Short-wavelength optoacoustic spectroscopy based on water muting.

Infrared (IR) optoacoustic spectroscopy can separate a multitude of molecules based on their absorption spectra. However, the technique is limited when measuring target molecules in aqueous solution by strong water absorption at IR wavelengths, which reduces detection sensitivity. Based on the dependence of optoacoustic signal on the temperature of the probed medium, we introduce cooled IR optoacoustic spectroscopy (CIROAS) to mute water contributions in optoacoustic spectroscopy. We showcase that spectral measurements of proteins, lipids, and glucose in the short-wavelength IR region, performed at 4 °C, lead to marked sensitivity improvements over conventional optoacoustic or IR spectroscopy. We elaborate on the dependence of optoacoustic signals on water temperature and demonstrate polarity changes in the recorded signal at temperatures below 4 °C. We further elucidate the dependence of the optoacoustic signal and the muting temperature on sample concentration and demonstrate that changes in these dependences enable quantification of the solute concentration. We discuss how CIROAS may enhance abilities for molecular sensing in the IR.

Spatial and Spectral Mapping and Decomposition of Neural Dynamics and Organization of the Mouse Brain with Multispectral Optoacoustic Tomography.

In traditional optical imaging, limited light penetration constrains high-resolution interrogation to tissue surfaces. Optoacoustic imaging combines the superb contrast of optical imaging with deep penetration of ultrasound, enabling a range of new applications. We used multispectral optoacoustic tomography (MSOT) for functional and structural neuroimaging in mice at resolution, depth, and specificity unattainable by other neuroimaging modalities. Based on multispectral readouts, we computed hemoglobin gradient and oxygen saturation changes related to processing of somatosensory signals in different structures along the entire subcortical-cortical axis. Using temporal correlation analysis and seed-based maps, we reveal the connectivity between cortical, thalamic, and sub-thalamic formations. With the same modality, high-resolution structural tomography of intact mouse brain was achieved based on endogenous contrasts, demonstrating near-perfect matches with anatomical features revealed by histology. These results extend the limits of noninvasive observations beyond the reach of standard high-resolution neuroimaging, verifying the suitability of MSOT for small-animal studies.

Extended Near-Infrared Optoacoustic Spectrometry for Sensing Physiological Concentrations of Glucose.

Glucose sensing is pursued extensively in biomedical research and clinical practice for assessment of the carbohydrate and fat metabolism as well as in the context of an array of disorders, including diabetes, morbid obesity, and cancer. Currently used methods for real-time glucose measurements are invasive and require access to body fluids, with novel tools and methods for non-invasive sensing of the glucose levels highly desired. In this study, we introduce a near-infrared (NIR) optoacoustic spectrometer for sensing physiological concentrations of glucose within aqueous media and describe the glucose spectra within 850-1,900 nm and various concentration ranges. We apply the ratiometric and dictionary learning methods with a training set of data and validate their utility for glucose concentration measurements with optoacoustics in the probe dataset. We demonstrate the superior signal-to-noise ratio (factor of ~3.9) achieved with dictionary learning over the ratiometric approach across the wide glucose concentration range. Our data show a linear relationship between the optoacoustic signal intensity and physiological glucose concentration, in line with the results of optical spectroscopy. Thus, the feasibility of detecting physiological glucose concentrations using NIR optoacoustic spectroscopy is demonstrated, enabling the sensing glucose with ±10 mg/dl precision.

Optoacoustic detection of tissue glycation.

Oxidative-based diseases including diabetes, chronic renal failure, cardiovascular diseases and neurological disorders are accompanied by accumulation of advanced glycation endproducts (AGE). Therefore, AGE-associated changes in tissue optical properties could yield a viable pathological indicator for disease diagnostics and monitoring. We investigated whether skin glycation could be detected based on absorption changes associated with AGE accumulation using spectral optoacoustic measurements and interrogated the optimal spectral band for skin glycation determination. Glycated and non-glycated skin was optoacoustically measured at multiple wavelengths in the visible region. The detected signals were spectrally processed and compared to measurements of skin auto-fluorescence and to second harmonic generation multiphoton microscopy images. Optoacoustic measurements are shown to be capable of detecting skin glycolysis based on AGE detection. A linear dependence was observed between optoacoustic intensity and the progression of skin glycation. The findings where corroborated by autofluorescence observations. Detection sensitivity is enhanced by observing normalised tissue spectra. This result points to a ratiometric method for skin glycation detection, specifically at 540 nm and 620 nm. We demonstrate that optoacoustic spectroscopy could be employed to detect AGE accumulation, and possibly can be employed as a non-invasive quick method for monitoring tissue glycation.

Characterizing the morphologic changes in collagen crosslinked-treated corneas by Fourier transform-second harmonic generation imaging.

PURPOSE: To evaluate the efficacy of using forward second harmonic generation (SHG) and 2-dimensional fast Fourier-transform (2D-FFT) analysis for the label-free characterization and quantification of morphologic changes in the corneal stroma after collagen crosslinking (CXL). SETTING: Department of Physics, National Taiwan University, Taipei, Taiwan. DESIGN: Experimental study. METHODS: En face forward SHG imaging and 2D-FFT analysis were performed on ex vivo porcine corneas at the depths of 100, 200, 400, and 800 µm. Morphologic changes in stromal collagen fiber in control, ultraviolet-A (UVA), riboflavin, and riboflavin-UVA treated porcine corneas were assessed. Hematoxylin-eosin staining and Sirius red staining were performed for comparison. RESULTS: Corneas after CXL treatment tended to have collagen fibers that were wavy compared with the linear pattern in normal corneas. Quantitative 2D-FFT analysis of forward SHG images also showed an increase in the standard deviations of the distribution of stromal collagen fiber orientations, which is indicative of the changed pattern of crosslinked stromal collagen fibers. CONCLUSIONS: Second harmonic generation imaging showed the morphologic changes in stromal collagen after CXL treatment. The linear collagen fibers in normal corneal stroma became wavy after treatment. With the introduction of 2D-FFT analysis, the morphologic changes can be quantified.

Imaging of biological tissues with pixel-level analysis of second-order susceptibility.

We discuss the recent advances in the development and applications of second-order susceptibility as a contrast mechanism in optical microscopy for biological tissues. We review nonlinear optical methods and approaches for differentiation of tissue structures and discrimination of normal and pathological skin tissues, which have been demonstrated for the potential use in clinical diagnosis. In addition, the potential of second-order susceptibility imaging, encompassing applications in differentiating various types of collagen molecules for clinical diagnosis, is demonstrated. Finally, we discuss future development and application of this technique.

Analysis of collagen fiber domain organization by Fourier second harmonic generation microscopy.

We present an automated and systematic two-dimensional discrete Fourier transform (2D-FFT) approach to analyze collagen fiber organization through the use of second harmonic generation (SHG) microscopy. Average orientations of individual domains and Ising-like order parameters introduced to characterize the correlation between orientations of adjacent domains may be used to quantitatively characterize fibrous tissues. Our approach was applied to analyze tissues including rat tail tendon, mouse skin, bovine corneas, and human corneas. We also show that collagen fiber organization in normal and keratokonus human corneas may be distinguished. The current approach may be used for the quantitative differentiation of SHG collagen fiber morphology in different tissues and may be applied for diagnostic purposes.

Quantitative analysis of multiphoton excitation autofluorescence and second harmonic generation imaging for medical diagnosis.

In recent years, two-photon excitation fluorescence and second harmonic generation microscopy has become an important tool in biomedical research. The ability of two-photon microscopy to achieve optical sectioning with minimal invasiveness is particularly advantageous for biomedical diagnosis. Advances in the miniaturization of the imaging system have increased its clinical potential, together with the development of quantitative technique for the analysis of data acquired using these imaging modalities. We present a review of the quantitative analysis techniques that have been used successfully with two-photon excitation fluorescence and SHG imaging. Specifically, quantification techniques using ratiometric, morphological, and structural differences to analyze two-photon images will be discussed, and their effectiveness at evaluating dermal and corneal pathologies and cancerous tumor growth will be described.

Fast Fourier transform-based analysis of second-harmonic generation image in keratoconic cornea.

PURPOSE: To qualify and quantify structural alterations in keratoconic corneas ex vivo by the use of second-harmonic generation (SHG) microscopy and two-dimensional fast Fourier transform (2D-FFT) analysis. METHODS: Two keratoconic and three normal human corneal specimens were imaged with a multiphoton microscope. Forward and backward second-harmonic generation images (FSHG and BSHG) of corneal stroma were obtained at high resolution and at different depths. The SHG images were further analyzed with a 2D-FFT algorithm to quantify the texture and orientation changes of collagen fibers. RESULTS: The results showed that the abnormality of collagen architecture was found through the whole layer of stroma. 2D-FFT analysis of SHG cornea images provided fiber orientation direction and an aspect ratio (AR) as a quantitative measure of fiber direction determination. It was found that for keratoconic cornea the average AR values are statistically greater than those of the normal cornea: 1.66 ± 0.42 (case I keratoconic cornea), 1.72 ± 0.44 (case II keratoconic cornea), and 1.34 ± 0.16 (average of three normal human corneas). Furthermore, the fiber directions determined by 2D-FFT analysis of BSHG and FSHG images were strongly correlated for large AR values (AR > 2). CONCLUSIONS: The high correlation between FSHG and BSHG fiber direction for large AR values shows that BSHG imaging can provide qualitative and quantitative information of the extent of structural changes in a keratoconic cornea. The ability to use BSHG for diagnosing and monitoring stroma abnormalities, even when cornea transparency is retained, demonstrates the clinical potential of this method:

Spatial and temporal analysis of skin glycation by the use of multiphoton microscopy and spectroscopy.

