Cone–shell Raman spectroscopy (CSRS) for depth‐sensitive measurements in layered tissue

We report the development of a depth‐sensitive Raman spectroscopy system using the configuration of cone–shell excitation and cone detection. The system uses a 785 nm diode laser and three identical axicons for Raman excitation of the target sample in the form of a hollow conic section. The Raman scattered light from the sample, passed through the same (but solid) conic section, is collected for detection. Apart from its ability of probing larger depths (? few mm), an important attraction of the system is that the probing depths can be varied by simply varying the separation between axicons in the excitation arm. Furthermore, no adjustment is required in the sample arm, which is a significant advantage for noncontact, depth‐sensitive measurement. Evaluation of the performance of the developed setup on nonbiological phantom and biological tissue sample demonstrated its ability to recover Raman spectra of layers located at depths of ?2–3 mm beneath the surface.

     

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white‐light laser source

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti‐Stokes Raman scattering (CARS), third‐order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously. Due to white‐light laser excitation, multiplex CARS and TSFG spectra were obtained. Combined multimodal and spectral analysis clearly elucidated the layered structure of rat cornea with molecular structural information. This study indicates that our multimodal nonlinear spectral microscope is a promising bioimaging method for tissue study.

Multimodal nonlinear spectral images of rat cornea at corneal epithelium and corneal stroma in the in‐plane (XY) direction. With use of the combinational analysis of different nonlinear optical processes, detailed molecular structural information is available without staining or labelling.     

Widefield multifrequency fluorescence lifetime imaging using a two‐tap complementary metal‐oxide semiconductor camera with lateral electric field charge modulators

Widefield frequency‐domain fluorescence lifetime imaging microscopy (FD‐FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD‐FLIM system based on a complementary metal‐oxide semiconductor camera equipped with two‐tap true correlated double sampling lock‐in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31‐harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean‐Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD‐FLIM system is a valuable tool in the biomedical field.      

Noninvasive assessments of skin glycated proteins by fluorescence and Raman techniques in diabetics and nondiabetics

Diabetes is a complex metabolic disease and has chronic complications. It has been considered a serious public health problem. The aim of the current study was to evaluate skin glycated proteins through fluorescence and Raman techniques. One hundred subjects were invited to participate in the study. Six volunteers did not attend due to exclusion criteria or a change of mind about participating. Therefore, 94 volunteers were grouped according to age range (20‐80 years), health condition (nondiabetic, with insulin resistance [IR] and/or diabetic) and Fitzpatrick skin type (I‐VI). The fluorescence spectrometer and the portable Raman spectroscopy system were used to measure glycated proteins from the skin. There was elevated skin autofluorescence in healthy middle‐aged and elderly subjects, as well as in patients with IR and/or diabetes. Regarding Raman spectroscopy, changes in the skin hydration state, degradation of type I collagen and greater glycation were related for diabetes and chronological aging. Weak and positive correlation between the skin autofluorescence and the Raman peaks ratio (855/876) related to the glycated proteins was also found. Raman spectroscopy shows several bands for spectral analyses, complementing the fluorescence data. Therefore, this study contributes to understanding of the optical of human skin for noninvasive diabetes screening.      

Identification and characterization of bladder cancer by low‐resolution fiber‐optic Raman spectroscopy

Raman spectroscopy has been proved to be a promising diagnostic technique for various cancers detection. A major drawback for its clinical translation is the intrinsic weakness of Raman effects. Highly sensitive equipment and optimal measurement conditions are generally applied to overcome this drawback. However, these equipment are usually bulky, expensive and may also be easily influenced by surrounding environment. In this preliminary work, a low‐resolution fiber‐optic Raman sensing system is applied to evaluate the diagnostic potential of Raman spectroscopy to identify different bladder pathologies ex vivo. A total number of 262 spectra taken from 32 bladder specimens are included in this study. These spectra are categorized into 3 groups by histopathological analysis, namely normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors. Principal component analysis fed artificial neural network are used to train a classification model for the spectral data with 10‐fold cross‐validation and an overall prediction accuracy of 93.1% is obtained. The sensitivities and specificities for normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors are 88.5% and 95.1%, 90.3% and 98%, and 97.5% and 96.4%, respectively. These results demonstrate the potential of using a low‐resolution fiber‐optic Raman system for in vivo bladder cancer diagnosis.      

