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

Abstract

Second harmonic generation (SHG) imaging is a well-suited multiphoton technique allowing visualization of biological tissues mainly composed of collagen with submicron resolution. Despite its inherent confocal properties, imaging of deeper layers within thick samples has still some limitations. Although the use of longer wavelengths might help to overcome this, the dependence between SHG signals and wavelength is still under discussion. We report here on the dependence with wavelength of SHG signals from collagen-based ocular tissues. The quality of SHG images for two commonly used excitation wavelengths (800 and 1045 nm) is studied. The analysis of the collagen structural information reveals that the information provided by both wavelengths is similar. It was also found that, independently of the depth location, 1045-nm SHG images presented always lower signal levels than those acquired with 800 nm. However, the contrast of the former images was higher, what may improve the visualization of certain features of interest. "Abnormalities of stromal structure in the bullous keratopathy cornea identified by second harmonic generation imaging microscopy," Invest.

Enhancement of second harmonic generation from three layers hybrid dielectric/metal/dielectric nanospheres

“…One of the critical NLO phenomena is the SHG, which includes the annihilation of two photons of the radiation beam at the fundamental frequency (FF) and the creation of one photon with a frequency equal double the FF. this phenomenon has always been attractive to researchers because of its application in various sciences such as spectroscopy [14], imaging [15], sensing [16], etc. In the dipole approximation, the second-order NLO phenomena are forbidden in the bulk of centrosymmetric materials.…”

Multiphoton Microscopy of Oral Tissues: Review

“…The MPM imaging modes are Two-Photon Excitation Fluorescence (2PEF), Three-Photon Excitation Fluorescence (3PEF), Second Harmonic Generation (SHG) and Third Harmonic Generation (THG), all of these being capable to image tissues in a label-free manner, based on their endogenous contrast. The use of infrared light sources allows deeper penetration into the tissues and reduced scattering [37,38], with reduced photodamage compared to other optical sectioning techniques working in the visible range, such as CLSM [39].…”

Calculations of second harmonic generation with radially polarized excitations by elliptical mirror focusing

“…There is no fluorescence label and energy absorption in SHG microscopy, which will cause no photodamage to biological samples (Stoller et al ., ,b; Chu et al ., ; Yasui et al ., ). SHG has been widely applied in microscopy imaging, such as rat‐tail tendon collagen (Stoller et al ., ,b), collagen‐based ocular tissues (Han et al ., ; Bueno et al ., ), tumours (Brown et al ., ), muscle (Both et al ., ) and the microstructure of nanodevices (Bautista et al ., ; Bautista et al ., ; Butet et al ., ). SHG only occurs within localized interaction region and has the ability to identify structural changes through the resolution of the tensor components of SHG emitting structure.…”

“…Tel: +860451-86412041; fax: + 860451-86402258; e-mail: wwbhit@hit.edu.cn 2002a,b; Chu et al, 2004;Yasui et al, 2004). SHG has been widely applied in microscopy imaging, such as rat-tail tendon collagen (Stoller et al, 2002a,b), collagen-based ocular tissues (Han et al, 2005;Bueno et al, 2017), tumours , muscle (Both et al, 2004) and the microstructure of nanodevices (Bautista et al, 2013;Bautista et al, 2015;Butet et al, 2015). SHG only occurs within localized interaction region and has the ability to identify structural changes through the resolution of the tensor components of SHG emitting structure.…”