405 nm versus 633 nm for protoporphyrin IX excitation in fluorescence‐guided stereotactic biopsy of brain tumors

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5‐aminolevulinic‐acid‐induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50‐fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well‐defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose‐gel phantoms. Even in direct contact, blood layers of 20–40 µm between probe and tissue must be expected, obscuring 405‐nm‐excited PpIX fluorescence almost completely, but reducing the 633‐nm‐excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX‐assisted detection of high‐grade gliomas in stereotactic biopsy.

PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation.     

Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near‐infrared spectrum can improve the present situation. Here, the capability of saturated two‐photon saturated excitation (TP‐SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering‐enlarged point spread function (PSF), we find that TP‐SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single‐photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering‐enlarged PSF, TP‐SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.      

Comparison of optically‐derived biomarkers in healthy and brain tumor tissue under one‐ and two‐photon excitation

The surgical outcome of brain tumor resection and needle biopsy is significantly correlated to the patient's prognosis. Brain tumor surgery is limited to resecting the solid portion of the tumor as current intraoperative imaging modalities are incapable of delineating infiltrative regions. For accurate delineation, in situ tissue interrogation at the submicron scale is warranted. Additionally, multimodal detection is required to remediate the genetically and molecularly heterogeneous nature of brain tumors, notably, that of gliomas, meningioma and brain metastasis. Multimodal detection, such as spectrally‐ and temporally‐resolved fluorescence under one‐ and two‐photon excitation, enables characterizing tissue based on several endogenous optical contrasts. In order to assign the optically‐derived parameters to different tissue types, construction of an optical database obtained from biopsied tissue is warranted. This report showcases the different quantitative and semi‐quantitative optical markers that may comprise the tissue discrimination database. These include: the optical index ratio, the optical redox ratio, the relative collagen density, spectrally‐resolved fluorescence lifetime parameters, two‐photon fluorescence imaging and second harmonic generation imaging.     

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).      

Intrinsic fluorescence and mechanical testing of articular cartilage in human patients with osteoarthritis

The degeneration of articular cartilage is the main cause of osteoarthritis (OA), a common cause of disability among elderly patients. The aim of this study is to understand the correlation between intrinsic fluorescence of articular cartilage and its biomechanical properties in patients with osteoarthritis. Cylindrical samples of articular cartilage 6 mm in diameter were extracted via biopsy punch from the femoral condyles of 6 patients with advanced OA undergoing knee replacement surgery. The mechanical stiffness and fluorescence of each cartilage plug were measured by indentation test and spectrofluorometry. Maps of fluorescence intensity, at excitation/emission wavelengths of 240–520/290‐530 nm, were used to identify wavelengths of interest. The mechanical stiffness and fluorescence intensity were correlated using a Spearman analysis. The excitation/emission maps demonstrated three fluorescence peaks at excitation/emission wavelength pairs 330/390, 350/430 and 370/460 nm. The best correlation between the fluorescence intensity and stiffness of cartilage was obtained for the 330 nm excitation band [R=0.82, p=0.04]. The intrinsic fluorescence of articular cartilage may have application in optically assessing the state of cartilage in patients with osteoarthritis.

     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.      

3‐photon fluorescence imaging of sulforhodamine B‐labeled elastic fibers in the mouse skin in vivo

Elastic fibers are key constituents of the skin. The commonly adopted optical technique for visualizing elastic fibers in the animal skin in vivo is 2‐photon microscopy (2 PM) of autofluorescence, which typically suffers from low signal level. Here we demonstrate a new optical methodology to image elastic fibers in animal models in vivo: 3‐photon microscopy (3 PM) excited at the 1700‐nm window combining with preferential labeling of elastic fibers using sulforhodamine B (SRB). First, we demonstrate that intravenous injection of SRB can circumvent the skin barrier (encountered in topical application) and preferentially label elastic fibers, as verified by simultaneous 2 PM of both autofluorescence and SRB fluorescence from skin structures. Then through 3‐photon excitation property characterization, we show that 3‐photon fluorescence can be excited from SRB at the 1700‐nm window, and 1600‐nm excitation is most efficient according to our 3‐photon action cross section measurement. Based on these results and using our developed 1600‐nm femtosecond laser source, we finally demonstrate 3 PM of SRB‐labeled elastic fibers through the whole dermis in the mouse skin in vivo, with only 3.7‐mW optical power deposited on the skin surface. We expect our methodology will provide novel optical solution to elastic fiber research.     

