Optical photon reassignment with increased axial resolution by structured illumination

Abstract

Fluorescent microscopy methods linked to the reassignment principle as image scanning microscopy (ISM), re-scan confocal (RSC), optical photon reassignment (OPRA) and instant structured illumination microscopy (iSIM) have the potential to replace confocal microscopy as the standard microscopy technique. Photon reassignment methods are known to link the most important properties in biological imaging as resolution, sensitivity, imaging speed and combinability with fluorophores in an elegant way. On the example of OPRA, we show how this method could be easily extended to the third dimension. If OPRA is used in combination with a structured illumination pattern the sectioning ability can be improved while maintaining the very high signal intensity. We present a detailed analysis about the imaging properties of OPRA in three dimensions and show experimental results on biological samples.

Movement of accessible plasma membrane cholesterol by the GRAMD1 lipid transfer protein complex

“…Spinning disc confocal (SDC) microscopy (Figures 3A, 4D, 7A,E, Figure 4—figure supplement 2A, Figure 4—figure supplement 3A, Figure 5—figure supplement 2A, Figure 7—figure supplement 1A, Figure 7—figure supplement 2A,E,G) and super-resolution SDC-structured illumination microscopy (SDC-SIM) (Figures 1B,C, 2C,H–I and Figure 1—figure supplement 1A,B) were performed on a setup built around a Nikon Ti2 inverted microscope equipped with a Yokogawa CSU-W1 confocal spinning head, a Plan-Apo objective (100 × 1.45 NA), a back-illuminated sCMOS camera (Prime 95B; Photometrics), and a super-resolution module (Live-SR; Gataca Systems) that was based on structured illumination with optical reassignment and image processing (Roth and Heintzmann, 2016). The method, known as multifocal structured illumination microscopy (York et al, 2012), makes it possible to double the resolution and the optical sectioning capability of confocal microscopy simultaneously.…”

Msps governs acentrosomal microtubule assembly and reactivation of quiescent neural stem cells

“…Super‐resolution Spinning disk confocal‐structured illumination microscopy (SDC‐SIM) were performed on a spinning disk system (Gataca Systems) based on an inverted microscope (Nikon Ti2‐E; Nikon) equipped with a confocal spinning head (CSU‐W; Yokogawa), a Plan‐Apo objective (100× 1.45‐NA), a back‐illuminated sCMOS camera (Prime95B; Teledyne Photometrics), and a super‐resolution module (Live‐SR; GATACA Systems) based on structured illumination with optical reassignment technique and online processing leading to a two‐time resolution improvement (Roth & Heintzmann, 2016 ). The maximum resolution is 128 nm with a pixel size in super‐resolution mode of 64 nm.…”

Biallelic PDE2A variants: a new cause of syndromic paroxysmal dyskinesia

“…Primary skin fibroblasts from patient#1 and #3 and a control individual were grown in DMEM supplemented with 10% fetal calf serum and 1% penicillin–streptomycin–glutamin at 37°C with 5% CO 2 . Cells incubated with 50 nM Mitotracker™ Red CMXRos (Invitrogen) for 30 min were fixed with paraformaldehyde 4% prior to observation on a fluorescence microscope (Zeiss Axio Observer D1) in Vectashield Antifade Mounting Medium bearing DAPI to stain the nucleus [ 10 ]. 100 cells were counted for each individual in three independent experiments, and the percentage of cells displaying aberrant mitochondria phenotype was determined, the mean was calculated and significance was determined using a T test.…”