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.      

Refractive index and pulse broadening characterization using oil immersion and its influence on three‐photon microscopy excited at the 1700‐nm window

Three‐photon microscopy excited at the 1700‐nm window enables deep‐tissue penetration. However, the refractive indices of commonly used immersion oils, and the resultant pulse broadening are not known, preventing imaging optimization. Here, we demonstrate detailed characterization of the refractive index, pulse broadening and distortion for excitation pulses at this window for commonly used immersion oils. On the physical side, we uncover that absorption, rather than material dispersion, is the main cause of pulse broadening and distortion. On the application side, comparative three‐photon imaging results indicate that 1600‐nm excitation yields 5 times higher three‐photon signal than 1690‐nm excitation.      

Imaging directed photothermolysis through two‐photon absorption demonstrated on mouse skin – a potential novel tool for highly targeted skin treatment

One‐photon absorption based traditional laser treatment may not necessarily be selective at the microscopic level, thus could result in un‐intended tissue damage. Our objective is to test whether two‐photon absorption (TPA) could provide highly targeted tissue alteration of specific region of interest without damaging surrounding tissues. TPA based laser treatments (785 nm, 140 fs pulse width, 90 MHz) were performed on ex vivo mouse skin using different average power levels and irradiation times. Reflectance confocal microscopy (RCM) and combined second‐harmonic‐generation (SHG) and two‐photon fluorescence (TPF) imaging channels were used to image before, during, and after each laser treatment. The skin was fixed, sectioned and H & E stained after each experiment for histological assessment of tissue alterations and for comparison with the non‐invasive imaging assessments. Localized destruction of dermal fibers was observed without discernible epidermal damage on both RCM and SHG + TPF images for all the experiments. RCM and SHG + TPF images correlated well with conventional histological examination. This work demonstrated that TPA‐based light treatment provides highly localized intradermal tissue alteration. With further studies on optimizing laser treatment parameters, this two‐photon absorption photothermolysis method could potentially be applied in clinical dermatology. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

Automatic and label‐free identification of blood vessels in gliomas using the combination of multiphoton microscopy and image analysis

Currently, the targeted treatment of tumor based on the tumor microenvironment is newly developed. Blood vessels are the key parts in the tumor microenvironment, which is taken as a new visible target for tumor therapy. Multiphoton microscopy (MPM), based on the second harmonic generation and two‐photon excited fluorescence, is available to make the label‐free analysis on the blood vessels in human gliomas. MPM can reveal the vascular morphological characteristics in gliomas, including vascular malformation, intense vascular proliferation, perivascular collagen deposition, perivascular lymphocytes aggregation and microvascular proliferation. In addition, the image analysis algorithms were developed to automatically calculate the perivascular collagen content, vascular cavity area, lumen area, wall area and vessel number. Thus, the vascular morphology, the perivascular collagen deposition and intense vascular proliferation degree can be further quantitatively characterized. Compared with the pathological analysis, the combination of MPM and image analysis has potential advantages in making a quantitative and qualitative analyzing on vascular morphology in glioma microenvironment. As micro‐endoscope and two‐photon fiberscope are technologically improved, this combined method will be a useful imaging way to make the real‐time research on the targeting tumor microenvironment in gliomas.     

Quantitative assessment of microenvironment characteristics and metabolic activity in glioma via multiphoton microscopy

Tumor microenvironment and metabolic activity in gliomas are the important biomarkers to evaluate the progression of gliomas. Many evidences have suggested that the targeting of metabolic activity and tumor microenvironment simultaneously can be more effective to take the tumor therapy. Therefore, the noninvasive, accurate assessment of tumor microenvironment and metabolic activity is quite important in clinical practice. Multiphoton microscopy (MPM), based on two‐photon‐excited fluorescence and second harmonic generation was performed on unstained glioma tissues. With our combined image analysis approaches, our research findings indicate that MPM is able to qualitatively and quantitatively describe the microenvironment characteristics in gliomas, such as collage deposition in extracellular matrix, lymphocyte infiltration and tumor angiogenesis, etc. Meanwhile, the metabolic activity can also be quantitatively evaluated by optical redox ratio, NADH and FAD intensity. With the microendoscope and fiberscope are portable, MPM technique can be used to perform in‐vivo studies and clinical examinations in gliomas.      

