Image Scanning Microscopy and Metal Induced Energy Transfer: Enhancing Microscopy Resolution in All Directions
Event details
| Date | 14.10.2014 |
| Hour | 11:00 |
| Speaker | Prof. Jörg Enderlein, Georg August University, Göttingen (D) |
| Location | |
| Category | Conferences - Seminars |
BIOENGINEERING SEMINAR
Abstract:
Classical fluorescence microscopy is limited in resolution by the wavelength of light (diffraction limit) restricting lateral resolution to ca. 200 nm, and axial resolution to ca. 500 nm (at typical excitation and emission wavelengths around 500 nm). However, recent years have seen a tremendous development in high- and super-resolution techniques of fluorescence microscopy, pushing spatial resolution to its diffraction-dictated limits and much beyond. One of these techniques is Structured Illumination Microscopy (SIM). In SIM, the sample is illuminated with a spatially modulated excitation intensity distribution, and the emerging fluorescence is imaged with a conventional wide-field imaging setup. By moving and rotating the excitation intensity distribution pattern in different positions and orientations, taking each time a wide-field image, a final fluorescence image is composed which has roughly double the resolution (laterally) of a conventional wide-field or a confocal laser scanning image alone. A similar technique is Image Scanning Microscopy (ISM). In ISM, the focus of a conventional laser-scanning confocal microscope (LCSM) is scanned over the sample, but instead of recording only the total fluorescence intensity for each scan position, as done in conventional operation of an LCSM, one records a small image of the illuminated region. The result is a four-dimensional stack of data: two dimensions refer to the lateral scan position, and two dimensions to the pixel position on the chip of the image-recording camera. This set of data can then be used to obtain a super-resolved image with doubled resolution, completely analogously to what is achieved with SIM. However, ISM is conceptually and technically much simpler, suffers less from sample imperfections like refractive index variations, and can easily be implemented into any existing LSCM.
A second, completely different approach which aims at achieving nanometer resolution along the optical axis is Metal Induced Energy Transfer or MIET. When placing a fluorescent molecule close to a metal, its fluorescence properties change dramatically. In particular, one observes a strongly modified lifetime of its excited state (Purcell effect). This is due to the efficient electromagnetic coupling of the excited state to surface plasmons in the metal, which is similar to Förster Resonance Energy Transfer (FRET), where the energy of an excited donor molecule is transferred into the excited state of an acceptor molecule. We call this effect metal-induced energy transfer or MIET. The MIET- coupling between an excited emitter and a metal film is strongly dependent on the emitter’s distance from the metal. We have used this effect for mapping the basal membrane of live cells with an axial accuracy of ~3 nm. The method is easy to implement and does not require any change to a conventional fluorescence lifetime microscope; it can be applied to any biological system of interest, and is compatible with most other super-resolution microscopy techniques which enhance the lateral resolution of imaging. Moreover, it is even applicable to localizing individual molecules, thus offering the prospect of using single-molecule localization microscopy for structural studies of biomolecules and biomolecular complexes.
Bio:
1981-86 Study of Physics at Ilya-Mechnikov-University Odessa
1991 PhD in Physical Chemistry (Humboldt-University Berlin)
1996-97 PostDoc at Los Alamos National Laboratory (USA)
1997-2000 Assistent Professor (C1) at University of Regensburg
2000 Habilitation in Physical Chemistry (University of Regensburg)
2001-2006 Heisenberg Fellow of the DFG at Forschungszentrum Jülich
2007-2008 Professor for Biophysical Chemistry at Eberhard-Karls-University Tübingen
Since 2008 Professor for Biophysics at Georg-August-University Göttingen
Abstract:
Classical fluorescence microscopy is limited in resolution by the wavelength of light (diffraction limit) restricting lateral resolution to ca. 200 nm, and axial resolution to ca. 500 nm (at typical excitation and emission wavelengths around 500 nm). However, recent years have seen a tremendous development in high- and super-resolution techniques of fluorescence microscopy, pushing spatial resolution to its diffraction-dictated limits and much beyond. One of these techniques is Structured Illumination Microscopy (SIM). In SIM, the sample is illuminated with a spatially modulated excitation intensity distribution, and the emerging fluorescence is imaged with a conventional wide-field imaging setup. By moving and rotating the excitation intensity distribution pattern in different positions and orientations, taking each time a wide-field image, a final fluorescence image is composed which has roughly double the resolution (laterally) of a conventional wide-field or a confocal laser scanning image alone. A similar technique is Image Scanning Microscopy (ISM). In ISM, the focus of a conventional laser-scanning confocal microscope (LCSM) is scanned over the sample, but instead of recording only the total fluorescence intensity for each scan position, as done in conventional operation of an LCSM, one records a small image of the illuminated region. The result is a four-dimensional stack of data: two dimensions refer to the lateral scan position, and two dimensions to the pixel position on the chip of the image-recording camera. This set of data can then be used to obtain a super-resolved image with doubled resolution, completely analogously to what is achieved with SIM. However, ISM is conceptually and technically much simpler, suffers less from sample imperfections like refractive index variations, and can easily be implemented into any existing LSCM.
A second, completely different approach which aims at achieving nanometer resolution along the optical axis is Metal Induced Energy Transfer or MIET. When placing a fluorescent molecule close to a metal, its fluorescence properties change dramatically. In particular, one observes a strongly modified lifetime of its excited state (Purcell effect). This is due to the efficient electromagnetic coupling of the excited state to surface plasmons in the metal, which is similar to Förster Resonance Energy Transfer (FRET), where the energy of an excited donor molecule is transferred into the excited state of an acceptor molecule. We call this effect metal-induced energy transfer or MIET. The MIET- coupling between an excited emitter and a metal film is strongly dependent on the emitter’s distance from the metal. We have used this effect for mapping the basal membrane of live cells with an axial accuracy of ~3 nm. The method is easy to implement and does not require any change to a conventional fluorescence lifetime microscope; it can be applied to any biological system of interest, and is compatible with most other super-resolution microscopy techniques which enhance the lateral resolution of imaging. Moreover, it is even applicable to localizing individual molecules, thus offering the prospect of using single-molecule localization microscopy for structural studies of biomolecules and biomolecular complexes.
Bio:
1981-86 Study of Physics at Ilya-Mechnikov-University Odessa
1991 PhD in Physical Chemistry (Humboldt-University Berlin)
1996-97 PostDoc at Los Alamos National Laboratory (USA)
1997-2000 Assistent Professor (C1) at University of Regensburg
2000 Habilitation in Physical Chemistry (University of Regensburg)
2001-2006 Heisenberg Fellow of the DFG at Forschungszentrum Jülich
2007-2008 Professor for Biophysical Chemistry at Eberhard-Karls-University Tübingen
Since 2008 Professor for Biophysics at Georg-August-University Göttingen
Practical information
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- Free