Quantitative biomarkers of human skin photoaging based on intrinsic second harmonic generation signal

Collagen change is a major feature in the photoaged human skin. Here, we present the use of intrinsic second harmonic generation (SHG) signal as a novel means to quantify collagen change with photoaging. We obtain the SHG images of the superficial dermis from ex vivo the cheek skin and the abdomen skin of eight patients aged 55–60 years. The results show that SHG signal can quantitatively reveal collagen change between normal and photoaged human skin in three dimensions. By comparing normal with photoaged dermis, there are significant differences in the collagen content and fine structure, providing substantial potential to be applied in vivo for the clinical diagnosis of human skin photoaging. SCANNING 35: 273‐276, 2013. © 2012 Wiley Periodicals, Inc.     

Sequential multitrack nonlinear ex vivo imaging of esophageal stroma based on backscattered second-harmonic generation and two-photon autofluorescence

We demonstrate the technique of subsequent multitrack nonlinear imaging based on backscattered second-harmonic generation (B-SHG) and two-photon autofluorescence (TPA) to obtain large-area, high-contrast, submicron-resolution image ex vivo of esophageal stroma. Our findings show that this technique is effective in improving the B-SHG/TPA image contrast. It was found that the method can quantitatively obtain microscopic structural and biochemical information on stroma. Our work suggests that the technique has the potential to provide accurate and comprehensive information in determining the physiological and pathological states of the esophagus.     

Multiphoton microscopic imaging of in vivo hair mouse skin based on two-photon excited fluorescence and second harmonic generation

Mouse is an important animal model to investigate skin physiological and pathological states. In this article, multiphoton microscopic imaging of in vivo hair mouse skin based on two-photon excited fluorescence and second harmonic generation was examined. Our results show that multiphoton microscopy can clearly display microstructure of stratum corneum, stratum spinosum, and dermis of in vivo mouse skin. The main components of epidermis and dermis such as corneocytes, spinosum cell, collagen fibers, and hair follicles can be distinctly identified in MPM images. Using the optional HRZ 200 fine focusing stage, thickness of different layers can be easily assessed. The results demonstrate that MPM can be regarded as an efficient method for in vivo investigation of skin physiological and pathological states by using hair mouse animal model.     

Near-field imaging of surface-enhanced second harmonic generation

Surface-enhanced second harmonic generation from individual topographical defects of an otherwise flat gold film and from metal-coated diffraction gratings was measured using a near-field optical microscope. Experimentally measured second harmonic field distributions were compared with theoretical calculations.     

Label-free discrimination of normal and fibroadenomal breast tissues using second harmonic generation imaging

Early detection of fibroadenoma (FA) is critical for preventing subsequent breast cancer. In this work, we show that label-free second harmonic generation (SHG) imaging is feasible and effective in quantitatively differentiating the fibroadenomal tissue from normal breast tissue. With the advent of the clinical portability of miniature SHG microscopy, we believe that the technique has great potential in offering a noninvasive in vivo imaging tool for early detection of FA and monitoring the treatment responses of FA in clinics.     

High second harmonic generation signal from muscles and fascia pig's muscles using the two-photon laser scanning microscope

I have provided update to our two photon laser scanning microscope by adding new technique which enables us to simultaneously measured the second harmonic generation signals in the forward and backward directions; in the meantime, one can measure the two photon excitations fluorescence if the materials produce fluorescence. In the present work, the fascia muscles, muscles of pig and pig's skin were used. I found that these materials produced high second harmonic generation signal in both directions. These measurements show that the second harmonic generation strongly depends on the state of the polarization of the laser light and the orientation of the dipole moment in the molecules that interact with the laser light. It is therefore advantageous to control the laser's state of polarization, to maximize second harmonic generation. The novelty of this work is to establish new multi-functional technique by combing three platforms of laser scanning microscopy – the fluorescence microscopy, harmonic generation microscopy and polarizing microscopy in which one can use the second harmonic imaging to investigate the true architecture of the sensitive samples and the samples which do not produce auto-fluorescence. Moreover investigation of the new sample needs to look at all details of the true architecture of the sample. Thereby the sample will be exposed to the laser radiation more than the well-known sample, and that will cause photo-bleaching and photo-damage. Since the second harmonic generation does not undergo from photo-bleaching and photo-damage it will be the promising technique for investigating the sensitive and new samples. Then one can move to acquire fluorescence images after good investigation of the true architecture of the sample by the SH imaging.     

In vivo spatio-temporal visualization of the human skin by real-time confocal microscopy

A modified tandem scanning confocal microscope is used to obtain in vivo images of the human skin in real time. Three-dimensional and temporal visualizations are demonstrated with volume reconstruction and blood flow images. Two image processing methods based on Fourier transform and logarithmic processing are presented. Their applications in noise removal of the scanning disk lines and of the heterogeneity of light are illustrated.     