BACKGROUND: Tissue glycation, the main cause of many diabetes-related complications, results in the accumulation of advanced glycation endproducts (AGE). OBJECTIVES: These AGEs are endogenous fluorophores that can serve as a viable pathological indicator for disease diagnostics. Here we explore the capabilities of multiphoton microscopy to non-invasively localize and quantify the skin glycation. METHODS: In our study, multiphoton microscopy and spectroscopy were used to investigate glycation events-induced changes in the intensities of autofluorescence and second harmonic generation on ex vivo human skin. RESULTS: Temporal and spatial dependence of degrees of glycation of the epidermis, collagen and elastin fibers of dermis were evaluated for their relevance to the changes in amplitudes of autofluorescence signals. We found that glycation drastically and linearly increases multiphoton autofluorescence intensity of epidermis and dermal collagen whereas changes in dermal elastin are moderate. We also found decrease in the level of second harmonic generation signal. CONCLUSION: Our study suggests that due to intrinsically weak autofluorescence the dermal collagen is the most sensitive skin tissue to be used for detecting changes in tissue glycation.

Photophysical mechanisms of collagen modification by 80 MHz femtosecond laser.

Photophysical mechanisms of collagen photomodification (CFP) by the use of a 80 MHz, 780 nm femtosecond titanium-sapphire laser were investigated. Our observation that the decrease in collagen second harmonic generation and increase in two-photon autofluorescence intensity occurred primarily at sites where photoproducts were present suggested that the photoproducts may act to facilitate the CFP process. Laser power study of CFP indicated that the efficiency of the process depended on the sixth power of the laser intensity. Furthermore, it was demonstrated that CFP can be used for bending and cutting of collagen fibers and creating 3D patterns within collagen matrix with high precision (~2 µm).

Multiphoton spectral microscopy for imaging and quantification of tissue glycation.

Tissue glycation from diabetes and aging can result in complications such as renal failure, blindness, nerve damage and vascular diseases. In this work, we applied multiphoton microscopy for imaging and characterizing the extent of tissue glycation. The characteristic features of multiphoton autofluorescence (MPAF) and second harmonic generation (SHG) images as well as MPAF spectra of glycated bovine skin, cornea and aorta were acquired. The analysis of MPAF intensity change accompanying the glycation process shows that collagen is more responsive to the formation of autofluorescent advanced glycation endproducts (AGE) than elastic fibers. Changes in spectral features were also used to estimate the rate of glycation in tissues with intrinsic AF. Our study shows that multiphton imaging may be used for the in vitro investigation of the effects of tissue glycation and that this approach may be used for monitoring AGE formation in the clinical setting.

Thermodynamics of interactions of water-soluble porphyrins with RNA duplexes.

We characterized the interactions of meso-tetrakis(4N-(2-hydroxyethyl)pyridinium-4-yl) porphyrin (TEtOHPyP4), meso-tetrakis(4N-allylpyridinium-4-yl) porphyrin (TAlPyP4), and meso-tetrakis(4N-metallylpyridinium-4-yl) porphyrin (TMetAlPyP4) with the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes between 18 and 45 degrees C by employing circular dichroism, light absorption, and fluorescence intensity spectroscopic measurements. Our results suggest that TEtOHPyP4 and TAlPyP4 intercalate into the poly(rA)poly(rU) and poly(rI)poly(rC) host duplexes, while TMetAlPyP4 associates with these RNA duplexes by forming outside-bound, self-stacked aggregates. We used our temperature-dependent absorption titration data to determine the binding constants and stoichiometry for each porphyrin-RNA binding event studied in this work. From the temperature dependences of the binding constants, we calculated the binding free energies, DeltaG(b), enthalpies, DeltaH(b), and entropies, DeltaS(b). For each RNA duplex, the binding enthalpy, DeltaH(b), is the most favorable for TEtOHPyP4 (an intercalator) followed by TAlPyP4 (an intercalator) and TMetAlPyP4 (an outside binder). On the other hand, for each duplex, external self-stacking of TMetAlPyP4 produces the most favorable change in entropy, DeltaS(b), followed by the intercalators TAlPyP4 and TEtOHPyP4. Thus, our results suggest that the thermodynamic profile of porphyrin-RNA binding may correlate with the binding mode. This correlation reflects the differential nature of molecular forces that stabilize/destabilize the two modes of binding-intercalation versus external self-stacking along the host duplex.