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.      

Oxygen‐dependent delayed fluorescence of protoporphyrin IX measured in the stomach and duodenum during upper gastrointestinal endoscopy

Protoporphyrin IX‐triplet state lifetime technique (PpIX‐TSLT) is a method used to measure oxygen (PO₂) in human cells. The aim of this study was to assess the technical feasibility and safety of measuring oxygen‐dependent delayed fluorescence of 5‐aminolevulinic acid (ALA)‐induced PpIX during upper gastrointestinal (GI) endoscopy. Endoscopic delayed fluorescence measurements were performed 4 hours after oral administration of ALA in healthy volunteers. The ALA dose administered was 0, 1, 5 or 20 mg/kg. Measurements were performed at three mucosal spots in the gastric antrum, duodenal bulb and descending duodenum with the catheter above the mucosa and while applying pressure to induce local ischemia and monitor mitochondrial respiration. During two endoscopies, measurements were performed both before and after intravenous administration of butylscopolamine. Delayed fluorescence measurements were successfully performed during all 10 upper GI endoscopies. ALA dose of 5 mg/kg showed adequate signal‐to‐noise ratio (SNR) values >20 without side effects. All pressure measurements showed significant prolongation of delayed fluorescence lifetime compared to measurements performed without pressure (P < .001). Measurements before and after administration of butylscopolamine did not differ significantly in the duodenal bulb and descending duodenum. Measurements of oxygen‐dependent delayed fluorescence of ALA‐induced PpIX in the GI tract during upper GI endoscopy are technically feasible and safe.     

Depth‐resolved multimodal imaging: Wavelength modulated spatially offset Raman spectroscopy with optical coherence tomography

A major challenge in biophotonics is multimodal imaging to obtain both morphological and molecular information at depth. We demonstrate a hybrid approach integrating optical coherence tomography (OCT) with wavelength modulated spatially offset Raman spectroscopy (WM‐SORS). With depth colocalization obtained from the OCT, we can penetrate 1.2‐mm deep into strong scattering media (lard) to acquire up to a 14‐fold enhancement of a Raman signal from a hidden target (polystyrene) with a spatial offset. Our approach is capable of detecting both Raman and OCT signals for pharmaceutical particles embedded in turbid media and revealing the white matter at depth within a 0.6‐mm thick brain tissue layer. This depth resolved label‐free multimodal approach is a powerful route to analyze complex biomedical samples.      

Anti‐Stokes fluorescence from endogenously formed protoporphyrin IX – Implications for clinical multiphoton diagnostics

Multiphoton imaging based on two‐photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two‐photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one‐photon anti‐Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

High‐resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO)

Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts benzyl benzoate) is easy in application and allows deep‐tissue imaging of a wide range of specimens. However, in both substances, optical clearing and storage times of enhanced green fluorescent protein (EGFP)‐expressing specimens are limited due to the continuous formation of peroxides and aldehydes, which severely quench fluorescence. Stabilisation of purified DBE or BABB by addition of the antioxidant propyl gallate efficiently preserves fluorescence signals in EGFP‐expressing samples for more than a year. This enables longer clearing times and improved tissue transparency with higher fluorescence signal intensity. The here introduced clearing protocol termed stabilised DISCO allows to image spines in a whole mouse brain and to detect faint changes in the activity‐dependent expression pattern of tdTomato.      

