Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

Abstract

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence.

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

“…The use of other DNA repairs foci like RAD51, MDC1 for DSBs and XRCC1, for example for non-DSBs maybe can help toward the optimization of detection according to the type of genotoxic stress [58]. In addition, very recent advances in fluorescence microscopy harnessing previously unused fluorescence or DNA autofluorescence by a 532-nm laser excitation [59] may improve the detection of protein distributions especially located in a complex form [60].…”

Accelerating iterative deconvolution and multiview fusion by orders of magnitude

“…In quadruple-view deconvolution, we start with the additive RLD update, finding as previously reported 11 that this method yields better reconstructions than the alternating joint deconvolution update used for diSPIM: Finally, setting b to be the unmatched WB filter appropriate for each view provides the fastest update, as for dual-view and single-view microscopes.…”

“…In addition to deblurring, deconvolution can be used to combine multiple independent measurements taken on the same sample to produce an improved overall estimate of the sample 5 . This approach is especially useful in reconstructing super-resolution images in structured illumination microscopy 6,7 or in performing joint deconvolution to improve spatial resolution in multiview light-sheet microscopy [8][9][10][11][12] .…”

An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy

“…A line of knock-in parasites was constructed in which the single genomic copy of TgAPR1 was replaced with TgAPR1-mCherryFP ( Fig. 11) by homologous recombination (Wu et al, 2016).…”