Multiphoton imaging of the dentine‐enamel junction

Multiphoton microscopy has been used to reveal structural details of dentine and enamel at the dentin‐enamel junction (DEJ) based on their 2‐photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). In dentine tubule 2PEF intensity varies due to protein content variation. Intertubular dentin produces both SHG and 2PEF signals. Tubules are surrounded by a thin circular zone with a lower SHG signal than the bulk dentine and the presence of collagen fibers perpendicular to the tubule longitudinal axis is indicated by strong SHG responses. The DEJ appears as a low intensity line on the 2PEF images and this was never previously reported. The SHG signal is completely absent for enamel and aprismatic enamel shows a homogeneous low 2PEF signal contrary to prismatic enamel. The SHG intensity of mantle dentine is increasing from the dentine‐enamel junction in the first 12 μm indicating a progressive presence of fibrillar collagen and corresponding to the more external part of mantle dentine where matrix metallo‐proteases accumulate. The high information content of multiphoton images confirms the huge potential of this method to investigate tooth structures in physiological and pathological conditions. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Slide‐free imaging of hematoxylin‐eosin stained whole‐mount tissues using combined third‐harmonic generation and three‐photon fluorescence microscopy

Intraoperative margin assessment of surgical tissues during cancer surgery is clinically important, especially in the case of tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is considered crucial. Frozen pathology is the gold standard of assessing excised tissues for signs of remaining cancerous lesions. The current protocol, however, is time‐consuming and labor‐intensive. Instead of the complex frozen sectioning, staining, and traditional white light microscopy imaging protocol, optically sectioned histopathological imaging of hematoxylin‐eosin stained whole‐mount skin tissues with a subfemtoliter resolution is demonstrated by using nonlinear microscopy in this study. With our proposed method, the reagents of staining and the contrast of imaging are fully consistent with the current clinical standard of frozen pathology, thus facilitating rapid intraoperative assessment of surgical tissues for future applications. Image: Slide‐free nonlinear microscopy imaging of H&E stained whole‐mount skin tissue showing the morphology of sweat glands.      

Two‐photon microscopy of healthy,infarcted and stem‐cell treated regenerating heart

Two‐photon excitation autofluorescence (produced in myocytes) and second‐harmonic generation (produced mainly by collagen) allow label‐free visualization of these two important components of myocardium. Because of their different emission wavelengths, these two signals can be separated spectrally. Here, we examine two‐photon microscopy images of healthy, infarcted and stem‐cell treated rat hearts. We find that in infarcted heart, regions distant from the site of infarct are similar to healthy tissue in composition (mostly myocytes, very little collagen) and organization (densely packed myocytes), but infarct regions are characterized by sparse myocytes and high collagen content indicative of scar tissue formation. Stem cell treated hearts, in contrast, show regions of intertwined myocytes and collagen throughout the infarct, suggesting reduced tissue damage. Finally, these results offer interesting insights into our ongoing polarized light studies of cardiac tissue anisotropy, and reveal that both tissue composition and tissue micro‐organization are reflected in polarization‐measured linear retardance values. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Assessment of the metabolism and morphology of the porcine cornea,lens and retina by 2‐photon imaging

Two‐photon imaging is a noninvasive imaging technique with increasing importance in the biological and medical fields since it allows intratissue cell imaging with high resolution. We demonstrate the feasibility of using a single 2‐photon instrument to evaluate the cornea, the crystalline lens and the retina based on their autofluorescence (AF). Image acquisition was performed using a custom‐built 2‐photon microscope for 5‐dimensional microscopy with a near infrared broadband sub‐15 femtosecond laser centered at 800 nanometers. Signals were detected using a spectral photomultiplier tube. The spectral ranges for the analysis of each tissue/layer AF were determined based on the spectra of each tissue as well as of pure endogenous fluorophores. The cornea, lens and retina are characterized at multiple depths with subcellular resolution based on their morphology and AF lifetime. Additionally, the AF lifetime of NAD(P)H was used to assess the metabolic activity of the cornea epithelium, endothelium and keratocytes. The feasibility to evaluate the metabolic activity of lens epithelial cells was also demonstrated, which may be used to further investigate the pathogenesis of cataracts. The results illustrate the potential of multimodal multiphoton imaging as a novel ophthalmologic technique as well as its potential as a diagnostic tool.      

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.      

Polarization‐dependent second‐harmonic generation for collagen‐based differentiation of breast cancer samples

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization‐dependent second‐harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second‐harmonic generation (SHG) anisotropy and the “ratio parameter” values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.     

Visualizing the “sandwich” structure of osteocytes in their native environment deep in bone in vivo

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3‐photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700‐nm window enables the highest optical transmittance through the skull. Using label‐free third‐harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140‐μm mouse skull and 155 μm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the “sandwich” structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.      

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.     

