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.     

Spectral characteristics of autofluorescence and second harmonic generation from ex vivo human skin induced by femtosecond laser and visible lasers

The spectral properties of one-photon, two-photon excited autofluorescence and second harmonic generation (SHG) from ex vivo human skin induced by a femtosecond (fs) laser and three visible lasers in backscattering geometry are systematically investigated. Our experimental results indicate that peak position of autofluorescence spectra from the dermis and epidermis shift toward long wavelengths, and the fluorescent intensity decreases when the excitation wavelength increases due to an effect of the excitation wavelength on autofluorescence signals. However, the intensity of the SHG signal in collagen has the maximal value of 800 nm excitation wavelength. This may be the result that the energy of the SHG signal is in resonance with an electronic absorption band. The two-photon excited autofluorescence and SHG intensity all obey a quadratical dependence on the excitation power. Compared with the two-photon excited fluorescence and SHG, the one-photon excited fluorescence in the dermis and epidermis exhibits different spectral characteristics. The investigation of the spectral characteristics of autofluorescence and SHG from ex vivo human skin can provide new insights into morphologic structures and biochemical components of tissues, which are vital for improving the application of laser-induced autofluorescence and SHG spectroscopy technique for noninvasive in vivo tissue diagnostics.     

Multiphoton microscopic imaging of adipose tissue based on second-harmonic generation and two-photon excited fluorescence

The fresh adipose tissue was investigated by the use of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second-harmonic generation (SHG). Microstructure of collagen and adipose cells in the adipose tissue is clearly imaged at a subcellular level with the excitation light wavelengths of 850 and 730 nm, respectively. The emission spectrum of collagen SHG signal and NADH and FAD fluorescence signal can also be obtained, which can be used to quantify the content of collagen and adipose cells and reflect the degree of pathological changes when comparing normal tissue with abnormal adipose tissue in the same condition. The results indicate that MPM has the potential to be applied to investigate the adipose tissue and can be used in the research field of lipid and connective tissues.     

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.     

Clinical two-photon microendoscopy

Two-photon medical imaging has found its way into dermatology as an excellent method for noninvasive skin cancer detection without need of contrast agents as well as for in situ drug screening of topically-applied cosmetical and pharmaceutical components. There is an increasing demand to apply the multiphoton technology also for deep-tissue skin imaging as well as for intracorporal imaging. We report on the first clinical use of multiphoton endoscopes, in particular of a miniaturized rigid two-photon GRIN lens endoscope. The microendoscope was attached to the multiphoton tomograph DermaInspect and employed to detect the extracellular matrix proteins collagen and elastin in the human dermis of volunteers and patients with ulcera by in vivo second harmonic generation and in vivo two-photon autofluorescence.     

Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging

The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435-700 nm) and second harmonic generation (SHG, 360-400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.     

Multiphoton microscopic imaging of normal human rectum tissue

In this paper, multiphoton microscopy (MPM), based on two‐photon excited fluorescence and second harmonic generation signals, was used to image microstructures of human rectal mucosa and submucosa. The morphology and distribution of the main components in mucosa layer, goblet cells, intestinal glands, and a little collagen fibers have been clearly monitored, and the content and distribution of collagen, elastic fibers, and blood vessels in submucosa layer have also been distinctly obtained. The variation of these components is very relevant to the pathology in gastrointestinal system, especially early rectal cancer. Our results indicate that the MPM technique has the potential application in vivo in the clinical diagnosis and monitoring of early rectal cancer. SCANNING 32: 347–350, 2010. © 2010 Wiley Periodicals, Inc.     

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.     

