Collagen is the most abundant protein in mammals and represents the main component of connective tissues, such as skin, cornea, artery or tendon. The three-dimensional multiscale organization of collagen is highly specific to every tissue and directly determines its physical and mechanical properties. This project aims at developing a new analytical method for in situ mapping of the fibrillar and denatured collagen multiscale structure in label-free biological tissues. To address this issue, infra-red nanospectroscopy (AFM-IR), which enables chemical mapping at nanometer scale, is combined to multiphoton microscopy based on Second Harmonic Generation (SHG) and 2PEF (two-photon excited fluorescence) signals, which probes collagen structure at micrometer scale. Optical signatures from multiphoton microscopy show that fibrillar collagen exhibits strong SHG signals and gelatin emits fluorescence signals. AFM-IR analysis shows that IR spectra exhibit amide I band and only in the case of gelatin an absorbing band around 1730 cm-1. Correlation of both techniques before and after denaturation on the same samples confirms this optical and chemical signatures of gelatinization process. The correlative imaging of IR nanospectroscopy and multiphoton microscopy of fibrillar collagen and gelatin structural states provide a calibration of the multiphoton signals that can be further used for the assessment of degradation of the collagen within tissues such as cornea or skin due to injuries or diseases.
Collagen is characterized by triple helical domains and plays a central role in the formation of fibrillar and
microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar
collagen exhibits efficient Second Harmonic Generation (SHG) and SHG microscopy proved to be a sensitive tool to
score fibrotic pathologies.
However, the nonlinear optical response of fibrillar collagen is not fully characterized yet and quantitative data are
required to further process SHG images. We therefore performed Hyper-Rayleigh Scattering (HRS) experiments and
measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large
considering that collagen presents no strong harmonophore in its amino-acid sequence. In order to get insight into the
physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple
helix and for a collagen-like short model peptide [(Pro-Pro-Gly)10]3. It showed that the collagen large nonlinear response
originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds,
resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we
successfully recorded SHG images in collagen liquid solutions by achieving liquid crystalline ordering of the collagen
triple helices.
We have performed Hyper-Rayleigh Scattering (HRS) experiments to measure the quadratic hyperpolarizability of
several natural amino acids, in particular tryptophan and tyrosine. Values of (29.6+/-0.4)x10-30 esu for tryptophan and
(25.7+/-0.03)x10-30 esu for tyrosine have been found. We have then investigated the dependence of the quadratic
hyperpolarizability of tryptophan-rich short peptides as a function of the number of tryptophans in the sequence. The
experimental findings indicate that the resulting quadratic hyperpolarizability in these peptides cannot be assumed as the
mere coherent superposition of the hyperpolarizabilities of the tryptophans contained in the peptide. Our results
unambiguously demonstrate that there must be strong interactions between the tryptophans contained in these short
peptides. We have also investigated the case of the collagen triple helix. A second order hyperpolarizability of (1.25+/-
0.05)x10-27 esu for rat-tail type I collagen has been measured. In this case, we have been able to model this effective
quadratic hyperpolarizability by summing coherently the nonlinear response of elementary moieties forming the triple
helix, as opposed to the previous case of the tryptophan-rich peptides.
Collagen is a major protein of the extracellular matrix that is characterized by triple helical domains. It plays a
central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of
the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) so that SHG
microscopy proved to be a sensitive tool to probe the
three-dimensional architecture of fibrillar collagen and to assess
the progression of fibrotic pathologies. We obtained sensitive and reproducible measurements of the fibrosis extent, but
we needed quantitative data at the molecular level to further process SHG images. We therefore performed Hyper-
Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail
type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its aminoacid
sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS
measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-
Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of
weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear
signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagenous
biomimetic matrices.
Two-photon microscopy is a key method for biological and medical research on cells and tissues mainly due to the submicronic spatial resolution. Unfortunately in its conventional form, this technique leads to long time recording for three-dimensional and fluorescence lifetime imaging because it requires a single point laser scanning. The most suitable way to improve acquisition time is to illuminate the biological sample with several excitation points simultaneously. We thus present a time-resolved multifocal multiphoton microscope. Besides the advantage of preserving biological samples by reducing by a factor 64 the exposition time, this method keeps also the possibility of measuring both intensity and lifetime images of the samples.
Urinary cytology is employed in diagnostic guidelines of bladder cancer in anatomo-pathological laboratories mostly for its ability to diagnose non detectable cancers using cystoscopy, but also because it is a non-invasive and non-constraining technique for a regular follow-up of the more exposed populations. The impossibility to detect such cancers is mainly due to their localization either in the bladder or in the upper urinary tract and the prostate. However, urinary cytology lacks sensitivity, especially for the detection of low grade low stage tumors due to inherent limitation of morphological criteria to distinguish low grade tumor cells from normal urothelial cells. For this purpose, we developed, in addition to urinary cytology, an original screening of these cytological slides by using spectrally-resolved and time-resolved fluorescence as a contrast factor, without changing any parameters in the cytological slide preparation. This method takes advantage of a femtosecond Ti:sapphire laser, continuously tunable in the spectral range 700-950 nm allowing the observation of most endogenous cellular chromophores by biphotonic excitation. A commercial confocal microscope was also used in the measurements allowing an excitation of the samples between 458 nm and 633 nm. We observed that the fluorescence emission is differentially distributed in normal and pathological urothelial cells. Spectral- and time-resolved measurements attested this difference over about one hundred cases which have been tested to confirm the high accuracy of this non-invasive technique.
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