Polarization dependence of aligned collagen tissues imaged with second harmonic generation microscopy

Abstract

A polarimetric second harmonic generation (SHG) microscope was used to analyze the dependence between polarization and SHG signal from collagen-based samples. A theoretical model was also developed to investigate the SHG intensity as a function of different polarization states for a set of quasiparallel fibers. Numerical simulations were compared to experimental SHG intensity values and a fairly good agreement was found. Linear polarized light produced periodical changes in the emitted SHG signal with a maximum of intensity corresponding to polarization parallel to the main orientation of the fibers, regardless the ratio of hyperpolarizabilities, ρρ . A similar behavior was found for elliptical states located along a vertical meridian on the Poincaré sphere (i.e., null azimuth) although the modulation of the SHG signal was different. Our numerical calculations described a dramatic change in this regular trend when ρρ changed from positive to negative values. Moreover, we provide an experimental method (based on the analysis of the modulation of the SHG signal) to determine the value of the ratio ρρ and, consequently, to obtain information about the internal organization of the collagen fibers.

Polarization response of second-harmonic images for different collagen spatial distributions

“…1). 11 In brief, a femtosecond laser (λ ω ¼ 760 nm, 76-MHz repetition rate) served as an illumination source. The PSG was composed of a fixed horizontal linear polarizer and a rotatory half-wave plate (λ∕2).…”

“…Although a strong polarization dependence in samples composed of quasialigned collagen fibers has been recently reported, 11 an indepth analysis of the sensitivity to polarization of collagen structures with different organization is lacking in the literature. In that sense, we explore the relationship between polarization and SHG signal for samples presenting different collagen arrangements.…”

“…This information can be extracted by computing the ratio of hyperpolarizabilities (ρ ¼ β xxx ∕β xyy ). 5,[21][22][23] It has been shown that the modulation of the SHG signal with polarization can be characterized by this ratio ρ, a parameter related to the internal collagen structure, 9,11 which decreases with collagen aging and loss of regular distribution (i.e., denaturation). 9,15,21,24,25 An objective tool to evaluate the external collagen organization (fiber distribution) in SHG images has recently been reported.…”

“…1,2 In particular, the dependence of second-harmonic generation (SHG) microscopy images of collagen-based tissues with incident polarization has been reported to better visualize certain features [3][4][5] and provide valuable information related to the structure and type of collagen fibers. [6][7][8] Although it is well known that polarization modulates the spatial distribution of the SHG signal from collagen fibers, this has only been analyzed in detail for regular patterns [9][10][11] (i.e., when the fibers are aligned along a preferential direction and the SHG signal is coherently produced). 12 However, the spatial distribution is not always uniform 13,14 and can be modified with aging, 3,15,16 mechanical damage, 17 and pathological processes [18][19][20] among others.…”

Comparison of second harmonic microscopy images of collagen-based ocular tissues with 800 and 1045 nm

“…A flip mirror (FM) allowed to select the desired beam and an aperture (AP) limited both beams to the same size. Both lasers were linearly polarized, that minimized any possible dependence between the SHG signal of collagen-based samples and the incident polarization [31][32][33]. For every analyzed specimen, the laser intensity at the sample's plane was set to be the same for both wavelengths (this was controlled with a variable neutral density filter, NDF).…”