Label‐free imaging characteristics of colonic mucinous adenocarcinoma using multiphoton microscopy

Colorectal carcinoma (CRC) has high mortality and increased incidence rates. An early detection of CRC is very important. Multiphoton microscopy (MPM) with high resolution and high sensitivity is used to effectively distinguish the microstructure changes of normal and mucinous adenocarcinoma slices of ex vivo human colonic tissues. In mucinous adenocarcinoma mucosa, the glands are distorted and elongated, the gland cavity is indistinct, and the mesh collagen fibers are diminished. In the submucosa, the collagens are seriously disordered, elongated, pushed aside, and sparsely visible, the content of elastic fibers is also broken and almost disappearing. Many cancer cells, some in cavity‐like shape full of mucus surrounded by some collagen fibers, occupied the submucosa, which are comparable to hematoxylin‐eosin (HE) stained images. Second harmonic generation and two‐photon excitation fluorescence (SHG/TPEF) intensity ratio can be used further to quantitatively evaluate normality and abnormality. The fast Fourier transform (FFT) images show that the normal collagen fibrils are dense and in random order, and the cancerous collagen is certainly organized. The exploratory results show that it has potential for the development of multiphoton mini‐endoscopy in real‐time early diagnosis of CRC. SCANNING 35: 277‐282, 2013. © 2012 Wiley Periodicals, Inc.     

Real-time in vivo imaging collagen in lymphedematous skin using multiphoton microscopy

Changes of dermal collagen are characteristic for chronic lymphedema. To evaluate these changes, a real-time imaging based on two-photon excited fluorescence and second-harmonic generation was developed for investigating collagen of lymphedematous mouse and rat tail skin in vivo. Our findings showed that the technique could image the morphological changes and distribution of collagen in lymphedematous mouse and rat tail skin in vivo. More importantly, it may allow visualization of dynamic collagen alteration during the progression of lymphedema. Our findings demonstrated that multiphoton microscopy may have potential in a clinical setting as an in vivo diagnostic and monitoring system for therapy in lymphology.     

Visualization of Epidermal and Dermal Alteration in Papulonodular Mucinosis by Multiphoton Microscopy

Papulonodular mucinosis (PM) is a cutaneous clue to the presence and activity of silent lupus erythematosus (LE), but the exact pathogenesis is still under secret. Moreover, the currently available treatments for PM are not satisfactory. To demonstrate the possibility of multiphoton microscopy (MPM) to trace the pathological state of PM and evaluate the treatment efficacy, epidermal and dermal alteration in skin lesion with PM before and after treatment was examined using MPM. Microstructure of epidermis as well as content and distribution of collagen and elastin in dermis were quantified to characterize the pathological states of PM. The results showed significant morphological difference between skin lesion before and after treatment, indicating the possibility of MPM to assess the therapeutic efficacy. With the advancement on MPM miniaturization and enhancement of contrast and depth of imaging, the MPM technique can be applied in in vivo tracking PM formation and progression, and leading the better understanding the PM pathogenesis and mechanism of response to any treatment, helping to establish novel effective therapies for PM. SCANNING 35:22‐27, 2013. © 2012 Wiley Periodicals, Inc.     

Combined in vivo multiphoton and CARS imaging of healthy and disease-affected human skin

We present combined epi-coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging with both chemical discrimination and subcellular resolution on human skin in vivo. The combination of both image modalities enables label-free imaging of the autofluorescence of endogenous fluorophores by two-photon excited fluorescence, as well as imaging of the distribution of intercellular lipids, topically applied substances and water by CARS. As an example for medical imaging, we investigated healthy and psoriasis-affected human skin with both image modalities in vivo and found indications for different lipid distributions on the cellular level.     

Real-time noninvasive optical diagnosis for colorectal cancer using multiphoton microscopy

In contrast to colonoscopy biopsy, which contains several disadvantages such as bleeding, sampling error, crush artifact, and time-consuming pathological procedure, multiphoton microscopy (MPM) enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents. We performed a proof-of-principle study to evaluate the feasibility of using MPM to make real-time noninvasive optical diagnosis of colorectal cancer by investigating 30 fresh, unfixed, and unstained full-thickness colorectal specimens. We found that MPM images demonstrated irregular tubular structures, reduced stroma, and cellular and nuclear pleomorphism in the cancerous tissues. Cancer cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, were clearly observed in MPM images, which were comparable to golden standard hematoxylin-eosin staining images. Our findings showed that MPM had the potential to make real-time noninvasive optical diagnosis of colorectal cancer. With miniaturization and integration of colonoscopy, MPM has a promising future in real-time noninvasive "optical biopsy" for colorectal cancer.     

