Structured illumination microscopy techniques use patterned excitation light and post-processing to double the resolution of a conventional microscope. Unlike other super-resolution techniques, they provide resolution enhancement at a relatively low illumination dose, thus enabling high resolution imaging over tens – hundreds of volumes. Previous implementations of SIM were limited to samples of < 10 um, but we have developed a multifocal version (MSIM)1 that permits resolution-doubling at depths exceeding 50 um. Most recently, we have extended the speed of MSIM 100x, facilitating super-resolution imaging at hundreds of frames per second.1
Selective plane illumination microscopy techniques combine a perpendicular excitation/detection geometry with light-sheet excitation, drastically reducing photobleaching and damage while providing higher signal-to-noise ratio and acquisition rates than confocal microscopy. Nevertheless, widespread adoption of SPIM has been slow because (1) the geometry is cumbersome, usually requiring special sample preparation; (2) Axial resolution is usually 3x worse than confocal microscopy. To address the usability issue, we have developed inverted selective plane illumination microscopy (iSPIM)2, a module that can be mounted onto an epifluorescence microscope, thus allowing conventional sample mounting on glass coverslips while retaining the advantages of SPIM. iSPIM offers an approximate 30-fold improvement over spinning disk confocal microscopy in terms of acquisition speed, photobleaching, and total acquisition length when imaging live samples. We have recently developed a variation of iSPIM that enables isotropic imaging with 330 nm spatial resolution (~4x better axial resolution than previous implementations) and temporal resolution down to 3 ms.
Along with extramural neuroscientists, scientists, and developmental biologists, we are using iSPIM to follow all neurons in the developing nematode embryo. We intend to create the first digital atlas of neurodevelopment. Further information on this project may be obtained at www.wormguides.org
1. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. York AG, Parekh SH, Dalle Nogare D, Fischer RS, Temprine K, Mione M, Chitnis AB, Combs CA, Shroff H. Nat Methods. 2012 May 13;9(7):749-54. doi: 10.1038/nmeth.2025.
2. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Wu Y, Ghitani A, Christensen R, Santella A, Du Z, Rondeau G, Bao Z, Colón-Ramos D, Shroff H. Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):17708-13. doi: 10.1073/pnas.1108494108. Epub 2011 Oct 17.
The Shroff lab currently houses:
- A photoactivated localization microscope (PALM) for 3D super-resolution (25-50 nm in XY and 100 nm in z).
- A multifocal structured illumination microscope with 150 nm lateral, 350 nm axial resolution and a time resolution of 5 ms.
- Two selective plane illumination microscopes, with isotropic spatial resolution down to 330 nm and temporal resolution of 5 ms.