Chemical imaging of biological tissue with synchrotron infrared light

Fourier transform infrared micro-spectroscopy (FTIRM) and imaging (FTIRI) have become valuable techniques for examining the chemical makeup of biological materials by probing their vibrational motions on a microscopic scale. Synchrotron infrared (S-IR) light is an ideal source for FTIRM and FTIRI due to the combination of its high brightness (i.e., flux density), also called brilliance, and broadband nature. Through a 10-μm pinhole, the brightness of a synchrotron source is 100-1000 times higher than a conventional thermal (globar) source. Accordingly, the improvement in spatial resolution and in spectral quality to the diffraction limit has led to a plethora of applications that is just being realized. In this review, we describe the development of synchrotron-based FTIRM, illustrate its advantages in many applications to biological systems, and propose some potential future directions for the technique.     

一种新型相干辐射--THz辐射在生物学中的应用

脉冲THz辐射是一种新型的远红外相干辐射源,近年来在不同的研究领域得到了广泛的应用。本文简要介绍THz辐射产生、探测的基本原理和方法;THz辐射的基本性质和它在生物学研究中应用的物理基础;对生物体系进行时域光谱分析和成像研究所取得的成果和最新进展,以及对该领域研究前景的展望。     

Biological and Biomedical Applications of Synchrotron Infrared Microspectroscopy

The high brightness of synchrotron light,which is about three orders of magnitudegreater than a thermal source, has beenexploited in biological and biomedicalapplications of infrared microspectroscopy. The potential of this analytical tool isdocumented in this article in the study ofhuman tissue (hair and skin) and individualcells: biochemical and bio-structuralchanges based on corresponding functionalgroups have been identified and imaged withunprecedented spatial resolution. Thistechnique also provides a new tool foranalysis of biochemical kinetics of samplesduring disease and treatment. In the future, the combination ofinfrared microspectroscopy with othersynchrotron-based microscopic techniques,such as X-ray microscopy, at the samesample location is discussed.     

A single molecule investigation of the photostability of quantum dots

Quantum dots (QDs) are very attractive probes for multi-color fluorescence imaging in biological applications because of their immense brightness and reported extended photostability. We report here however that single QDs, suitable for biological applications, that are subject to continuous blue excitation from a conventional 100 W mercury arc lamp will undergo a continuous blue-switching of the emission wavelength eventually reaching a permanent dark, photobleached state. We further show that β-mercaptoethanol has a dual stabilizing effect on the fluorescence emission of QDs: 1) by increasing the frequency of time that a QD is in its fluorescent state, and 2) by decreasing the photobleaching rate. The observed QD color spectral switching is especially detrimental for multi-color single molecule applications, as we regularly observe spectral blue-shifts of 50 nm, or more even after only ten seconds of illumination. However, of significant importance for biological applications, we find that even small, biologically compatible, concentrations (25 μM) of β-mercaptoethanol has a significant stabilizing effect on the emission color of QDs, but that greater amounts are required to completely abolish the spectral blue shifting or to minimize the emission intermittency of QDs.     

Improvement of low-level light imaging performance using optical clearing method

Low-level light-emitting imaging technique often detects the light emerged at the tissue surface that is generated internally from a specific target. However, in most cases, the high scattering nature of biological tissue limits the sensitivity and spatial resolution of this imaging modality. In this paper, we report that a significant improvement of chemiluminescence (CL) imaging performance in terms of both sensitivity and spatial resolution can be achieved by use of the topical application of glycerol solution onto tissue sample, i.e. optical clearing approach. Monte Carlo (MC) simulation of internally-launched point source shows that the decrease of scattering coefficient of turbid medium, which can be achieved by optical tissue clearing approach, causes stronger peak intensity with a narrower full-width at half-maximum (FWHM). The improvement becomes more significant with the source depth increasing from 1 to 5 mm. The experimental results shows that tissue clearing with 50% glycerol solution could largely improve the brightness and the spatial resolution of CL imaging when the target is covered by biological tissue with a thickness of either 1 or 3mm. This method could have potential applications for the in vivo low-level light imaging techniques.     