Multiphoton microscopic imaging of human normal and cancerous oesophagus tissue

In this paper, microstructures of human oesophageal submucosa are evaluated using multiphoton microscopy, based on two‐photon excited fluorescence and second harmonic generation. The content and distribution of collagen, elastic fibers and cancer cells in normal and cancerous submucosa layer have been distinctly obtained and briefly discussed. The variation of these components is very relevant to the pathology in oesophagus, especially in early oesophageal cancer. Our results further indicate that the multiphoton microscopy technique has the potential application in vivo in clinical diagnosis and monitoring of early oesophageal cancer.     

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.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Mapping piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation microscopy

Taking advantage of the electric field-enhanced second-harmonic generation effect in bulk gallium nitride (GaN) and indium gallium nitride (InGaN) quantum wells, we demonstrated the piezoelectric field distribution mapping in bulk GaN and InGaN multiple-quantum-well (MQW) samples using scanning second-harmonic generation (SHG) microscopy. Scanning SHG microscopy and the accompanying third-harmonic generation (THG) microscopy of the bulk GaN sample were demonstrated using a femtosecond Cr:forsterite laser at a wavelength of 1230 nm. Taking advantage of the off-resonant electric field-enhanced SHG effect and the bandtail state-resonance THG effect, the second- and third-harmonic generation microscopic images obtained revealed the piezoelectric field and bandtail state distributions in a GaN sample. Combined with 720 nm wavelength excited two-photon fluorescence microscopy in the same sample, the increased defect density around the defect area was found to suppress bandedge photoluminescence, to increase yellow luminescence, to increase bandtail state density, and to decrease residue piezoelectric field intensity. Scanning SHG microscopy of the InGaN MQW sample was resonant excited with 800 nm femtosecond pulses from a Ti:sapphire laser in order to suppress SHG contribution from the bulk GaN substrate. Taking advantage of the strong piezoelectric field inside the InGaN quantum well, the wavelength resonant effect, and the electric field-enhanced SHG effect of InGaN quantum wells, resonant scanning SHG microscopy revealed the piezoelectric field distribution inside the wells. Combined with accompanying three-photon fluorescence microscopy from the bulk GaN substrate underneath the quantum wells, the direct correspondence between the piezoelectric field strength inside the quantum well and the substrate quality can be obtained. According to our study, the GaN substrate area with bright bandedge luminescence corresponds to the area with strong SHG signals indicating a higher stained-induced piezoelectric field. These scanning harmonic generation microscopies exhibit superior images of the piezoelectric field and defect state distributions in GaN and InGaN MQWs not available before. Combining with scanning multiphoton fluorescence microscopy, these techniques open new ways for the physical property study of this important material system and can provide interesting details that are not readily available by other microscopic techniques.     

Imaging tissue engineering scaffolds using multiphoton microscopy

In this study, we combined two-photon autofluorescence and second harmonic generation imaging to investigate the three-dimensional microstructure and nonlinear optical properties of tissue engineering scaffolds. We focused on five different types of scaffold materials commonly used in tissue engineering, including: open-cell polylactic acid, polyglycolic acid, collagen composite scaffold, collagraft bone graft matrix strip, and nylon. By the use of multiphoton microscopy and a motorized stage, we obtained high resolution, spectrally resolved structural information of the scaffolds over large areas or in three-dimensions. Our results show that the nonlinear optical properties of the scaffolds will enable us to spectrally and morphologically distinguish the different types of scaffold materials investigated. We envision multiphoton microscopy to be a useful technique in tissue engineering applications in understanding the interplay between cultured cells and the scaffold materials.     

Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser

With its output wavelength covering the infrared penetrating window of most biological tissues at 1,200-1,250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti:sapphire laser. In this paper, we study the capability of using a femtosecond Cr:forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photoluminescence spectrum measurement on several commonly used bioprobes using the 1,230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two-photon or three-photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1,230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two-photon photoluminescence from chlorophyll, we demonstrated the two-photon scanning paradermal and cross-sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.     

Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy

Highly optically active nonlinear bio‐photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through second‐harmonic generation (SHG) interactions, behaving similarly to man‐made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third‐harmonic generation, and multiphoton‐induced fluorescence. With no energy release during harmonic generation processes, the nonlinear‐photonic‐crystal‐like SHG activity is useful for investigating the dynamics of structure–function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.