Resolution Doubling in Live, Multicellular Organisms Via Multifocal Structured Illumination Microscopy

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2012 May 15

PMID 22581372

Citations 144

Authors

Andrew G York

Sapun H Parekh

Damian Dalle Nogare

Robert S Fischer

Kelsey Temprine

Marina Mione

Ajay B Chitnis

Christian A Combs

Hari Shroff

Affiliations

Soon will be listed here.

Abstract

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 μm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.

Citing Articles

Expanding super-resolution imaging versatility in organisms with multi-confocal image scanning microscopy.

Ren W, Guan M, Liang Q, Li M, Jin B, Duan G Natl Sci Rev. 2025; 11(9):nwae303.

PMID: 40040644 PMC: 11879394. DOI: 10.1093/nsr/nwae303.

Keratin intermediate filaments mechanically position melanin pigments for genome photoprotection.

Benito-Martinez S, Salavessa L, Mace A, Lardier N, Fraisier V, Sires-Campos J bioRxiv. 2025; .

PMID: 39868182 PMC: 11761041. DOI: 10.1101/2025.01.15.632531.

Narrowband photoblinking InP/ZnSe/ZnS quantum dots for super-resolution multifocal structured illumination microscopy enhanced by optical fluctuation.

Zhou L, Cao H, Huang L, Jing Y, Wang M, Lin D Nanophotonics. 2024; 12(9):1777-1785.

PMID: 39634114 PMC: 11501639. DOI: 10.1515/nanoph-2023-0033.

Bi-Plane Multicolor Scanning Illumination Microscopy with Multispot Excitation and a Distorted Diffraction Grating.

Li S, Zhang Y, Liao Z, Tian Z, Hashim H, Zeng Y Biosensors (Basel). 2024; 14(11).

PMID: 39590009 PMC: 11592293. DOI: 10.3390/bios14110550.

Extended-depth of field random illumination microscopy, EDF-RIM, provides super-resolved projective imaging.

Mazzella L, Mangeat T, Giroussens G, Rogez B, Li H, Creff J Light Sci Appl. 2024; 13(1):285.

PMID: 39384765 PMC: 11479626. DOI: 10.1038/s41377-024-01612-0.

References

Aman A, Piotrowski T . Wnt/beta-catenin and Fgf signaling control collective cell migration by restricting chemokine receptor expression. Dev Cell. 2008; 15(5):749-61. DOI: 10.1016/j.devcel.2008.10.002. View

Fiolka R, Shao L, Rego E, Davidson M, Gustafsson M . Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proc Natl Acad Sci U S A. 2012; 109(14):5311-5. PMC: 3325651. DOI: 10.1073/pnas.1119262109. View

Gustafsson M . Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 2000; 198(Pt 2):82-7. DOI: 10.1046/j.1365-2818.2000.00710.x. View

Rankin B, Moneron G, Wurm C, Nelson J, Walter A, Schwarzer D . Nanoscopy in a living multicellular organism expressing GFP. Biophys J. 2011; 100(12):L63-5. PMC: 3123922. DOI: 10.1016/j.bpj.2011.05.020. View

Pasapera A, Schneider I, Rericha E, Schlaepfer D, Waterman C . Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation. J Cell Biol. 2010; 188(6):877-90. PMC: 2845065. DOI: 10.1083/jcb.200906012. View

Shroff H, Galbraith C, Galbraith J, White H, Gillette J, Olenych S . Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci U S A. 2007; 104(51):20308-13. PMC: 2154427. DOI: 10.1073/pnas.0710517105. View

Chitnis A, Dalle Nogare D, Matsuda M . Building the posterior lateral line system in zebrafish. Dev Neurobiol. 2011; 72(3):234-55. PMC: 3376715. DOI: 10.1002/dneu.20962. View

Huang B, Jones S, Brandenburg B, Zhuang X . Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods. 2008; 5(12):1047-52. PMC: 2596623. DOI: 10.1038/nmeth.1274. View

Pampaloni F, Reynaud E, Stelzer E . The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 2007; 8(10):839-45. DOI: 10.1038/nrm2236. View

10.

Yi J, Wu X, Crites T, Hammer 3rd J . Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells. Mol Biol Cell. 2012; 23(5):834-52. PMC: 3290643. DOI: 10.1091/mbc.E11-08-0731. View

11.

Betzig E, Patterson G, Sougrat R, Lindwasser O, Olenych S, Bonifacino J . Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006; 313(5793):1642-5. DOI: 10.1126/science.1127344. View

12.

Rust M, Bates M, Zhuang X . Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006; 3(10):793-5. PMC: 2700296. DOI: 10.1038/nmeth929. View

13.

Donnert G, Keller J, Medda R, Andrei M, Rizzoli S, Luhrmann R . Macromolecular-scale resolution in biological fluorescence microscopy. Proc Natl Acad Sci U S A. 2006; 103(31):11440-5. PMC: 1518808. DOI: 10.1073/pnas.0604965103. View

14.

Berning S, Willig K, Steffens H, Dibaj P, Hell S . Nanoscopy in a living mouse brain. Science. 2012; 335(6068):551. DOI: 10.1126/science.1215369. View

15.

Mattila P, Lappalainen P . Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol. 2008; 9(6):446-54. DOI: 10.1038/nrm2406. View

16.

Muller C, Enderlein J . Image scanning microscopy. Phys Rev Lett. 2010; 104(19):198101. DOI: 10.1103/PhysRevLett.104.198101. View

17.

Nechiporuk A, Raible D . FGF-dependent mechanosensory organ patterning in zebrafish. Science. 2008; 320(5884):1774-7. DOI: 10.1126/science.1156547. View

18.

Hell S, Wichmann J . Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett. 2009; 19(11):780-2. DOI: 10.1364/ol.19.000780. View

19.

Shao L, Kner P, Rego E, Gustafsson M . Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Methods. 2011; 8(12):1044-6. DOI: 10.1038/nmeth.1734. View

20.

Jones S, Shim S, He J, Zhuang X . Fast, three-dimensional super-resolution imaging of live cells. Nat Methods. 2011; 8(6):499-508. PMC: 3137767. DOI: 10.1038/nmeth.1605. View