Bond‐selective imaging of deep tissue through the optical window between 1600 and 1850 nm

We report the employment of an optical window between 1600 nm and 1850 nm for bond‐selective deep tissue imaging through harmonic vibrational excitation and acoustic detection of resultant pressure waves. In this window where a local minimum of water absorption resides, we found a 5 times enhancement of photoacoustic signal by first overtone excitation of the methylene group CH₂ at 1730 nm, compared to the second overtone excitation at 1210 nm. The enhancement allows 3D mapping of intramuscular fat with improved contrast and of lipid deposition inside an atherosclerotic artery wall in the presence of blood. Moreover, lipid and protein are differentiated based on the first overtone absorption profiles of CH₂ and methyl group CH₃ in this window. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Analyzing the feasibility of discriminating between collagen types I and II using polarization‐resolved second harmonic generation

According to previous studies, the nonlinear susceptibility tensor ratio χ₃₃/χ₃₁ obtained from polarization‐resolved second harmonic generation (P‐SHG) under the assumption of cylindrical symmetry can be used to distinguish between fibrillar collagen types. Discriminating between collagen fibrils of types I and II is important in tissue engineering of cartilage. However, cartilage has a random organization of collagen fibrils, and the assumption of cylindrical symmetry may be incorrect. In this study, we simulated the P‐SHG response from different collagen organizations and demonstrated a possible method to exclude areas where cylindrical symmetry is not fulfilled and where fibrils are located in the imaging plane. The χ₃₃/χ₃₁‐ratio for collagen type I in tendon and collagen type II in cartilage was estimated to be 1.33 and 1.36, respectively, using this method. These ratios are now much closer than what has been reported previously in the literature, and the larger reported differences between collagen types can be explained by variation in the structural organization.      

‘Turn‐on’ fluorescent chemosensors based on naphthaldehyde‐2‐pyridinehydrazone compounds for the detection of zinc ion in water at neutral pH

A series of naphthaldehyde‐2‐pyridinehydrazone derivatives were discovered to display interesting ‘turn‐on’ fluorescence response to Zn²⁺ in 99% water/DMSO (v/v) at pH 7.0. Mechanism study indicated that different substituent groups in the naphthaldehyde moiety exhibited significant influence on the detection of Zn²⁺. The electron rich group resulted in longer fluorescence wavelengths but smaller fluorescence enhancement for Zn²⁺. Among these compounds, 1 showed the highest fluorescence enhancement of 19‐fold with the lowest detection limit of 0.17 μmol/L toward Zn²⁺. The corresponding linear range was at least from 0.6 to 6.0 μmol/L. Significantly, 1 showed an excellent selectivity toward Zn²⁺ over other metal ions including Cd²⁺.     

Morphological and functional characteristics of aging kidneys based on two‐photon microscopy in vivo

Age‐related kidney disease, which is chronic and naturally occurring, is a general term for a set of heterogeneous disorders affecting kidney structures and characterized by a decline in renal function. Age‐related renal insufficiency has important implications with regard to body homeostasis, drug toxicity and renal transplantation. In our study, two‐photon microscopy was used to image kidney morphological and functional characteristics in an age‐related rat model in vivo. The changes in morphology are analyzed based on autofluorescence and Hoechst 33342 labeling in rats with different ages. Structural parameters including renal tubular diameter, cell nuclei density, size and shape are studied and compared with Hematoxylin and Eosin histological analysis. Functional characteristics, such as blood flow, and glomerular filtration rate are studied with high‐molecular weight (MW) 500‐kDa dextran‐fluorescein and low‐MW 10‐kDa dextran‐rhodamine. Results indicate that morphology changes significantly and functional characteristics deteriorate with age. These parameters are potential indicators for evaluating age‐related renal morphology and function changes. Combined analyses of these parameters could provide a quantitative, novel method for monitoring kidney diseases and/or therapeutic effects of kidney drugs.     

Rapid identification of human ovarian cancer in second harmonic generation images using radiomics feature analyses and tree‐based pipeline optimization tool

Ovarian cancer is currently one of the most common cancers of the female reproductive organs, and its mortality rate is the highest among all types of gynecologic cancers. Rapid and accurate classification of ovarian cancer plays an important role in the determination of treatment plans and prognoses. Nevertheless, the most commonly used classification method is based on histopathological specimen examination, which is time‐consuming and labor‐intensive. Thus, in this study, we utilize radiomics feature extraction methods and the automated machine learning tree‐based pipeline optimization tool (TOPT) for analysis of 3D, second harmonic generation images of benign, malignant and normal human ovarian tissues, to develop a high‐efficiency computer‐aided diagnostic model. Area under the receiver operating characteristic curve values of 0.98, 0.96 and 0.94 were obtained, respectively, for the classification of the three tissue types. Furthermore, this approach can be readily applied to other related tissues and diseases, and has great potential for improving the efficiency of medical diagnostic processes.