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.     

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.     

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.     

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.     

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.     

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 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.     

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.     

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.     

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.     

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.     

In vivo three-dimensional imaging of plants with optical coherence microscopy

Achieving the ability to non‐destructively, non‐invasively examine subsurface features of living multicellular organisms at a microscopic level is currently a challenge for biologists. Optical coherence microscopy (OCM) is a new photonics‐based technology that can be used to address this challenge. OCM takes advantage of refractive properties of biological molecules to generate three‐dimensional images that can be viewed with a computer. We describe new data processing techniques and a different visualization algorithm that substantially improve OCM images. We have applied OCM imaging, in conjunction with these improvements, to a variety of structures of plants, including leaves, flowers, ovules and germinating seeds, and describe the visualization of cellular and subcellular structures within intact plants. We present evidence, based on detailed examination of our OCM images, comparisons to classical plant anatomy studies, and current knowledge of light scattering by cells and their components, that we can distinguish nuclei, organelles and vacuoles. Detailed examination of vascular tissue, which contains cells with elaborate wall structure, shows that cell walls produce no significant OCM signal. These improvements to the visualization process, together with the powerful non‐invasive, non‐destructive aspects of the technology, will broaden the application of OCM to questions in studies of plants as well as animals.     

A deep tissue fluorescence imaging system with enhanced SHG detection capabilities

We describe a novel two‐photon fluorescence microscopy system capable of producing high‐quality second harmonic generation (SHG) images in thick turbid media by using an innovative detection system. This novel detection system is capable of detecting photons from a very large surface area. This system has proven effective in providing images of thick turbid samples, both biological and artificial. Due to its transmission detection geometry, the system is particularly suitable for detecting SHG signals, which are generally forward directed. In this article, we present comparative data acquired simultaneously on the same sample with the forward and epidetection schemes. Microsc. Res. Tech. 77:368–373, 2014. © 2014 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.     

Frequency domain lifetime and spectral imaging microscopy

In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus.