Measure and model a 3-D space-variant PSF for fluorescence microscopy image deblurring

Abstract

Conventional deconvolution methods assume that the microscopy system is spatially invariant, introducing considerable errors. We developed a method to more precisely estimate space-variant point-spread functions from sparse measurements. To this end, a space-variant version of deblurring algorithm was developed and combined with a total-variation regularization. Validation with both simulation and real data showed that our PSF model is more accurate than the piecewise-invariant model and the blending model. Comparing with the orthogonal basis decomposition based PSF model, our proposed model also performed with a considerable improvement. We also evaluated the proposed deblurring algorithm. Our new deblurring algorithm showed a significantly better signal-to-noise ratio and higher image quality than those of the conventional space-invariant algorithm.

3D test sample for the calibration and quality control of super-resolution and confocal microscopes

“…This enables quality control of the microscope, with this particular setup being found to maintain a compact 3D STED PSF at depths of up to 80 µm. Furthermore, measurements of the PSFs at different heights enables accurate deconvolution and high precision analysis of images captured deep inside thick specimens [8][9][10] .…”

“…Calibration and alignment have become even more critical with the advent of super-resolution microscopy techniques, such as stimulated emission depletion (STED) microscopy [2][3][4] . Additionally, it has been demonstrated that image restoration with an experimentally measured point spread function (PSF) allows for better image restoration than deconvolution with a theoretical PSF 1,[5][6][7][8] . Measurements of the PSF in 3D is also of importance if one wants to achieve the maximum possible accuracy in the analysis of microscopy images 9,10 .…”

“…In recent work using (3D) STED in combination with glycerol objectives, super-resolution imaging far from the cover glass was demonstrated in life science specimens, where the refractive index is close to 1.45 1,22,23 . In addition, knowledge of the depth dependent PSF has been shown to be important for accurate 3D image reconstruction 8 and high accuracy image analysis 9,10 .…”

Gold/Silica Particles for Various Applications in (CLEM) Microscopy

“…Calibration and alignment have become even more critical with the advent of super-resolution microscopy techniques, such as stimulated emission depletion (STED) microscopy [122,123,124]. Additionally, it has been demonstrated that image restoration with an experimentally measured 'point response function', referred to as the point spread function (PSF), allows for better image restoration than deconvolution with a theoretical PSF [23,24,121,125,126,127]. Therefore, is it also important to be able to reliably measure the PSF of a setup.…”

“…With recent work using (3D) STED in combination with glycerol objectives, sub-resolution imaging far from the cover glass was demonstrated in life science specimens, where the refractive index is close to 1.45 [25,121,136]. In addition, knowledge of the depth dependent PSF has shown to be important for accurate 3D image reconstruction [127].…”