Two‐photon excited lasing of Coumarin 307 for lysozyme amyloid fibrils detection

Amyloid fibrils are a well‐recognized hallmark of neurodegeneration. A common approach to detect amyloid fibrils is staining with organic molecules and monitoring optical properties using fluorescence spectroscopy. However, the structural diversity of amyloids necessitates new sensitive methods and probes that can be reliably used to characterize them. Here, Coumarin 307 is applied for lysozyme fibrils detection by observation of laser action in the process of two‐photon excited stimulated emission. It is shown that the lasing threshold and spectrum significantly depend on the adopted structure (α‐helix or β‐sheet) of the lysozyme protein, whereas fluorescence spectrum is insensitive to the protein structure. The applications of coherent stimulated emission light that can be emitted deep inside a scattering medium can be particularly promising for imaging and therapeutic purposes in the neurodegeneration field. Two‐photon excitation with the near‐infrared light, which allows the deepest penetration of tissues, is an important advantage of the method.     

Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

Fluorescence imaging in the second near‐infrared optical window (NIR‐II, 900‐1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR‐II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR‐II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5‐mm‐thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR‐1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR‐II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR‐1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 μm, with a superb spatial resolution of around 2 μm.      

Assessment of ALA‐induced PpIX production in porcine skin pretreated with microneedles

Photodynamic therapy (PDT) is used for skin treatments of premalignant and cancer lesions and recognized as a non‐invasive technique that combines tissue photosensitization and subsequent exposure to light to induce cell death. However, it is limited to the treatment of superficial lesions, mainly due to the low cream penetration. Therefore, the improvement of transdermal distribution of aminolevulinic acid (ALA) is needed. In this study, the kinetics and homogeneity of production of ALA‐induced PpIX after the skin pre‐treatment with microneedles rollers of 0.5, 1.0 and 1.5 mm length were investigated. An improvement in homogeneity and production of PpIX was shown in a porcine model.

Widefield fluorescence imaging three hours after the topical application of ALA‐cream in the combined treatment with microeedles rollers.     

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short‐lifetime near‐infrared dyes and time‐gated imaging

We introduce a simple new approach for time‐resolved multiplexed analysis of complex systems using near‐infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user‐friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time‐gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely‐available software, has the advantage of time‐resolved NIR imaging, including better tissue penetration and background‐free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image‐guided surgery or optical tomography.      

Label‐free imaging and spectroscopy for early detection of cervical cancer

The label‐free imaging and spectroscopy method was studied on cervical unstained tissue sections obtained from 36 patients. The native fluorescence spectra of tissues are analyzed by the optical redox ratio (ORR), which is defined as fluorescence intensity ratio between NADH and FAD, and indicates the metabolism change with the cancer development. The ORRs of normal tissues are consistently higher than those of precancer or cancerous tissues. A criterion line of ORR at 5.0 can be used to discriminate cervical precancer/cancer from normal tissues. The sensitivity and specificity of the native fluorescence spectroscopy method for cervical cancer diagnosis are determined as 100% and 91%. Moreover, the native fluorescence spectroscopy study is much more sensitive on the healthy region of cervical precancer/cancer patients compared with the traditional clinical staining method. The results suggest label‐free imaging and spectroscopy is a fast, highly sensitive and specific method on the detection of cervical cancer.      

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)     

Development of a molecular bioswitch using fluorescence lifetime imaging: Incremental activation of fluorescein diacetate

Molecular bioswitches offer an invaluable asset in the shift from systemic to targeted treatments. Within the growing arsenal of switches are imaging probes that functionalize only in given locations or situations. Acetate esters are a common fluorescent example, known to activate upon interaction with esterases. Fluorescein diacetate (FDA) is one such fluorophore used in cell viability assays. These assays rely on the fact that the compound begins colorless and with no fluorescent signature whatsoever, and only after internalization into cells it is possible to detect a fluorescence signal. In this study, using fluorescence intensity (FI) and fluorescence lifetime (FLT) imaging, FDA is shown to be fluorescent even when unactivated. Furthermore, the FLT is shown to change with pH. Finally, the ability to image FDA in different environments simulated by tissue‐imitating phantoms is explored. Altogether, the ability of FDA to serve as a bioswitch when measured using FLT imaging microscopy (FLIM) is assessed. The combination of a spectrum of FDA activation and FLIM serves as a bioswitch, where biologically relevant stimulation can generate detectable and incremental variations.