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.     

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 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.     

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.     

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two‐photon excitation microscopy including 2‐photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2‐photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in‐tandem combination of 2‐photon fluorescence and second harmonic generated signal microscopy as two‐modality microscopy allows for in situ co‐localization imaging of various microstructural components in the whole‐mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2‐photon‐controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two‐modal 2‐photon microscopy/tomography, acting as an efficient and sensitive non‐injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non‐linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.     

A simple introduction to multiphoton microscopy

Multiphoton microscopy is a powerful technique based on complex quantum mechanical effects. Thanks to the development of turnkey mode-locked laser systems, multiphoton microscopy is now available for everyone to use without extreme complexity. In this short introduction, we describe qualitatively the important concepts underlying the most commonly used type of multiphoton microscopy (two-photon excitation). We elucidate how those properties lead to the powerful results that have been achieved using this technique. As with any technique, two-photon excitation microscopy has limitations that we describe, and we provide examples of particular classes of experiments where two-photon excitation microscopy is advantageous over other approaches. Finally, we briefly describe other useful multiphoton microscopy approaches, such as three-photon excitation and second harmonic generation imaging.     

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.     

Second‐harmonic imaging microscopy for identifying colorectal intraepithelial neoplasia

In this study, second‐harmonic imaging microscopy was used to monitor precancerous colorectal lesions at different stages. It was found that the morphology of glands and lamina propria in mucosa changes with the progression of colorectal diseases from normal to low‐grade intraepithelial neoplasia to high‐grade intraepithelial neoplasia and this microscopy has the ability of direct visualization of these warning symptoms. Furthermore, two morphologic variables were quantified to determine the changes of glands and collagen in lamina propria during the development of colorectal intraepithelial neoplasia. These results suggest that second‐harmonic imaging microscopy has the potential in label‐freely and effectively distinguishing between normal and precancerous colorectal tissues, and will be helpful for early diagnosis and treatment of colorectal diseases.     

Multiphoton microscopy in life sciences

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm^?2 to GW cm^?2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells. Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm^?2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.     

Imaging cellular responses to mechanical stimuli within three-dimensional tissue constructs

The cellular response to environmental cues is complex, involving both structural and functional changes within the cell. Our understanding of this response is facilitated by microscopy techniques, but has been limited by our ability to image cell structure and function deep in highly-scattering tissues or 3D constructs. A novel multimodal microscopy technique that combines coherent and incoherent imaging for simultaneous visualization of structural and functional properties of cells and engineered tissues is demonstrated. This microscopic technique allows for the simultaneous acquisition of optical coherence microscopy and multiphoton microscopy data with particular emphasis for applications in cell biology and tissue engineering. The capability of this technique is shown using representative 3D cell and tissue engineering cultures consisting of primary fibroblasts from transgenic green fluorescent protein (GFP) mice and GFP-vinculin transfected fibroblasts. Imaging is performed following static and dynamic mechanically-stimulating culture conditions. The microscopy technique presented here reveals unique complementary data on the structure and function of cells and their adhesions and interactions with the surrounding microenvironment.     

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.     

Combination of multiphoton and reflective confocal imaging of cornea

We combine reflective confocal microscopy with multiphoton microscopy to form a minimally invasive technique to observe the cornea. The two imaging modalities allow detection of complementary information from the cornea. The autofluorescence signal shows the cytoplasm of epithelial cells, and the second harmonic generation signal is used to detect collagen, found mostly in the stroma of the cornea. The reflective confocal imaging allows detection of epithelial cells and keratocytes in the stroma. The system is first tested on bovine cornea. Assessment of the result on the bovine eye will be used to evaluate the potential of the system as a technique for in vivo clinical application.     

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.     

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non‐destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four‐dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567–582, 2016. © 2016 Wiley Periodicals, Inc.     

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.     

Real‐time histological imaging of kidneys stained with food dyes using multiphoton microscopy

We have developed a real‐time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model‐mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real‐time diagnosis of kidney diseases. Microsc. Res. Tech. 78:847–858, 2015. © 2015 Wiley Periodicals, Inc.     

A component‐by‐component characterisation of high‐risk atherosclerotic plaques by multiphoton microscopic imaging

Aims: Atherosclerotic plaques vulnerable to rupture are almost always inflamed, and carry a large lipid core covered by a thin fibrous cap. The other components may include neovascularisation, intraplaque haemorrhage and spotty calcification. In contrast, stable plaques are characterised by a predominance of smooth muscle cells and collagen, and lipid core is usually deep seated or absent. This study is a proof of principle experiment to evaluate the feasibility of multiphoton microscopy (MPM) to identify aforementioned plaque components. Methods and Results: MPM is a nonlinear optical technique that allows imaging based on intrinsic tissue signals including autofluorescence and higher‐order scattering. In our study, MPM imaging was performed on morphologically diverse aortic and coronary artery plaques obtained during autopsy. Various histologically verified plaque components including macrophages, cholesterol crystals, haemorrhage, collagen and calcification were recognised by MPM. Conclusions: Recognition of the distinct signatures of various plaque components suggests that MPM has the potential to offer next‐generation characterisation of atherosclerotic plaques. The higher lateral resolution (comparable to histology) images generated by MPM for identifying plaque components might complement larger field of view and greater imaging depth currently available with optical coherence tomography imaging. As the next step MPM would need to be evaluated for intact vessel imaging ex vivo and in vivo.     

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.     

Correlation of two‐photon in vivo imaging and FIB/SEM microscopy

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two‐photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long‐term two‐photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three‐dimensional high‐resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.