Visualizing astrocytes in the deep mouse brain in vivo

Astrocytes play a key role in the central nervous system. However, methods of visualizing astrocytes in the deep brain in vivo have been lacking. 3‐photon fluorescence imaging of astrocytes labeled by sulforhodamine 101 (SR101) is demonstrated in deep mouse brain in vivo. Excitation wavelength selection was guided by wavelength‐dependent 3‐photon action cross section (ησ ₃) measurement of SR101. 3‐photon fluorescence imaging of the SR101‐labeled vasculature enabled an imaging depth of 1340‐μm into the mouse brain. This justifies the deep imaging capability of the technique and indicates that the imaging depth is not determined by the signal‐to‐background ratio limit encountered in 2‐photon fluorescence imaging. Visualization of astrocytes 910 μm below the surface of the mouse brain in vivo is demonstrated, 30% deeper than that using 2‐photon fluorescence microscopy. Through quantitative comparison of the signal difference between the SR101‐labeled blood vessels and astrocytes, the challenges of visualizing astrocytes below the white matter is further elucidated.      

Comparison of higher‐order multiphoton signal generation and collection at the 1700‐nm window based on transmittance measurement of objective lenses

One benefit of excitation at the 1700‐nm window is the more accessible modalities of multiphoton signal generation. It is demonstrated here that the transmittance performance of the objective lens is of vital importance for efficient higher‐order multiphoton signal generation and collection excited at the 1700‐nm window. Two commonly used objective lenses for multiphoton microscopy (MPM) are characterized and compared, one with regular coating and the other with customized coating for high transmittance at the 1700‐nm window. Our results show that, fourth harmonic generation imaging of mouse tail tendon and 5‐photon fluorescence of carbon quantum dots using the regular objective lens shows an order of magnitude signal higher than those using the customized objective lens. Besides, the regular objective lens also enables a 3‐photon fluorescence imaging depth of >1600 μm in mouse brain in vivo. Our results will provide guidelines for objective lens selection for MPM at the 1700‐nm window.     

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.     

Deep‐brain three‐photon microscopy excited at 1600 nm with silicone oil immersion

Three‐photon microscopy excited at the 1700‐nm window (roughly covering 1600‐1840 nm) is especially suitable for deep‐brain imaging in living animals. To match the brain refractive index, D₂O has been exclusively used as the immersion medium. However, the hygroscopic property of D₂O leads to a decrease of transmittance of the excitation light and as a result a decrease in three‐photon signals over time. Solutions such as replacing D₂O from time to time, wrapping both the objective lens and the immersion D₂O, and sealing D₂O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D₂O for deep‐brain imaging. Excited at 1600 nm, our comparative deep‐brain imaging using both D₂O and silicone oil immersion show that silicone oil immersion yields 17% higher three‐photon signal in third‐harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three‐photon fluorescence imaging of vasculature up to 1460 μm (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long‐span three‐photon brain imaging excited at the 1700‐nm window.      

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.      

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.      

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness,cell‐specific multiphoton fluorescence brain imaging

We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.     

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.      

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

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)     

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.      

Slow Organic‐to‐Inorganic Sub‐Lattice Thermalization in Methylammonium Lead Halide Perovskites Observed by Ultrafast Photoluminescence

Carrier dynamics in methylammonium lead halide (CH₃NH₃PbI_3–xCl_x) perovskite thin films, of differing crystal morphology, are examined as functions of temperature and excitation wavelength. At room temperature, long‐lived (>nanosecond) transient absorption signals indicate negligible carrier trapping. However, in measurements of ultrafast photoluminescence excited at 400 nm, a heretofore unexplained, large amplitude (50%–60%), 45 ps decay process is observed. This feature persists for temperatures down to the orthorhombic phase transition. Varying pump photon energy reveals that the fast, band‐edge photoluminescence (PL) decay only appears for excitation ≥2.38 eV (520 nm), with larger amplitudes for higher pump energies. Lower photon‐energy excitation yields slow dynamics consistent with negligible carrier trapping. Further, sub‐bandgap two‐photon pumping yields identical PL dynamics as direct absorption, signifying sensitivity to the total deposited energy and insensitivity to interfacial effects. Together with first principles electronic structure and ab initio molecular dynamics calculations, the results suggest the fast PL decay stems from excitation of high energy phonon modes associated with the organic sub‐lattice that temporarily enhance wavefunction overlap within the inorganic component owing to atomic displacement, thereby transiently changing the PL radiative rate during thermalization. Hence, the fast PL decay relates a characteristic organic‐to‐inorganic sub‐lattice equilibration timescale at optoelectronic‐relevant excitation energies.     

Non‐Invasive Multi‐Dimensional Two‐Photon Microscopy enables optical fingerprinting (TPOF) of immune cells

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non‐invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two‐photon microscopy to non‐invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence‐based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients.

Two photon based autofluorescence features of immune cells enables non‐invasive differentiation.     