Validation of a laser driven plasma X-ray microfocus source for high resolution radiography imaging

Hard X-ray radiation with high brightness and high fluxes is nowadays available on the fourth generation of synchrotrons and X-FELs, but the large size and complexity of these sources makes its use difficult for widespread applications. New table top X-ray sources driven by ultrashort high power lasers offer a compelling route to expand the availability of hard X-ray sources. They can be used for advanced imaging techniques, due to its small source size and spatial coherence. We present in this paper the validation of a compact laser-driven X-ray microfocus source for high-resolution radiography imaging. This novel device was built at the Laser Laboratory for Acceleration and Applications (L2A2) at the University of Santiago de Compostela. This paper describes the laser-plasma X-ray source with improved stability and characterize some of its properties. We demonstrate the high-contrast and resolution of the images obtained with this source by using masks with well known geometries, and detailed analysis by using the modulation transfer function. Finally, we discuss the properties of this source in comparison to other compact microfocus X-ray sources.     

Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease

Alzheimer's disease (AD) is characterized by the misfolding and plaque-like accumulation of a naturally occurring peptide in the brain called amyloid beta (Abeta). Recently, this process has been associated with the binding of metal ions such as iron (Fe), copper (Cu), and zinc (Zn). It is thought that metal dyshomeostasis is involved in protein misfolding and may lead to oxidative stress and neuronal damage. However, the exact role of the misfolded proteins and metal ions in the degenerative process of AD is not yet clear. In this study, we used synchrotron Fourier transform infrared micro-spectroscopy (FTIRM) to image the in situ secondary structure of the amyloid plaques in brain tissue of AD patients. These results were spatially correlated with metal ion accumulation in the same tissue sample using synchrotron X-ray fluorescence (SXRF) microprobe. For both techniques, a spatial resolution of 5-10 microm was achieved. FTIRM results showed that the amyloid plaques have elevated beta-sheet content, as demonstrated by a strong amide I absorbance at 1625cm(-1). Using SXRF microprobe, we find that AD tissue also contains "hot spots" of accumulated metal ions, specifically Cu and Zn, with a strong spatial correlation between these two ions. The "hot spots" of accumulated Zn and Cu were co-localized with beta-amyloid plaques. Thus for the first time, a strong spatial correlation has been observed between elevated beta-sheet content in Abeta plaques and accumulated Cu and Zn ions, emphasizing an association of metal ions with amyloid formation in AD.     

Spectral attenuation of brain and retina tissues in the near‐infrared range measured using a fiber‐based supercontinuum device

A novel setup for the efficient constant optical measurements of biological tissues in the near infrared is presented. The system combines the use of a fiber‐based supercontinuum source with a simple optics fiber collimator. This configuration allows a wide spectral range of measurement and, at the same time, can efficiently filter the straightforward transmitted light while avoiding scattered light. As a performance example, the optical characterization of rat brain and retina tissues are shown. The attenuation coefficient for both tissues in the near infrared region is also obtained. This technique could be applied in clinical research as a noninvasive method with several potential practical applications.     

Single-molecule detection technologies in miniaturized high-throughput screening: fluorescence intensity distribution analysis

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence.     

Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.     

Molecular and morphological adaptations in compressed articular cartilage by polarized light microscopy and Fourier-transform infrared imaging

Fifteen articular cartilage-bone specimens from one canine humeral joint were compressed in the strain range of 0-50%. The deformation of the extracellular matrices in cartilage was preserved and the same tissue sections were studied using polarized light microscopy (PLM) and Fourier-transform infrared imaging (FTIRI). The PLM results show that the most significant changes in the apparent zone thickness due to 'reorganization' of the collagen fibrils based on the birefringence occur between 0% and 20% strain values, where the increase in the superficial zone and decrease in the radial zone thicknesses are approximately linear with the applied strain. The FTIRI anisotropy results show that the two amide components with bond direction perpendicular to the external compression retain anisotropy (amide II in the superficial zone and amide I in the radial zone). In contrast, the measured anisotropy from the two amide components with bond direction parallel to the external compression changes their anisotropy significantly (amide I in the superficial zone and amide II in the radial zone). Statistical analysis shows that there is an excellent correlation (r=0.98) between the relative depth of the minimum retardance in PLM and the relative depth of the amide II anisotropic cross-over. The changes in amide anisotropies in different histological zones are explained by the strain-dependent tipping angle of the amide bonds. These depth-dependent adaptations to static loading in cartilage's morphological structure and chemical distribution could be useful in the future studies of the early diseased cartilage.     

Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy

Synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy is a powerful bioanalytical technique for the simultaneous analysis of lipids, proteins, carbohydrates, and a variety of phosphorylated molecules within intact cells. SR-FTIR microspectroscopy can be used in the imaging mode to generate biospectroscopic maps of the distribution and intensity profiles of subcellular biomolecular domains at diffraction-limited spatial resolution. However, the acquisition of highly spatially resolved IR images of cells is not only a function of instrumental parameters (source brightness, sampling aperture size) but also the cell preparation method employed. Additionally, for the IR data to be biochemically relevant the cells must be preserved in a life-like state without introducing artefacts. In the present study we demonstrate, for the first time, the differences in biomolecular localizations observed in SR-FTIR images of cells fixed by formalin, formalin-critical point drying (CPD), and glutaraldehyde-osmium tetroxide-CPD, using the PC-3 prostate cancer cell line. We compare these SR-FTIR images of fixed cells to unfixed cells. The influence of chemical fixatives on the IR spectrum is discussed in addition to the biological significance of the observed localizations. Our experiments reveal that formalin fixation at low concentration preserves lipid, phosphate, and protein components without significantly influencing the IR spectrum of the cell.     

Transmission FT-IR microspectroscopy of mineral phases in calcified tissues

Fourier-transform infrared microspectroscopy (FT-IRM) was used to study bone mineralization processes in an in vivo model and in enamel in osteogenesis imperfecta. Finally, the ability of FT-IRM to map new bone formed in implanted macroporous calcium phosphate biomaterial from sections was reported for the first time. FTIRM allowed the correlation of the microstructure of bone formation in the in vivo model with modifications in carbonate and phosphate environments of the mineral phases during maturation. FT-IRM analysis on enamel sections revealed changes in the mineral environment of carbonate and phosphate ions and probably in the size of enamel crystals. These modifications contributed to the fragility of enamel in osteogenesis imperfecta. The infrared functional group imaging of a part of implanted biomaterial and the bone ingrowth provided the visualization of chemical modifications occurring in biomaterial implants at 20 μm spatial resolution. The use of FT-IRM, in conjunction with appropriate sampling methods and data analysis should provide further insight into the molecular structure of mineral phases of calcified tissues and help to elucidate mineralization processes, skeletal disorders and properties of the biomaterials used as bone substitute.     

In situ examination of the time-course for secondary mineralization of Haversian bone using synchrotron Fourier transform infrared microspectroscopy.

At the tissue level it is well established that the rate of remodeling is related to the degree of mineralization. However, it is unknown how long it takes for an individual bone structural unit (BSU) to become fully mineralized during secondary mineralization. Using synchrotron Fourier transform infrared microspectroscopy (FTIRM) we examined the time required for newly formed bone matrix to reach a physiological mineralization limit. Twenty-six, four-month old female New Zealand white rabbits were administered up to four different fluorochrome labels at specific time points to evaluate the chemical composition of labeled osteons from the tibial diaphysis that had mineralized for 1, 8, 18, 35, 70, 105, 140, 175, 210, 245, 280, 315, 350, and 385 days. Interstitial bone from 505 day old rabbits was used as a reference value for the physiological limit to which bone mineralizes. Using synchrotron FTIRM, area integrations were carried out on protein (Amide I: 1688-1623 cm(-1)), carbonate (v(2)CO(3)(2-): 905-825 cm(-1)), and phosphate (v(4)PO(4)(3-): 650-500 cm(-1)) IR bands. IR spectral data are presented as ratios of phosphate/protein (overall matrix mineralization) and carbonate/protein. The rate of mineralization of osteonal bone proceeded rapidly between day 1 and 18, reaching 67% of interstitial bone levels. This was followed by a slower, more progressive accumulation of mineral up to day 350. By 350 days the rate of increase plateaued. The ratio of carbonate/protein also increased rapidly during the first 18 days, reaching 73% of interstitial bone levels. The ratio of carbonate/protein plateaued by day 315, reaching levels not significantly different to interstitial bone levels. In conclusion, our data demonstrate that bone accumulates mineral rapidly during the first 18 days (primary mineralization), followed by a more gradual increase in the accumulation of mineral (secondary mineralization) which we found to be completed in 350 days.     

