Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)—nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices.     

Label-Free Detection of Breast Masses Using Multiphoton Microscopy

Histopathology forms the gold standard for the diagnosis of breast cancer. Multiphoton microscopy (MPM) has been proposed to be a potentially powerful adjunct to current histopathological techniques. A label-free imaging based on two- photon excited fluorescence and second-harmonic generation is developed for differentiating normal breast tissues, benign, as well as breast cancer tissues. Human breast biopsies (including human normal breast tissues, benign as well as breast cancer tissues ) that are first imaged (fresh, unfixed, and unstained) with MPM and are then processed for routine H-E histopathology. Our results suggest that the MPM images, obtained from these unprocessed biopsies, can readily distinguish between benign lesions and breast cancers. In the tissues of breast cancers, MPM showed that the tumor cells displayed marked cellular and nuclear pleomorphism. The tumor cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, infiltrated into disrupted connective tissue, leading to the loss of second-harmonic generation signals. For breast cancer, MPM diagnosis was 100% correct because the tissues of breast cancers did not have second-harmonic generation signals in MPM imaging. On the contrary, in benign breast masses, second-harmonic generation signals could be seen easily in MPM imaging. These observations indicate that MPM could be an important potential tool to provide label-free noninvasive diagnostic impressions that can guide surgeon in biopsy and patient management.     

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

Background

Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression.

Methodology/Principal Findings

We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells.

Conclusions/Significance

NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.     

In vivo cytometry: a spectrum of possibilities.

BACKGROUND: We investigate whether optical imaging can reliably detect abnormalities in tissue, in a range of specimens (live cells in vitro; fixed, fresh ex-vivo and in vivo tissue), without the use of added contrast agents, and review our promising spectral methods for achieving quantitative, real-time, high resolution intrasurgical optical diagnostics. METHODS: We use reflectance, fluorescence, two-photon, and Mie scattering imaging, performed with instrumentation we developed or modified, to detect intrinsic tissue signatures. Emphasis is on spectral/hyperspectral imaging approaches allowing the equivalent of in vivo pathology. RESULTS: With experimental focus on unstained specimens, we demonstrate the ability to segment tissue images for cancer detection. Spectral reflectance imaging, coupled with advanced analysis, typically yields 90% specificity and sensitivity. Autofluorescence is also shown to be diagnostically useful, with lymph nodes results highlighted here. Elastic scattering hyperspectral imaging endoscopy, using a new instrument we designed and built, shows promise in bronchoscopic detection of dysplasia and early cancer in patients. CONCLUSIONS: The results demonstrate that advanced optical imaging can detect and localize cellular signatures of cancer in real-time, in vivo, without the use of contrast agents, in animals and humans. This is an important step towards tight spatio-temporal coupling between such detection and clinical intervention.     

Second harmonic generation imaging of endogenous structural proteins

We show that structural protein arrays consisting largely of collagen, myosin, and tubulin, and their associated proteins can be imaged in three dimensions with high contrast and resolution by laser-scanning second harmonic generation (SHG) microscopy. SHG is a nonlinear optical scheme and this form of microscopy shares several common advantages with multiphoton excited fluorescence, namely, intrinsic three-dimensionality and reduced out-of-plane photobleaching and phototoxicity. SHG does not arise from absorption and in-plane photodamage considerations are therefore also greatly reduced. In particular, structural protein arrays that are highly ordered and birefringent produce large SHG signals without the need for any exogenous labels. We demonstrate that thick tissues including muscle and bone can be imaged and sectioned through several hundred micrometers of depth. Combining SHG with two-photon excited green fluorescent protein (GFP) imaging allows inference of the molecular origin of the SHG contrast in Caenorhabditis elegans sarcomeres. Symmetry and organization of microtubule structures in dividing C. elegans embryos are similarly studied by comparing the endogenous tubulin contrast with that of GFP::tubulin fluorescence. It is found that SHG provides molecular level data on radial and lateral symmetries that GFP constructs cannot. The physical basis of SHG is discussed and compared with that of two-photon excitation as well as that of polarization microscopy. Due to the intrinsic sectioning, lack of photobleaching, and availability of molecular level data, SHG is a powerful tool for in vivo imaging.     

Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm². The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 × 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman’s layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.Dedicated on the occasion of the 66th birthday of Professor Dr. Karl-Juergen Halbhuber     

Nonlinear laser imaging of skin lesions

We investigated different kinds of human ex‐vivo skin samples by combined two‐photon intrinsic fluorescence (TPE), second‐harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two‐photon emission detection (MTPE). Morphological and spectroscopic differences were found between healthy and pathological skin samples, including tumors. In particular, we examined tissue samples from normal and pathological scar tissue (keloid), and skin tumors, including basal cell carcinoma (BCC), and malignant melanoma (MM). By using combined TPE‐SHG microscopy we investigated morphological features of different skin regions. Further comparative analysis of healthy skin and neoplastic samples was performed using FLIM, and MTPE. Finally, we demonstrated the use of methyl‐aminolevulinate as a contrast agent to increase the contrast in BCC border detection. The results obtained represent further support for in‐vivo noninvasive imaging of diseased skin. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Polarization‐dependent second‐harmonic generation for collagen‐based differentiation of breast cancer samples

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization‐dependent second‐harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second‐harmonic generation (SHG) anisotropy and the “ratio parameter” values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.     

