Calcium silicate cement‐induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate‐based restorative material (Biodentine^TM) and a glass ionomer cement (GIC:?Fuji?IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate‐free tetracycline, and tetracycline‐free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8‐week storage at 37 °C, dentine samples were imaged using two‐photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine^TM in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra‐ and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement‐free or PBS‐free media, or with GIC. It could therefore be concluded that Biodentine^TM induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate‐rich media, which was selectively detectable using the tetracycline labelling.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Spectra and lifetimes of fluorescence resonance energy transfer fluorophores under two-photon excitation

We show two-photon spectra and lifetimes acquired using conventional confocal microscopes equipped with an ultra-short pulsed laser and a time-gated intensified charge coupled device. We report on the two-photon spectra and lifetimes of Alexa350, enhanced green fluorescent protein (EGFP), EGFP-CD46, and Cy3 labelled antibodies. Cellular and extracellular EGFP two-photon spectra and lifetimes are compared.     

The effects of spherical aberration on multiphoton fluorescence excitation microscopy

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.     

Second harmonic generation imaging of the deep shade plant Selaginella erythropus using multifunctional two‐photon laser scanning microscopy

Background : Multifunctional two‐photon laser scanning microscopy provides attractive advantages over conventional two‐photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. Results : These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two‐photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. Conclusions : With the multifunctional two‐photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.     

Multiphoton imaging with a direct‐diode pumped femtosecond Ti:sapphire laser

A direct‐diode pumped Ti:sapphire femtosecond oscillator is used to perform multiphoton imaging for the first time.     

Label‐free identification of the microstructure of rat spinal cords based on nonlinear optical microscopy

The spinal cord is a vital link between the brain and the body and mainly comprises neurons, glial cells and nerve fibres. In this work, nonlinear optical (NLO) microscopy based on intrinsic tissue properties was employed to label‐freely analyze the cells and matrix in spinal cords at a molecular level. The high‐resolution and high‐contrast NLO images of unstained spinal cords demonstrate that NLO microscopy has the ability to show the microstructure of white and grey matter including ventral horn, intermediate area, dorsal horns, ventral column, lateral column and dorsal column. Neurons with various sizes were identified in grey matter by dark spots of nonfluorescent nuclei encircled by cytoplasm‐emitting two‐photon excited fluorescence signals. Nerve fibres and neuroglias were observed in white matter. Besides, the spinal arteries were clearly presented by NLO microscopy. Using spectral and morphological information, this technique was proved to be an effective tool for label‐freely imaging spinal cord tissues, based on endogenous signals in biological tissue. With future development, we foresee promising applications of the NLO technique for in vivo, real‐time assessment of spinal cord diseases or injures.     

The effects of refractive index heterogeneity within kidney tissue on multiphoton fluorescence excitation microscopy

Although multiphoton fluorescence excitation microscopy has improved the depth at which useful fluorescence images can be collected in biological tissues, the reach of multiphoton fluorescence excitation microscopy is nonetheless limited by tissue scattering and spherical aberration. Scattering can be reduced in fixed samples by mounting in a medium whose refractive index closely matches that of the fixed material. Using optical 'clearing', the effects of refractive index heterogeneity on signal attenuation with depth are investigated. Quantitative measurements show that by mounting kidney tissue in a high refractive index medium, less than 50% of signal attenuates in 100 μm of depth.     

Differentiating the extent of cartilage repair in rabbit ears using nonlinear optical microscopy

Nonlinear optical microscopy (NLOM) was used as a noninvasive and label‐free tool to detect and quantify the extent of the cartilage recovery. Two cartilage injury models were established in the outer ears of rabbits that created a different extent of cartilage recovery based on the presence or absence of the perichondrium. High‐resolution NLOM images were used to measure cartilage repair, specifically through spectral analysis and image texture. In contrast to a wound lacking a perichondrium, wounds with intact perichondria demonstrated significantly larger TPEF signals from cells and matrix, coarser texture indicating the more deposition of type I collagen. Spectral analysis of cells and matrix can reveal the matrix properties and cell growth. In addition, texture analysis of NLOM images showed significant differences in the distribution of cells and matrix of repaired tissues with or without perichondrium. Specifically, the decay length of autocorrelation coefficient based on TPEF images is 11.2 ± 1.1 in Wound 2 (with perichondrium) and 7.5 ± 2.0 in Wound 1 (without perichondrium), indicating coarser image texture and faster growth of cells in repaired tissues with perichondrium (p < 0.05). Moreover, the decay length of autocorrelation coefficient based on collagen SHG images also showed significant difference between Wound 2 and 1 (16.2 ± 1.2 vs. 12.2 ± 2.1, p < 0.05), indicating coarser image texture and faster deposition of collagen in repaired tissues with perichondrium (Wound 2). These findings suggest that NLOM is an ideal tool for studying cartilage repair, with potential applications in clinical medicine. NLOM can capture macromolecular details and distinguish between different extents of cartilage repair without the need for labelling agents.     

In vivo monitoring of intracellular chloroplast movements in intact leaves of c4 plants using two‐photon microscopy

Dynamic changes in the spatial distribution of chloroplasts are essential for optimizing photosynthetic capacity under changing light conditions. Light‐induced movement of chloroplasts has been widely investigated, but most studies were conducted on isolated tissues or protoplasts. In this study, a two‐photon microscopy (TPM) system was adapted to monitor the intracellular 3‐dimensional (3D) movements of chloroplasts in intact leaves of plants during dark to light transitions. The TPM imaging was based on autofluorescence of chlorophyll generated by a femto‐second Ti:Sapphire laser. All chloroplasts did not exhibit the same motion in response to irradiation variation. In the sub‐epidermal mesophyll cells, chloroplasts generally moved away from the surface following blue light treatment, however many chloroplasts did not show any movement. Such spatial heterogeneity in chloroplast motility underlines the importance of monitoring intracellular orientation and movement of individual chloroplasts across intact leaves. Our investigation shows that the 3D imaging of chloroplasts using TPM can help to understand the changes in local photosynthetic capacity in intact leaves under changing environmental conditions. Microsc. Res. Tech. 77:806–813, 2014. © 2014 Wiley Periodicals, Inc.     

Automatic quantification of mitochondrial fragmentation from two‐photon microscope images of mouse brain tissue

The morphology of mitochondria can inform about their functional state and, thus, about cell vitality. For example, fragmentation of the mitochondrial network is associated with many diseases. Recent advances in neuronal imaging have enabled the observation of mitochondria in live brains for long periods of time, enabling the study of their dynamics in animal models of diseases. To aid these studies, we developed an automatic method, based on supervised learning, for quantifying the degree of mitochondrial fragmentation in tissue images acquired via two‐photon microscopy from transgenic mice, which exclusively express Enhanced cyan fluorescent protein (ECFP) under Thy1 promoter, targeted to the mitochondrial matrix in subpopulations of neurons. We tested the method on images prior to and after cardiac arrest, and found it to be sensitive to significant changes in mitochondrial morphology because of the arrest. We conclude that the method is useful in detecting morphological abnormalities in mitochondria and, likely, in other subcellular structures as well.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.     

Multifunctional imaging of endogenous contrast by simultaneous nonlinear and optical coherence microscopy of thick tissues

A variety of high resolution optical microscopy techniques have been developed in recent years for basic and clinical studies of biological systems. We demonstrate a trimodal microscope combining optical coherence microscopy (OCM) with two forms of nonlinear microscopy, namely two-photon excited fluorescence (2PF) and second harmonic generation (SHG), for imaging turbid media. OCM combines the advantages of confocal detection and coherence gating for structural imaging in highly scattering tissues. Nonlinear microscopy enables the detection of biochemical species, such as elastin, NAD(P)H, and collagen. While 2PF arises from nonlinear excitation of fluorescent species, SHG is a form of nonlinear scattering observed in materials that lack a center of inversion symmetry, such as type I collagen. Characterization of the microscope showed nearly diffraction-limited spatial resolution in all modalities. Images were obtained in fish scales and excised human skin samples. The primary endogenous sources of contrast in the dermis were due to elastin autofluorescence and collagen SHG. Multimodal microscopy allows the simultaneous visualization of structural and functional information of biological systems.     