Imaging of mouse experimental melanoma in vivo and ex vivo by combination of confocal and nonlinear microscopy

We investigated possibilities of the combination of the one- and two-photon excitation microscopy for examination of the experimental melanoma tissue in vivo, in mice under general anesthesia, and ex vivo on freshly harvested specimens. Our aim was to obtain sufficiently informative images of unstained tumor tissues and their modifications after hyperthermia treatment. The mouse experimental melanoma structure was studied and compared with normal tissue from the same animal by using confocal and nonlinear microscopy techniques based on (i) one-photon excitation (1PE) fluorescence, (ii) 1PE reflectance, (iii) second harmonic generation imaging, and (iv) two-photon excitation autofluorescence. We checked different spectral conditions and other settings of image acquisition, as well as combinations of the above imaging modalities, to fully exploit the potential of these techniques in the evaluation of treated and untreated cancer tissue morphology. Our approach enabled to reveal the collagen fiber network in relation with the other tissues, and to identify invasive tumor cells. It also proved to be useful for the examination of interrelationships between functional and morphological aspects based on optical properties of the tissues, especially in studies of changes between the tumor and control tissue, as well as changes induced by physical treatments, e.g., delivery of microwave hyperthermia treatment. These differences were also evaluated quantitatively, when we found out that the maximum Euler–Poincaré characteristic reflects well the melanoma morphological structure. The results showed that the proposed investigative approach could be suitable also for a direct evaluation of tissue modifications induced by clinical interventions. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

In vivo confocal microscopy of human skin: A new design for cosmetology and dermatology

In-depth exploration of cellular structures in living human skin in situ is possible with the tandem scanning microscope (TSM). However, the rigid design of the microscope limited observations to the arms, hands, and fingers. A mobile version allowing the investigation of any parts of the body has been designed. The head containing the Nipkow disk and the optical path were the only part saved from the original TSM. This prototype can be used to observe, in real time, the different skin structures down to a depth of 200 μm and to measure the thickness of the different layers with micron precision level. The hydration of the stratum corneum (SC) could be assessed. For example, lengthy immersion of the hand in water led to an increase in SC thickness without affecting that of the living epidermis. Occlusive patch tests also showed that water and, even more so, propylene glycol, led to transient swelling of the SC. In dermatology, the example of psoriasis illustrated the value of the TSM for describing, measuring, and assessing pathologic skin changes. The availability of this noninvasive method for observing changes with time in a given skin site should prove useful for monitoring treatment efficacy. This tool opens up new insight for the investigation of cutaneous pathophysiology.     

Optical analysis of surfaces by second‐harmonic generation: Possible applications to tribology

Optical second‐harmonic generation is a recently developed technique in surface science, the range of applications of which has been steadily broadening. It allows, among other things, the direct probing of molecular adsorption on to a solid substrate from a liquid or gaseous environment. This paper reports on the possibility of applying it to tribological studies. A set of possible experiments that could offer information, in particular, on the working principle of those oil additives, commonly used in the lubricant industry, whose effect derives from surface adsorption, are discussed briefly. In addition, the preliminary results of a first experiment are described.     

The use of optical parametric oscillator for harmonic generation and two-photon UV fluorescence microscopy

Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1,050-1,300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.     

Two-photon fluorescence and second-harmonic generation imaging of collagen in human tissue based on multiphoton microscopy

Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. In this study, two-photon fluorescence and second-harmonic generation (SHG) imaging of collagen in human skin dermis and submucosa of colon and stomach tissues were investigated based on multiphoton microscopy (MPM). Our results show that multiphoton microscopic image of collagen bundles exhibits apparently different pattern in human tissues. The collagen bundles can simultaneously reveal its SHG and two-photon excited fluorescence images in the submucosa of colon and stomach, whereas it solely emit SHG signal in skin dermis. The intensity spectral information from tissues further demonstrated the above results. This indicates that collagen bundles have completely different space arrangement in these tissues. Our experimental results bring more detailed information of collagen for the application of MPM in human noninvasive imaging.     

Application of an advanced maximum likelihood estimation restoration method for enhanced‐resolution and contrast in second‐harmonic generation microscopy

Second‐harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label‐free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two‐photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two‐photon processes like two‐photon fluorescence, it is challenging to apply the various PSF‐engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ～480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ～26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro‐nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.     

In vivo second‐harmonic generation and ex vivo coherent anti‐stokes raman scattering microscopy to study the effect of obesity to fibroblast cell function using an Yb‐fiber laser‐based CARS extension unit

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near‐infrared spectral range (680–1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti‐Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes‐related skin disorders in the near future. Microsc. Res. Tech. 78:823–830, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope

A new noninvasive microscopic technique of three-dimensional optical biopsy from in vivo human skin based on real-time confocal microscopy and computer reconstruction is demonstrated. A tandem scanning confocal microscope is a prototype of a mobile, flexible design for the in-depth microscopic exploration of the skin on the human body. The various skin layers were observed in real-time, at the subcellular level down to a depth of 200 μm with a vertical resolution of 2 μm. Rapid video recording of the Z-series through the ventral aspect of the forearm avoided shifts caused by subject movement and blood flow pulsations. Two video frames were averaged, and the average was digitized, providing a stack of 64 optical sections in 1-μm vertical steps. Three-dimensional reconstructions of in vivo human skin were obtained with sets of orthogonal slices, and slices at arbitrary planes through a volume containing the stack of slices. This method clearly shows the spatial relationships between the different cell layers. The use of orthogonal cutting planes is preferred because of its analogy with classical vertical sections of histopathology. Linear structures (surface lines) within the stratum corneum are described and their global orientations were determined by the use of Fourier transform analysis. En face optical sections constitute unusual views of this tissue, since typical pathohistological studies are based on sagittal (vertical) slices. The noninvasive optical microscopic technique provides a three-dimensional optical biopsy of in vivo human skin.