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.     

A Fiber-Optic Fluorescence Microscope Using a Consumer-Grade Digital Camera for In Vivo Cellular Imaging

Background

Early detection is an essential component of cancer management. Unfortunately, visual examination can often be unreliable, and many settings lack the financial capital and infrastructure to operate PET, CT, and MRI systems. Moreover, the infrastructure and expense associated with surgical biopsy and microscopy are a challenge to establishing cancer screening/early detection programs in low-resource settings. Improvements in performance and declining costs have led to the availability of optoelectronic components, which can be used to develop low-cost diagnostic imaging devices for use at the point-of-care. Here, we demonstrate a fiber-optic fluorescence microscope using a consumer-grade camera for in vivo cellular imaging.

Methods

The fiber-optic fluorescence microscope includes an LED light, an objective lens, a fiber-optic bundle, and a consumer-grade digital camera. The system was used to image an oral cancer cell line labeled with 0.01% proflavine. A human tissue specimen was imaged following surgical resection, enabling dysplastic and cancerous regions to be evaluated. The oral mucosa of a healthy human subject was imaged in vivo, following topical application of 0.01% proflavine.

Findings

The fiber-optic microscope resolved individual nuclei in all specimens and tissues imaged. This capability allowed qualitative and quantitative differences between normal and precancerous or cancerous tissues to be identified. The optical efficiency of the system permitted imaging of the human oral mucosa in real time.

Conclusion

Our results indicate this device as a useful tool to assist in the identification of early neoplastic changes in epithelial tissues. This portable, inexpensive unit may be particularly appropriate for use at the point-of-care in low-resource settings.     

Label-free imaging of basement membranes differentiates normal, precancerous, and cancerous colonic tissues by second-harmonic generation microscopy

Since changes in the basement membranes are the critical indicators for differentiating normal, precancerous, and cancerous colonic tissues, direct visualization of these warning signs is essential for the early diagnosis and treatment of colonic cancer. Here, we present that second harmonic generation (SHG) microscopy can probe the changes of basement membranes in different colonic cancer stages. Our results also show the capability of using the quantitative analyses of images for quantifying these changes in different cancer stages. These results suggest that SHG microscopy has the potential in label-freely imaging the changes of basement membranes for effectively distinguishing between normal, precancerous, and cancerous colonic tissues. To our knowledge, this is the first demonstration of the dynamics of basement membrane changes in different colonic cancer stages using entirely intrinsic source of contrast.     

生物活组织的背向二次谐波成像

光学二次谐波成像技术由于具有三维高分辨率、不需要荧光标记、对生物样品的杀伤效应小等特点,在生物医学研究上具有广阔的应用前景.在双光子荧光成像基础上,实现了适合对厚组织样品观测的背向光学二次谐波成像,探讨了背向二次谐波成像的特点和影响因素.通过对多种生物组织样品进行大量实验,发现胶原纤维和肌肉纤维均可以产生很强的背向二次谐波,并成功地将背向二次谐波成像技术应用于糖尿病患者皮肤的观测.背向二次谐波成像技术可望推广到病理检查等临床应用中.     

Second- and third-harmonic generation microscopies for the structural imaging of intact tissues

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.     

Two-Photon Microscopy for Non-Invasive,Quantitative Monitoring of Stem Cell Differentiation

Background

The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed.

Methodology/Principal Findings

Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress.

Conclusions/Significance

In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner.     

A novel multiphoton microscopy images segmentation method based on superpixel and watershed

Multiphoton microscopy (MPM) imaging technique based on two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG) shows fantastic performance for biological imaging. The automatic segmentation of cellular architectural properties for biomedical diagnosis based on MPM images is still a challenging issue. A novel multiphoton microscopy images segmentation method based on superpixels and watershed (MSW) is presented here to provide good segmentation results for MPM images. The proposed method uses SLIC superpixels instead of pixels to analyze MPM images for the first time. The superpixels segmentation based on a new distance metric combined with spatial, CIE Lab color space and phase congruency features, divides the images into patches which keep the details of the cell boundaries. Then the superpixels are used to reconstruct new images by defining an average value of superpixels as image pixels intensity level. Finally, the marker‐controlled watershed is utilized to segment the cell boundaries from the reconstructed images. Experimental results show that cellular boundaries can be extracted from MPM images by MSW with higher accuracy and robustness.

