnostic tool to analyze living cells during colonoscopy, thereby enabling virtual histology of neoplastic changes with high accuracy. These newly discovered diagnostic possibilities may be of crucial importance in clinical practice and lead to an optimized rapid diagnosis of neoplastic changes during ongoing colonoscopy.
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