Tracing molecular dephasing in biological tissue

Abstract

Abstract.We demonstrate the quantitative spectroscopic characterization and imaging of biological tissue using coherent time-domain microscopy with femtosecond resolution. We identify tissue constituents and perform dephasing time (T 2 ) measurements of characteristic Raman active vibrations. This was shown in subcutaneous mouse fat embedded within collagen rich areas of the dermis and the muscle connective tissue. The demonstrated equivalent spectral resolution (<0.3 cm -1 ) is an order of magnitude better compared to commonly used frequency-domain methods for characterization of biological media. This provides with the important dimension and parameter in biological media characterization and can become an effective tool in detecting minute changes in bio-molecular composition and environment that is critical for molecular level diagnosis. 2Fundamental nonlinear optical phenomena have been shown to be useful in applications related to noninvasive characterization of biological media [1][2][3][4]. Raman scattering based techniques, both spontaneous and coherent versions, are of particular interest since their spectroscopic power can deliver molecular sensitive information that can become a key in early diagnosis of diseases. Absolute majority of the relevant applications of the techniques is, for natural reasons, in frequency domain. The coherent Raman microscopy studies were primarily applied to highlight tissue and cells constituent by producing high-contrast images at targeted Raman active vibration [5,6]. Spontaneous Raman version has been applied with greater focus towards detection of spectral features within cells and tissue [7]. However, the reported results have been limited to obtaining characteristic multi-line spectra and detecting relative changes in the intensities and spectral shifts with a goal to correlate those with biomolecular alterations occurring on sub-cellular level [8]. The true spectroscopic strength, that would ultimately include resolution of molecular vibration damping rates Γ (or linewidths, Δν=1/Γ) and line shapes, has not been enabled and demonstrated. It is worth noting that the damping rate is directly affected by inter-and intra-molecular interactions. Therefore, ability to measure Raman line shapes with a precision is absolutely crucial from that point of view. Depending on the immediate molecular environment (e.g. density, viscosity) and composition of tissue and cells, linewidths for the investigated Raman active modes can vary broadly. For instance, solute-solvent interactions in aqueous solutions can result in 0.3-7.5 cm -1 linewidth range that depend on the molar ratio [9].Both spontaneous Raman and frequency-domain have the best possible resolution of ~ 3-7 cm -1 .The limits are imposed by the detection sensitivity in spontaneous Raman spectroscopy. Laser pulse bandwidths (~3-10 cm -1 ) employed in coherent Raman microscopy does not provide a better resolution either. In addition, for the latter case the need to adjust laser wavelength in 3 point-by-point spectral measu...