Safety assessment of near infrared light emitting diodes for diffuse optical measurements

Background  

Near infrared (NIR) light has been used widely to monitor important hemodynamic parameters in tissue non-invasively. Pulse oximetry, near infrared spectroscopy, and diffuse optical tomography are examples of such NIR light-based applications. These and other similar applications employ either lasers or light emitting diodes (LED) as the source of the NIR light. Although the hazards of laser sources have been addressed in regulations, the risk of LED sources in such applications is still unknown.     

太赫兹(THz)光谱在生物大分子研究中的应用

太赫兹(THz)辐射是一种新型的远红外相干辐射源,近年来,在生物大分子研究中得到了广泛的应用,特别是在生物分子的结构和动力学特性等方面有着巨大的应用潜力.结合THz光谱的特点,介绍了利用THz光谱对蛋白质、糖类及DNA等生物大分子的探索研究,以及THz技术在测定水环境与生物分子相互作用等方面的应用.探讨了该技术在生物学领域应用中有待解决的问题及发展前景.     

The ratio 1660/1690 cm(-1) measured by infrared microspectroscopy is not specific of enzymatic collagen cross-links in bone tissue

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm(-1) area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm(-1) by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm(-1) by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm(-1) area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm(-1) ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm(-1) was unmodified. In conclusion, the 1660/1690 cm(-1) is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process.     

Altered expression and localization of N-myristoyltransferase in experimentally induced rat model of ischemia-reperfusion

Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate in vitro to form a cartilaginous structure analogous to the epiphyseal growth plate. When inorganic phosphate, Pi, is included in the medium such that the total Pi concentration is 4 mM, apatite mineral precipitates around the "hypertrophic" chondrocytes. These hypertrophic chondrocytes are characterized by their increased expression of type X collagen, alkaline phosphatase activity, and apoptosis, as well as by the ability of their extracellular matrices to support mineral deposition. Under standard mineralizing conditions (0.8 x 10(6)cells/micromass; 4 mM Pi, 1.3 mM Ca(2+), 10% FCS, and antibiotics) mineralization does not commence until day 14-16. Based on the ability of bone morphogenic protein 6 (BMP-6) to stimulate chondrocyte maturation in other systems, 100 ng/ml BMP-6 was added to chick limb-bud mesenchymal cell cultures 2 and 5 days after plating, and the effects of this addition on mineral accretion and the characteristics of the mineral and matrix determined. Addition of BMP-6 accelerated the differentiation of the mesenchymal cells to hypertrophic chondrocytes. In the presence of BMP-6 added on both days 2 and 5, mineralization (assessed on basis of (45)Ca uptake) commenced by day 12. Fourier transform infrared imaging (FTIRI) was used to monitor the mineral content and mineral crystallinity as a function of time from day 9 to 21 in cultures with and without exogenous BMP-6. While BMP-6 accelerated the rate of mineral accretion, and the crystals that were formed in the BMP-6 cultures were initially more mature, by day 21 the crystal size distribution in experimental and control cultures were not significantly different. This study, the first to report the detailed application of FTIRI to cell cultures, indicates the importance of the extracellular matrix in the control of crystal maturation.     

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants <Emphasis Type="Italic">in vitro</Emphasis>

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)₂ from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)₂ were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.     

Vision in the edible snail. The morphology and total electrical activity of the retina]

Recording of the integrative electrical activity of the retina of the snail and electron microscopic study of its eye have shown the following. The electrical activity of the photoreceptors is a source of the retinogram (ERG) of the snail. ERG reaction form is characterized by two phases of a response: the initial spike and the following slow fading. For each given value of the photometric brightness of a light signal there exists a low limit of its length, beginning from which the ERG reaction of the snail assumes the form described. The value of the ERG response is a logarithmic function of brightness of the light stimulus.