     

High-Resolution Optical Molecular Imaging of Changes in Choline Metabolism in Oral Neoplasia

This study was aimed at developing an optical molecular imaging approach to measure differences in uptake and intracellular retention of choline in clinically isolated tissue biopsies from head and neck cancer patients. An optically detectable analogue of choline (propargyl choline) was synthesized and evaluated in 2D and 3D models and clinically isolated paired biopsies (n = 22 biopsies). Fluorescence contrast between clinically abnormal and normal tissues based on uptake and intracellular retention of propargyl choline was measured and correlated with pathologic diagnosis. Results in 2D and 3D models demonstrated a rapid uptake of propargyl choline in cancer cells, uniform permeation in tissue models, and specific detection of intracellular entrapped propargyl choline using the click chemistry reaction with an azide-modified Alexa 488 dye. Fluorescence imaging measurements following topical delivery of propargyl choline in clinically isolated biopsies showed that the mean fluorescence intensity (MFI) of neoplastic tissues was four-fold to five-fold higher than the MFI of clinically and pathologically normal samples. This difference in fluorescence contrast was measured on the basis of comparison of paired biopsy sets isolated from individual patients as well as comparison of clinically abnormal and normal biopsies independent of anatomic locations in the head and neck cavity and across diverse patients. In conclusion, a novel imaging approach based on monoalkyne-modified choline was developed and validated using cell and tissue models. Results in clinically isolated tissue biopsies demonstrate a significant fluorescent contrast between neoplastic and normal tissues and illustrate high specificity of the optical imaging approach.     

Image correlation spectroscopy of multiphoton images correlates with collagen mechanical properties

Multiphoton microscopy (MPM) holds promise as a noninvasive imaging technique for characterizing collagen structure, and thus mechanical properties, through imaging second harmonic generation (SHG) and two-photon fluorescence in engineered and real connective tissues. Controlling polymerization pH to manipulate collagen gel microstructure, we quantified pore and fiber dimensions using both standard methods and image correlation spectroscopy (ICS) on MPM, scanning electron, and darkfield microscopy images. The latter two techniques are used to confirm microstructural measurements made from MPM images. As polymerization pH increased from 5.5 to 8.5, mean fiber diameter decreased from 3.7 ± 0.7 μm to 1.6 ± 0.3 μm, the average pore size decreased from 81.7 ± 3.7 μm² to 7.8 ± 0.4 μm², and the pore area fraction decreased from 56.8% ± 0.8% to 18.0% ± 1.3% (measured from SHG images), whereas the storage modulus G′ and loss modulus G′, components of the shear modulus, increased ∼33-fold and ∼16-fold, respectively. A characteristic length scale measured using ICS, W_ICS, correlates well with the mean fiber diameter from SHG images (R² = 0.95). Semiflexible network theory predicts a scaling relationship of the collagen gel storage modulus (G′) depending upon mesh size and fiber diameter, which are estimated from SHG images using ICS. We conclude that MPM and ICS are an effective combination to assess bulk mechanical properties of collagen hydrogels in a noninvasive, objective, and systematic fashion and may be useful for specific in vivo applications.     

Wavelength- and Time-Dependence of Potentiometric Non-linear Optical Signals from Styryl Dyes

Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, complementing existing one- and two-photon fluorescence (2PF) methods. A non-linear phenomenon employing light from mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry, a constraint that is readily satisfied by lipid membranes with only one leaflet stained by a dye. Of particular interest is “resonance-enhanced” SHG from styryl dyes in cellular membranes and the possibility that SHG is sensitive to transmembrane potential. We have previously confirmed this, using simultaneous voltage-clamping and non-linear imaging of cells to find that SHG is up to four times more sensitive to potential than fluorescence. In this work, we have extended these results in two directions. First, with a range of wavelengths available from a mode-locked Ti:sapphire laser and a fiber-based laser, we have more fully investigated SHG and 2PF voltage-sensitivity from ANEP and ASTAP chromophores, obtaining SHG sensitivity spectra that are consistent with resonance enhancements. Second, we have modified our system to coordinate the application of voltage-clamp steps with non-linear image acquisition to more precisely characterize the time dependence of SHG and 2PF voltage sensitivity, finding that, at least for some dyes, SHG responds more slowly than fluorescence to changes in transmembrane potential.     

Quantitative second harmonic generation imaging of cartilage damage

Cartilage damage was studied using non-invasive multiphoton-excited autofluorescence and quantitative second harmonic generation (SHG) microscopy. Two cryopreservation techniques based upon freezing and vitrification methods, respectively, were employed to determine whether or not the collagen fiber structure of full thickness porcine articular cartilage was affected by cryopreservation and whether the level of collagen damage could be determined quantitatively in non-processed (non-fixed, non-sliced, non-stained) tissues. Multiphoton-induced autofluorescence imaging revealed the presence of chondrocytes, as well as collagenous structures in all fresh, vitrified and frozen cryopreserved cartilage samples. SHG imaging of the frozen cryopreserved specimens showed a dramatic loss of mean gray value intensities when compared to both fresh and vitrified tissues (P < 0.05), indicating structural changes of the extracellular matrix, in particular the deformation and destruction of the collagen fibers in the analyzed articular cartilage. A 0.9974 correlation coefficient was observed between the metabolic cell activity assessed by the alamarBlue technique, and retention of collagen structure between the three experimental groups. These studies suggest that multiphoton-induced autofluorescence imaging combined with quantitative SHG signal profiling may prove to be useful tools for the investigation of extracellular matrix changes in preserved cartilage, giving insights on the structural quality prior to implantation.