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

Selective cellular imaging with lanthanide‐based upconversion nanoparticles

Upconversion nanoparticles (UCNPs) with sodium yttrium fluoride, NaYF₄ (host lattice) doped with Yb³⁺ (sensitizer) and Er³⁺ (activator) were synthesized via hydrothermal route incorporating polyethyleneimine (PEI) for their long‐term stability in water. The cationic PEI‐modified UCNPs with diameter 20 ± 4 nm showed a zeta potential value of +36.5 mV and showed an intense, visible red luminescence and low‐intensity green emission with 976 nm laser excitation. The particles proven to be nontoxic to endothelial cells, with a 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, showing 90% to 100% cell viability, across a wide range of UCNP concentrations (0.3 ng/mL‐0.3 mg/mL) were used in multiphoton imaging. Multiphoton cellular imaging and emission spectroscopy data reported here prove that the UCNPs dispersed in cell culture media are predominantly concentrated in the cytoplasm than the cell nucleus. The energy transfer from PEI‐coated UCNPs to surrounding media for red luminescence in the biological system is also highlighted with spectroscopic measurements. Results of this study propose that UCNPs can, therefore, be used for cytoplasm selective imaging together with multiphoton dyes (eg, 4′,6‐diamidino‐2‐phenylindole (DAPI)) that are selective to cell nucleus.      

The whither of bacteriophytochrome‐based near‐infrared fluorescent proteins: Insights from two‐photon absorption spectroscopy

We present one‐ and two‐photon‐absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore–based single‐domain near‐infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV‐based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red‐shift tunability of currently available BV‐based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins. The absolute value of the two‐photon absorption (TPA) cross section of a fluorescent calcium sensor based on the studied iRFP is found to be significantly larger than the TPA cross sections of other widely used genetically encodable fluorescent calcium sensors.      

Lasing transition at 1.06 μm emission in Nd3+‐doped borate‐based tellurium calcium zinc niobium oxide glasses for high‐power solid‐state lasers

The spectroscopic properties of Tellurium Calcium Zinc Niobium oxide Borate (TCZNB) glasses of composition (in mol%) 10TeO₂ + 15CaO + 5ZnO + 10 Nb₂O₅ + (60 – x)B₂O₃ + Nd₂O₃ (x = 0.1, 0.5, 1.0 or 1.5 mol%) have been investigated experimentally. The three phenomenological intensity parameters Ω₂, Ω_4, Ω₆ have been calculated using the Judd–Ofelt theory and in turn radiative properties such as radiative transition probabilities, emission cross‐sections, branching ratios and radiative lifetimes have been estimated. The trend found in the JO intensity parameter is Ω₂ > Ω₆ > Ω₄ If Ω₆ > Ω₄, the glass system is favourable for the laser emission ⁴F_3/2 → ⁴I_11/2 in the infrared (IR) wavelength. The experimental values of branching ratio of ⁴F_3/2 → ⁴I_11/2 transition indicate favourable lasing action with low threshold power. The evaluated total radiative transition probabilities (A_T), stimulated emission cross‐section (σ_e) and gain bandwidth parameters (σ_e × Δλ_p) were compared with earlier reports. An energy level analysis has been carried out considering the experimental energy positions of the absorption and emission bands.     

Retracted: Single layer molybdenum disulfide as an optical nanoprobe for 2 photon luminescence and second harmonic generation cell imaging

More recently, tremendous progress has been achieved in the development of two‐dimensional semiconductor materials applied in catalyst, energy application, sensor device and bioengineering since the birth of graphene isolated from graphite. Layered molybdenum disulfide (MoS₂) as an indirect gap semiconductor can efficiently emit photoluminescence (PL) excited by visible light, which shows a great potential in adaptive biological imaging. However, 1 photon PL of MoS₂ for cell imaging purposes suffers from strong autofluorescence and ion‐induced PL quenching. Herein, we report single layer small chitosan decorated MoS₂ nanosheets as a nonbleaching, nonblinking optical nanoprobe under near infrared femtosecond laser excitation and their applications for strong 2 photon luminescence (TPL) and strong second harmonic generation (SHG) bioimaging. Furthermore, the TPL can resist the ion‐induced quenching on the cellular membrane. The proposed TPL and SHG of single‐layer MoS₂ show great potential for real‐time, deep, multiphoton and three‐dimensional bioimaging under low‐power laser excitation.      

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)     

Mechanism of dihydrofolate reductase downregulation in melanoma by 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐epicatechin

In our search to improve the stability and cellular absorption of tea polyphenols, we synthesized 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(?)‐epicatechin (TMECG), which showed high antiproliferative activity against melanoma. TMECG downregulates dihydrofolate reductase (DHFR) expression in melanoma cells and we detail the sequential mechanisms that result from this even. TMECG is specifically activated in melanoma cells to form a stable quinone methide (TMECG‐QM). TMECG‐QM has a dual action on these cells. First, it acts as a potent antifolate compound, disrupting folate metabolism and increasing intracellular oxidized folate coenzymes, such as dihydrofolate, which is a non‐competitive inhibitor of dihydropterine reductase, an enzyme essential for tetrahydrobiopterin (H₄B) recycling. Such inhibition results in H₄B deficiency, endothelial nitric oxide synthase (eNOS) uncoupling and superoxide production. Second, TMECG‐QM acts as an efficient superoxide scavenger and promotes intra‐cellular H₂O₂ accumulation. Here, we present evidence that TMECG markedly reduces melanoma H₄B and NO bioavailability and that TMECG action is abolished by the eNOS inhibitor N_ω‐nitro‐L ‐arginine methyl ester or the H₂O₂ scavenger catalase, which strongly suggests H₂O₂‐dependent DHFR downregulation. In addition, the data presented here indicate that the simultaneous targeting of important pathways for melanoma survival, such as the folate cycle, H₄B recycling, and the eNOS reaction, could represent an attractive strategy for fighting this malignant skin pathology. J. Cell. Biochem. 110: 1399–1409, 2010. © 2010 Wiley‐Liss, Inc.