title: Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy.
abstract: Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼ 4-µm-deep volume, with lateral and axial localization precisions of ∼ 20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.
authors.affiliation: Laboratoire Physico-Chimie Curie, Institut Curie, CNRS UMR168, Université Pierre et Marie Curie-Paris 6, 75005 Paris, France; and Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147.
authors.forename: Bassam
authors.initials: B
authors.lastname: Hajj
authors.affiliation: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147.
authors.forename: Jan
authors.initials: J
authors.lastname: Wisniewski
authors.affiliation: Laboratoire Physico-Chimie Curie, Institut Curie, CNRS UMR168, Université Pierre et Marie Curie-Paris 6, 75005 Paris, France; and Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147.
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Beheiry
authors.affiliation: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147.
authors.forename: Jiji
authors.initials: J
authors.lastname: Chen
authors.affiliation: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147.
authors.forename: Andrey
authors.initials: A
authors.lastname: Revyakin
authors.affiliation: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147.
authors.forename: Carl
authors.initials: C
authors.lastname: Wu
authors.affiliation: Laboratoire Physico-Chimie Curie, Institut Curie, CNRS UMR168, Université Pierre et Marie Curie-Paris 6, 75005 Paris, France; and Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147 maxime.dahan@curie.fr.
authors.forename: Maxime
authors.initials: M
authors.lastname: Dahan
mesh_terms: D002138:Calibration; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins
keywords: 3D localization; microscopy; multiplane imaging; single-molecule fluorescence; superresolution