Abstract
Structured Illumination Microscopy (SIM) is a super-resolution microscopy method that has significantly advanced studies of cellular structures. It relies on projection of illumination patterns onto a fluorescently labelled biological sample. The information derived from the sample is then shifted to a detectable band, and in the process of image calculation in Fourier space the resolution is doubled. Refractive index homogeneity along the optical path is crucial to maintain a highly modulated illumination pattern necessary for high-quality SIM. This applies in particular to thick samples consisting of large cells and tissues. Surprisingly, sample mounting media for SIM have not undergone a significant evolution for almost a decade. Through identification and systematic evaluation of a number of non-hazardous, water-soluble chemical components of mounting media, we demonstrate an unprecedented improvement in SIM-image quality. Mounting solutions presented in this research are capable of reducing abundant light scattering which constitutes the limiting factor in 3D-SIM imaging of large Hodgkin’s lymphoma and embryonic stem cells as well as 10 µm tissue sections. Moreover, we demonstrate usefulness of some of the media in single molecule localisation microscopy. The results presented here are of importance for standardisation of 3D-SIM data acquisition pipelines for an expanding community of users.
Highlights
Structured Illumination Microscopy (SIM) is a fluorescence-based super-resolution microscopy method providing isotropic resolution improvement by a factor of 2 over diffraction-limited microscopy[1,2]
The nuclear DNA was stained using Hoechst 33258 DNA-binding dye. This dye was chosen for its short excitation and emission wavelengths that are refracted more than longer visible wavelengths. These wavelengths are highly absorbed by biological material, making them especially challenging in imaging thick samples[23]
We find the following explanation of low modulation contrast-to-noise ratio (MCNR) values, corresponding to low pattern quality (Supplementary Fig. S3) in Hodgkin’s lymphoma (HL) cells - the immersion-oil based adjustment aims at matching the optical properties of the sample with the properties encoded in the optical transfer function (OTF) used for the reconstruction
Summary
Structured Illumination Microscopy (SIM) is a fluorescence-based super-resolution microscopy method providing isotropic resolution improvement by a factor of 2 over diffraction-limited microscopy[1,2] It has been broadly recognised in cell biology research and applied in various studies including investigations of architecture of the cell nucleus[3,4], DNA repair processes[5], applied to microbiology[6] and many others. Highly contrasted illumination patterns in the imaged location of a sample are desired These aspects make SIM extremely sensitive to various inherent sample imperfections including refractive index (RI) discontinuity leading to scattering, spherical aberrations, and low photon numbers[9]. For instance wave-front was measured in scattering media and corrected with feedback-based algorithms to produce close to ideal SIM illumination patterns adapted to inherent sample imperfections[13] This approach enabled correction of the aberrations and restoration of the highly modulated illumination pattern within biological samples[14]. This approach, does not aim to reduce the effects of light scattering at refracting interfaces but rather to compensate for an overall mismatch between RI of the mounting medium and glass
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