The Gustafsson Lab was dedicated to creating new forms of light microscopy for biological imaging.
The nature of its work was highly interdisciplinary - drawing from the toolboxes of physics, mathematics, engineering, computer science, and cell & molecular biology to develop and apply advanced imaging techniques. The Lab concentrated primarily on two areas:
- Developing microscopes with spatial resolution beyond the diffraction limit; such microscopes have been demonstrated to be compatible with live-cell imaging.
- Finding new ways of rapidly imaging three-dimensional specimens
Mats G. L. Gustafsson passed away in April 2011 after a courageous battle with brain cancer. In his honor, The Mats Gustafsson Memorial Symposium on High Resolution Imaging was held May 20 – 23, 2012 at Janelia Farm.
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Images and movies of the PNAS publication: Fiolka et al, Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination, http://www.pnas.org/content/early/2012/03/14/1119262109.full.pdf+html
Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.Nature methods 2013
S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. Darzacq, X. Darzacq, C. Wu, C. I. Bargmann, D. A. Agard, M. Dahan, and M. G L. Gustafsson Nature methods, 10:60-63 (2013)
Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.
Prior Publications (6)
Fast live simultaneous multiwavelength four-dimensional optical microscopy.Proceedings of the National Academy of Sciences of the United States of America 2010
P. M. Carlton, J. Boulanger, C. Kervrann, J. Sibarita, J. Salamero, S. Gordon-Messer, D. Bressan, J. E. Haber, S. Haase, L. Shao, L. Winoto, A. Matsuda, P. Kner, S. Uzawa, M. Gustafsson, Z. Kam, D. A. Agard, and J. W. Sedat Proceedings of the National Academy of Sciences of the United States of America, 107:16016-22 (2010)
Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.
Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.
Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.Science 2008
L. Schermelleh, P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, C. M. Cardoso, D. A. Agard, M. G L. Gustafsson, H. Leonhardt, and J. W. Sedat Science, 320:1332-6 (2008)
Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.
A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions.
Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.Biophysical Journal 2008
M. G L. Gustafsson, L. Shao, P. M. Carlton, R. C J. Wang, I. N. Golubovskaya, Z. W. Cande, D. A. Agard, and J. W. Sedat Biophysical Journal, 94:4957-70 (2008)
Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.