Strategic and Practical Guidelines for Successful Structured Illumination Microscopy

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2017 Apr 14

PMID 28406496

Citations 116

Authors

Justin Demmerle

Cassandravictoria Innocent

Alison J North

Graeme Ball

Marcel Muller

Ezequiel Miron

Atsushi Matsuda

Ian M Dobbie

Yolanda Markaki

Lothar Schermelleh

Affiliations

Soon will be listed here.

Abstract

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d.

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