Application of an advanced maximum likelihood estimation restoration method for enhanced‐resolution and contrast in second‐harmonic generation microscopy

Abstract

Second-harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label-free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two-photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two-photon processes like two-photon fluorescence, it is challenging to apply the various PSF-engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ∼480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ∼26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro-nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.

Contemporary Circulating Enterovirus D68 Strains Infect and Undergo Retrograde Axonal Transport in Spinal Motor Neurons Independent of Sialic Acid

“…Neuron distance was measured linearly from the closest edge of the microgrooves to the center of the neuron nucleus using Olympus cellSens software. Immunofluorescent images taken for publication were imaged as a z-series and processed using advanced maximum likelihood constrained iterative deconvolution (52). In-focus planes were extracted and combined in a maximum z-stack projection for the final image.…”

Multiview Deconvolution Two-photon Laser Scanning Microscopy

“…The PSF of SHG depends strongly on the local sample structure making its de nitions challenging, and for this reason deconvolution applications have been limited 27 . SHG is a coherent phenomenon, emission is not isotropic as uorescence, and is mainly in the direction of the excitation beam.…”

A Confocal Reflection Super‐Resolution Technique to Image Golgi‐Cox Stained Neurons

“…Images of GNPs and GC speckles were deconvolved using the Autoquant blind deconvolution algorithm to test the impact of deconvolution in addition to the smaller pinholes, where the PSF is estimated from the supplied image using 20 constrained iterations, medium noise setting as shown in Figure and described in detail in Sivaguru et al . () and references cited in that article. Full width at half maximum (FWHM) values of individual GC speckles or gold‐nanoparticles in both XY and XZ dimensions were generated by conducting line profile analysis in Zeiss’ Zen software and/or ImageJ (NIH open source software; Rueden et al ., ).…”

“…Although diffraction limit is surpassed in fluorescence imaging (Sheppard, ; Hell & Wichmann, ; Gustafsson, ;), resolution improvement in other conventional optical techniques such as reflection and other modalities are constantly attempted (Ploem, ; Brakenhoff et al ., ; Koerten et al ., ; ; Cox et al ., ; Cox et al ., ; Brakenhoff et al ., ; Cornelesetenvelde et al ., ; Prins et al ., ; Brakenhoff & Muller, ; Prins et al ., ; Azeredo et al ., ; Ploem & Prins, ; Sivaguru et al ., ; Sivaguru et al ., ). Superresolution techniques such as stimulated emission and depletion (Hell & Wichmann, ), structured illumination (Gustafsson, ), Airyscan (Sheppard, ; Huff, ) and PALM/STORM (Betzig et al ., ) have recently been commercialised to go beyond diffraction limit of resolution in biological samples labelled with fluorescent targets.…”