Abstract
Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.
Highlights
Super-resolution optical fluorescence microscopy is rapidly gaining significant interest in the biomedical sciences [1]
We demonstrate the process of raw image data collection by 2P-LSM striped illumination and the reconstruction of super-resolved images by two popular open-source image reconstruction packages, fairSIM and SIM Toolbox [20,21]
We used three different resolution estimation methods, including full width at half maximum (FWHM) estimation, image decorrelation analysis [22] and circular average power spectral density (PSDca) analysis [23] in order to obtain a mostly unbiased determination of the gain in spatial resolution provided by our illumination scheme
Summary
Super-resolution optical fluorescence microscopy is rapidly gaining significant interest in the biomedical sciences [1] It permits the observation of biological objects and processes in their native environment on a length scale well below the diffraction limit. The known periodicity and orientation of these patterns can be used to compute images with approximately twice the spatial resolution in all directions. Even multi-photon fluorescence excitation and nonlinear optical microscopies have been realized in this way [16,17] These instruments are still complex and rather specialized, this clearly demonstrates that it should, in principle, be possible to simplify this form of SR-SIM even further and to achieve super-resolution microscopy even with unmodified laser scanning microscopes
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