3D Live Fluorescence Imaging of Cellular Dynamics Using Bessel Beam Plane Illumination Microscopy

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2014 Apr 12

PMID 24722406

Citations 88

Authors

Liang Gao

Lin Shao

Bi-Chang Chen

Eric Betzig

Affiliations

Soon will be listed here.

Abstract

3D live imaging is important for a better understanding of biological processes, but it is challenging with current techniques such as spinning-disk confocal microscopy. Bessel beam plane illumination microscopy allows high-speed 3D live fluorescence imaging of living cellular and multicellular specimens with nearly isotropic spatial resolution, low photobleaching and low photodamage. Unlike conventional fluorescence imaging techniques that usually have a unique operation mode, Bessel plane illumination has several modes that offer different performance with different imaging metrics. To achieve optimal results from this technique, the appropriate operation mode needs to be selected and the experimental setting must be optimized for the specific application and associated sample properties. Here we explain the fundamental working principles of this technique, discuss the pros and cons of each operational mode and show through examples how to optimize experimental parameters. We also describe the procedures needed to construct, align and operate a Bessel beam plane illumination microscope by using our previously reported system as an example, and we list the necessary equipment to build such a microscope. Assuming all components are readily available, it would take a person skilled in optical instrumentation ∼1 month to assemble and operate a microscope according to this protocol.

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References

Friedrich M, Gan Q, Ermolayev V, Harms G . STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution. Biophys J. 2011; 100(8):L43-5. PMC: 3077687. DOI: 10.1016/j.bpj.2010.12.3748. 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

Gao L, Shao L, Higgins C, Poulton J, Peifer M, Davidson M . Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens. Cell. 2012; 151(6):1370-85. PMC: 3615549. DOI: 10.1016/j.cell.2012.10.008. View

Neil M, Juskaitis R, Wilson T . Method of obtaining optical sectioning by using structured light in a conventional microscope. Opt Lett. 2008; 22(24):1905-7. DOI: 10.1364/ol.22.001905. View

Belanger P, Rioux M . Ring pattern of a lens-axicon doublet illuminated by a Gaussian beam. Appl Opt. 2010; 17(7):1080-8. DOI: 10.1364/AO.17.001080. View

Keller P, Schmidt A, Wittbrodt J, Stelzer E . Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science. 2008; 322(5904):1065-9. DOI: 10.1126/science.1162493. View

Ahrens M, Orger M, Robson D, Li J, Keller P . Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat Methods. 2013; 10(5):413-20. DOI: 10.1038/nmeth.2434. View

Krzic U, Gunther S, Saunders T, Streichan S, Hufnagel L . Multiview light-sheet microscope for rapid in toto imaging. Nat Methods. 2012; 9(7):730-3. DOI: 10.1038/nmeth.2064. View

Greger K, Swoger J, Stelzer E . Basic building units and properties of a fluorescence single plane illumination microscope. Rev Sci Instrum. 2007; 78(2):023705. DOI: 10.1063/1.2428277. View

10.

Zanacchi F, Lavagnino Z, Perrone Donnorso M, Del Bue A, Furia L, Faretta M . Live-cell 3D super-resolution imaging in thick biological samples. Nat Methods. 2011; 8(12):1047-9. DOI: 10.1038/nmeth.1744. View

11.

Keller P, Schmidt A, Santella A, Khairy K, Bao Z, Wittbrodt J . Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat Methods. 2010; 7(8):637-42. PMC: 4418465. DOI: 10.1038/nmeth.1476. View

12.

Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer E . Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. 2004; 305(5686):1007-9. DOI: 10.1126/science.1100035. View

13.

Schermelleh L, Heintzmann R, Leonhardt H . A guide to super-resolution fluorescence microscopy. J Cell Biol. 2010; 190(2):165-75. PMC: 2918923. DOI: 10.1083/jcb.201002018. View

14.

Durnin J, Miceli Jr J, Eberly J . Comparison of Bessel and Gaussian beams. Opt Lett. 2009; 13(2):79. DOI: 10.1364/ol.13.000079. View

15.

Majoul I, Gao L, Betzig E, Onichtchouk D, Butkevich E, Kozlov Y . Fast structural responses of gap junction membrane domains to AB5 toxins. Proc Natl Acad Sci U S A. 2013; 110(44):E4125-33. PMC: 3816413. DOI: 10.1073/pnas.1315850110. View

16.

Roh-Johnson M, Shemer G, Higgins C, McClellan J, Werts A, Tulu U . Triggering a cell shape change by exploiting preexisting actomyosin contractions. Science. 2012; 335(6073):1232-5. PMC: 3298882. DOI: 10.1126/science.1217869. View

17.

Planchon T, Gao L, Milkie D, Davidson M, Galbraith J, Galbraith C . Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat Methods. 2011; 8(5):417-23. PMC: 3626440. DOI: 10.1038/nmeth.1586. View

18.

Zanacchi F, Lavagnino Z, Faretta M, Furia L, Diaspro A . Light-sheet confined super-resolution using two-photon photoactivation. PLoS One. 2013; 8(7):e67667. PMC: 3699622. DOI: 10.1371/journal.pone.0067667. 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.

Habib S, Chen B, Tsai F, Anastassiadis K, Meyer T, Betzig E . A localized Wnt signal orients asymmetric stem cell division in vitro. Science. 2013; 339(6126):1445-8. PMC: 3966430. DOI: 10.1126/science.1231077. View