Label‐free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy

Glioblastoma multiforme (GBM‐WHO grade IV) is the most common and the most aggressive form of brain tumors in adults with the median survival of 10–12 months. The diagnostic detection of extracellular matrix (ECM) component in the tumour microenvironment is of prognostic value. In this paper, the fibrillar collagen deposition associated with vascular elements in GBM were investigated in the fresh specimens and unstained histological slices by using multiphoton microscopy (MPM) based on two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our study revealed the existence of fibrillar collagen deposition in the adventitia of remodelled large blood vessels and in glomeruloid vascular structures in GBM. The degree of fibrillar collagen deposition can be quantitatively evaluated by measuring the adventitial thickness of blood vessels or calculating the ratio of SHG pixel to the whole pixel of glomeruloid vascular structure in MPM images. These results indicated that MPM can not only be employed to perform a retrospective study in unstained histological slices but also has the potential to apply for in vivo brain imaging to understand correlations between malignancy of gliomas and fibrillar collagen deposition.     

Extension of imaging depth in two-photon fluorescence microscopy using a long-wavelength high-pulse-energy femtosecond laser source

We experimentally demonstrate, for the first time to the best of our knowledge, two-photon fluorescence imaging with a femtosecond optical parametric amplifier. In particular, we systematically compare the imaging depths of two-photon fluorescence microscopes based on three different excitation sources, including a femtosecond oscillator, a femtosecond regenerative amplifier and the optical parametric amplifier. The results show that the optical parametric amplifier can greatly extend the penetration depth by approximately 227% as compared with that obtained with the femtosecond oscillator due to effective suppression of scattering at longer wavelength and enhanced excitation efficiency enabled by higher pulse energy.     

Detailed characterisation of heavy alkylated benzene fluids by multipulse one‐ and two‐dimensional NMR spectroscopy

In this paper, the results of a characterisation of heavy alkylated benzenes (HAB) using multipulse NMR spectroscopy are presented. Four commercial samples of heavy alkylated benzene have been characterised in detail in terms of various structural parameters, such as percentage aromatic carbon (Ca), degree of aromatic substitution, average alkyl chain length, type of alkyl chain and their proportions. Three of the HAB samples were very similar in structure, whereas, in the fourth, the structures present were entirely different. The latter had an abundance of 2‐phenyl alkane‐type structures, and did not contain methyl substituted tetralin or naphthalene type molecules. The influence of the structures on lubricating oil properties, including viscosity index, pour point, and viscosity, as well as Freon^TM floc point, are also discussed.     

Torsion estimation of particle paths through porous media observed by in‐situ time‐resolved microtomography

The torsion distribution of cell paths in two‐phase flow of a cell suspension through a porous medium is an important quantity for chromatographic processes. It can be estimated from cell tracking in sequences of three‐dimensional images of a suspension pumped through the medium, where the images are acquired by in‐situ time‐resolved microtomography using synchrotron radiation and inline phase contrast modus. This contribution presents a new method for estimating path torsion from discrete positions, where the estimation is based on an appropriate discretization of the differential‐geometric formula of the torsion of a space curve. Investigations performed for an alcoholic suspension of silica gel particles pumped through a partially open polyurethane foam show that the shape of the inner surface of the foam has a considerable impact on the particle paths. It turns out that the variance of the torsion for slow particle moving close to the surface is a half order of magnitude smaller than that of fast particles, which underlines the importance of torsion for filter efficiency.