Fast multicolor 3D imaging using aberration-corrected multifocus microscopy

Abrahamsson, Sara; Chen, Jiji; Hajj, Bassam; Stallinga, Sjoerd; Katsov, Alexander Y.; Wiśniewski, Jan; Mizuguchi, Gaku; Soule, Pierre; Mueller, Florian; Dugast-Darzacq, Claire; Darzacq, Xavier; Wu, Carl; Bargmann, Cornelia I.; Agard, David A.; Dahan, Maxime; Gustafsson, Mats

doi:10.1038/nmeth.2277

Cited by 411 publications

(436 citation statements)

References 18 publications

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“…High-speed light sheet microscopy, light field microscopy, multifocus microscopy, and two-photon structured illumination microscopy have proved effective for rapidly recording large numbers of neurons in immobilized, intact, transparent animals like larval zebrafish and nematodes (15)(16)(17)(18)(19). However, these methods are problematic when attempting to track many neurons within the bending and moving body of a behaving animal.…”

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confidence: 99%

Pan-neuronal imaging in roaming Caenorhabditis elegans

Venkatachalam

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

221

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We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.C. elegans | Drosophila | calcium imaging | thermotaxis U nderstanding how brain dynamics creates behaviors requires quantifying the flow and transformation of sensory information to motor output in behaving animals. Optical imaging using genetically encoded calcium or voltage fluorescent probes offers a minimally invasive method to record neural activity in intact animals. The nematode Caenorhabditis elegans is particularly ideal for optical neurophysiology owing to its small size, optical transparency, compact nervous system, and ease of genetic manipulation. Imaging systems for tracking the activity of small numbers of neurons have been effective in determining their role during nematode locomotion and navigational behaviors like chemotaxis, thermotaxis, and the escape response (1-6).Recordings from large numbers of interconnected neurons are required to understand how neuronal ensembles carry out the systematic transformations of sensory input into motor patterns that build behavioral decisions.Several methods for fast 3D imaging of neural activity in a fixed imaging volume have been developed for different model organisms (7)(8)(9)(10)(11)(12)(13)(14). High-speed light sheet microscopy, light field microscopy, multifocus microscopy, and two-photon structured illumination microscopy have proved effective for rapidly recording large numbers of neurons in immobilized, intact, transparent animals like larval zebrafish and nematodes (15)(16)(17)(18)(19). However, these methods are problematic when attempting to track many neurons within the bending and moving body of a behaving animal. Panneuronal recording in moving animals poses higher demands on spatial and temporal resolution. Furthermore, extracting neuronal signals from recordings in a behaving animal requires an effective analysis pipel...

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confidence: 99%

Pan-neuronal imaging in roaming Caenorhabditis elegans

Venkatachalam

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

221

216

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“…AFM cantilever lightsheet (by Gebhardt, J.C. et al [11]), lattice light-sheet (by Chen B.C. et al [56]), multi-focus (by Abrahamsson S. et al [57]. ), remote focusing (by Yang et al [58].…”

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confidence: 99%

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Leake

2018

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High-speed single-molecule fluorescence microscopy in vivo shows that transcription factors in eukaryotes can act in oligomeric clusters mediated by molecular crowding and intrinsically disordered protein. This finding impacts on the longstanding puzzle of how transcription factors find their gene targets so efficiently in the complex, heterogeneous environment of the cell.Abbreviations CDF - cumulative distribution function; FRAP - fluorescence recovery after photobleaching; GFP - Green fluorescent protein; STORM - stochastic optical reconstruction microscopy; TF - Transcription factor; YFP - Yellow fluorescent protein

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“…The distortion of the MFG acts on the emission wavefront to counterbalance this phase-shift profile (23). The induced phase shift scales as ð2π=λÞn λ aΔz (a is an integer between −4 and +4 specific to the order of diffraction).…”

Section: Resultsmentioning

confidence: 99%

“…Our method relies on the recently developed multifocus microscope (MFM) (23), which achieves simultaneous acquisition of nine equally spaced focal planes ( Fig. 1 C and D) on a single camera through the combination of a specialized diffractive grating and chromatic correction elements placed in the microscope emission path (Fig.…”

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confidence: 99%

Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy

Hajj

Wiśniewski

Beheiry

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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102

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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.superresolution | 3D localization | microscopy | multiplane imaging | single-molecule fluorescence

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Fast multicolor 3D imaging using aberration-corrected multifocus microscopy

Cited by 411 publications

References 18 publications

Pan-neuronal imaging in roaming Caenorhabditis elegans

Pan-neuronal imaging in roaming Caenorhabditis elegans

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy

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