Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

Abstract

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

Computational methods in super-resolution microscopy

“…In spinning disk ISM, multiple focal points are scanned in parallel with the help of a microlens array, so the image speed can be significantly enhanced (Schulz et al, 2013). Two-photon ISM uses longer wavelength excitation for better sectioning ability (Ingaramo et al, 2014).…”

Post-processing strategies in image scanning microscopy

“…This approach offers an improved signal to noise ratio and increased resolution with relatively little modification to the existing hardware of a laser--scanning microscope. There are now several different implementations of ISM, underpinned by the concept of 'pixel reassignment' [1--3], achieved either by optical (ISIM [4,5], OPRA [6]) or computational means (MSIM [7] & spinning disk ISM [8]. This manuscript will explore the potential of computational approaches utilizing methods drawn from single molecule localization microscopy for a posteriori pixel reassignment.…”

“…Assuming that both emission and excitation point spread functions are identical, as would be the case for single photon fluorescence with no Stokes shift, the probability of an excitation and detection event is maximal at the position midway between the excitation and detection maxima. Because the detected light is therefore most likely to have originated from this position, it can be 'reassigned' to a location half the original distance from the excitation focus [1,6,8]. Performing this for each pixel corresponds to scaling the image by a factor of ½ around the excitation focus.…”

“…Approximating the PSFs as Gaussian, the appropriate theoretical scaling factor is given by equation 1 [6]. In practice, a factor of ½ is used [see 5,6,8].…”

“…Consequently a uniformly fluorescing sample, such as a thin layer of fluorescein solution, is typically used as a reference to determine the positions of the excitation foci [7,8].…”