Multimodal optical analysis of meningioma and comparison with histopathology

Meningioma is the most frequent primary central nervous system tumor. The risk of recurrence and the prognosis are correlated with the extent of the resection that ideally encompasses the infiltrated dura mater and, if required, the infiltrated bone. No device can deliver real‐time intraoperative histopathological information on the tumor environment to help the neurosurgeon to achieve a gross total removal. This study assessed the abilities of nonlinear microscopy to provide relevant and real‐time data to help resection of meningiomas. Nine human meningioma samples (four World Health Organization Grade I, five Grade II) were analyzed using different optical modalities: spectral analysis and imaging, lifetime measurements, fluorescence lifetime imaging microscopy, fluorescence emitted under one‐ and two‐photon excitation and the second‐harmonic generation signal imaging using a multimodal setup. Nonlinear microscopy produced images close to histopathology as a gold standard. The second‐harmonic generation signal delineated the collagen background and two‐photon fluorescence underlined cell cytoplasm. The matching between fluorescence images and Hematoxylin and Eosin staining was possible in all cases. Grade I meningioma emitted less autofluorescence than Grade II meningioma and Grade II meningioma exhibited a distinct lifetime value. Autofluorescence was correlated with the proliferation rates and seemed to explain the observed differences between Grade I and II meningiomas. This preliminary multimodal study focused on human meningioma samples confirms the potential of tissue autofluorescence analysis and nonlinear microscopy in helping intraoperatively neurosurgeons to reach the actual boundaries of the tumor infiltration.

Correspondence between H&E staining (top pictures) and the two‐photon fluorescence imaging (bottom pictures)     

Multimodal imaging to explore endogenous fluorescence of fresh and fixed human healthy and tumor brain tissues

To complement a project toward label‐free optical biopsy and enhanced resection which the overall goal is to develop a multimodal nonlinear endomicroscope, this multimodal approach aims to enhance the accuracy in classifying brain tissue into solid tumor, infiltration and normal tissue intraoperatively. Multiple optical measurements based on one‐ and two‐photon spectral and lifetime autofluorescence, including second harmonic generation imaging, were acquired. As a prerequisite, studying the effect of the time of measurement postexcision on tissue's spectral/lifetime fluorescence properties was warranted, so spectral and lifetime fluorescences of fresh brain tissues were measured using a point‐based linear endoscope. Additionally, a comparative study on tissue's optical properties obtained by multimodal nonlinear optical imaging microscope from fresh and fixed tissue was necessary to test whether clinical validation of the nonlinear endomicroscope is feasible by extracting optical signatures from fixed tissue rather than from freshly excised samples. The former is generally chosen for convenience. Results of this study suggest that an hour is necessary postexcision to have consistent fluorescence intensities\lifetimes. The fresh (a,b,c) vs fixed (d,e,f) tissue study indicates that while all optical signals differ after fixation. The characteristic features extracted from one‐ and two‐photon excitation still discriminate normal brain (a,d) cortical tissue, glioblastoma (GBM) (b,e) and metastases (c,f).      

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to < 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.Download video file.^{(95M, mov)}     

Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm^?1, is demonstrated.     

Development and first in‐human use of a Raman spectroscopy guidance system integrated with a brain biopsy needle

Navigation‐guided brain biopsies are the standard of care for diagnosis of several brain pathologies. However, imprecise targeting and tissue heterogeneity often hinder obtaining high‐quality tissue samples, resulting in poor diagnostic yield. We report the development and first clinical testing of a navigation‐guided fiberoptic Raman probe that allows surgeons to interrogate brain tissue in situ at the tip of the biopsy needle prior to tissue removal. The 900 μm diameter probe can detect high spectral quality Raman signals in both the fingerprint and high wavenumber spectral regions with minimal disruption to the neurosurgical workflow. The probe was tested in three brain tumor patients, and the acquired spectra in both normal brain and tumor tissue demonstrated the expected spectral features, indicating the quality of the data. As a proof‐of‐concept, we also demonstrate the consistency of the acquired Raman signal with different systems and experimental settings. Additional clinical development is planned to further evaluate the performance of the system and develop a statistical model for real‐time tissue classification during the biopsy procedure.      

Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy

Using the shifted-excitation Raman difference spectroscopy technique and an optical fibre featuring a negative curvature excitation core and a coaxial ring of high numerical aperture collection cores, we have developed a portable, background and fluorescence free, endoscopic Raman probe. The probe consists of a single fibre with a diameter of less than 0.25 mm packaged in a sub-millimetre tubing, making it compatible with standard bronchoscopes. The Raman excitation light in the fibre is guided in air and therefore interacts little with silica, enabling an almost background free transmission of the excitation light. In addition, we used the shifted-excitation Raman difference spectroscopy technique and a tunable 785 nm laser to separate the fluorescence and the Raman spectrum from highly fluorescent samples, demonstrating the suitability of the probe for biomedical applications. Using this probe we also acquired fluorescence free human lung tissue data.     

In vivo full‐field functional optical hemocytometer

We present an in vivo lab‐free full‐field functional optical hemocytometer (FFOH) for application to the capillaries of a live biological specimen, based on the absorption intensity fluctuation modulation (AIFM) effect. Because of the absorption difference between the red blood cells (RBCs) and background tissue under low‐coherence light illumination, an endogenous instantaneous intensity fluctuation is generated by the AIFM effect when RBCs discontinuously traverse the capillary. The AIFM effect is used to highlight the RBC signal relative to the background tissue by computing the real‐time modulation depth. FFOH can simultaneously provide a flow video, the flow velocity and the RBC count. Ourexperimental results can potentially be applied to study the physiological mechanisms of the blood circulation systems of near‐transparent live biological samples.      

Raman spectroscopic identification of Mycobacterium tuberculosis

In this study, Raman microspectroscopy has been utilized to identify mycobacteria to the species level. Because of the slow growth of mycobacteria, the per se cultivation‐independent Raman microspectroscopy emerges as a perfect tool for a rapid on‐the‐spot mycobacterial diagnostic test. Special focus was laid upon the identification of Mycobacterium tuberculosis complex (MTC) strains, as the main causative agent of pulmonary tuberculosis worldwide, and the differentiation between pathogenic and commensal nontuberculous mycobacteria (NTM). Overall the proposed model considers 26 different mycobacteria species as well as antibiotic susceptible and resistant strains. More than 8800 Raman spectra of single bacterial cells constituted a spectral library, which was the foundation for a two‐level classification system including three support vector machines. Our model allowed the discrimination of MTC samples in an independent validation dataset with an accuracy of 94% and could serve as a basis to further improve Raman microscopy as a first‐line diagnostic point‐of‐care tool for the confirmation of tuberculosis disease.

     

Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies

We demonstrate a novel bio‐spectroscopic technique, “simultaneous Raman/GFP microspectroscopy”. It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti‐Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two‐component multivariate curve resolution with alternate least squares (MCR‐ALS) analysis in the anti‐Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR‐ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm^?1, previously reported as “Raman spectroscopic signature of life of yeast.”     

Quantitative subsurface spatial frequency‐domain fluorescence imaging for enhanced glioma resection

The rate of complete resection of glioma has improved with the introduction of 5‐aminolevulinic acid‐induced protoporphyrin IX (PpIX) fluorescence image guidance. Surgical outcomes are further enhanced when the fluorescence signal is decoupled from the intrinsic tissue optical absorption and scattering obtained from diffuse reflectance measurements, yielding the absolute PpIX concentration, [PpIX]. Spatial frequency domain imaging was used previously to measure [PpIX] in near‐surface tumors under blue fluorescence excitation. Here, we extend this to subsurface [PpIX] fluorescence under red‐light excitation. The decay rate of the modulation amplitude of the fluorescence signal was used to calculate the PpIX depth, which was then applied in a forward diffusion model to estimate [PpIX] at depth. For brain‐like optical properties in phantoms with PpIX fluorescent inclusions, the depth can be recovered up to depths of 9.5 mm ± 0.4 mm, with [PpIX] ranging from 5 to 15 μg/mL within an average deviation of 15% from the true [PpIX] value.      

Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam

The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular‐shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut‐shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.      

Ultra‐low background Raman sensing using a negative‐curvature fibre and no distal optics

Measuring Raman spectra through an optical fibre is usually complicated by the high intrinsic Raman scatter of the fibre material. Common solutions such as the use of multiple fibres and distal optics are complex and bulky. We demonstrate the use of single novel hollow‐core negative‐curvature fibres (NCFs) for Raman and surface‐enhanced Raman spectroscopy (SERS) sensing using no distal optics. The background Raman emission from the silica in the NCF was at least 1000× smaller than in a conventional solid fibre, while maintaining the same collection efficiency. We transmitted pump light from a 785‐nm laser through the NCF, and we collected back the weak Raman spectra of different distal samples, demonstrating the fibre probe can be used for measurements of weak Raman and SERS signals that would otherwise overlap spectrally with the silica background. The lack of distal optics and consequent small probe diameter (<0.25 mm) enable applications that were not previously possible.      

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

Ex and in vivo characterization of the wavelength‐dependent 3‐photon action cross‐sections of red fluorescent proteins covering the 1700‐nm window

Multiphoton action cross‐sections are the prerequisite for excitation light selection. At the 1700‐nm window suitable for deep‐tissue imaging, wavelength‐dependent 3‐photon action cross‐sections ησ₃ for RFPs are unknown, preventing wavelength selection. Here we demonstrate: (1) ex vivo measurement of wavelength‐dependent ησ₃ for purified RFPs; (2) a multiphoton imaging guided measurement system for in vivo measurement; and (3) in vivo measurement of wavelength‐dependent ησ₃ in RFP labeled cells. These fundamental results will provide guidelines for excitation wavelength selection for 3‐photon fluorescence imaging of RFPs at the 1700‐nm window, and augment the existing database of multiphoton action cross‐sections for fluorophores.      

Absolute quantum yield measurement of powder samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry. Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material. For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. Measurement of quantum yield in powder samples is performed following these steps: 1. Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations. 2. Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements. 3. Reference and Sample measurement using direct excitation and indirect excitation. 4. Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation. 5. Corrected quantum yield calculation. 6. Chromaticity coordinates calculation using Report Generator program. The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this application, as follows: High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate. Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. Measurement of quantum yield over a wide wavelength range from 240 to 800 nm. Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample. Large selection of calculated parameters provided by dedicated and easy to use software. During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.