Superauflösungsmikorskopie durch Einzelmoleküllokalisierung bei kryogenen Temperaturen
Biophysik
Zusammenfassung der Projektergebnisse
Investigations at cryogenic temperatures are especially interesting for biological applications, as rapid freezing induces vitrification, allowing the fixation of samples without any structural changes commonly caused by chemical techniques. One of the most exciting topics is correlative microscopy, the combination of electron and light microscopy. Fluorescence microscopy is highly specific, but exhibits a resolution that is two orders of magnitude lower than the former one. Super-resolution cryo-fluorescence microscopy could diminish this resolution gap and holds the potential for correlative microscopy that reveals ultra-structural details with the specificity of fluorescence labeling. A few years ago, we developed and built a cryo‐fluorescence microscope optimized for single‐molecule wide-field imaging. We were able to demonstrate that, with this instrument, it is possible to localize single molecules with Ångström accuracy, due to the tremendously increased photo‐stability of dyes at cryogenic temperatures. Nowadays, a major problem of super-resolution microscopy implementation at 90 K is still the absence of fluorescent molecules (organic dyes, fluorescent proteins) that still photo‐switch at cryogenic temperatures. Moreover, due to the low numerical aperture of the air objective required by most cryo‐fluorescence setups, the axial localization using standard schemes like astigmatic imaging becomes very challenging. In the project, we developed potential solutions for both problems, the lack of fluorescent labels that still photo‐switch at cryogenic temperatures by introducing polarization microscopy, and the axial localization problem, by implementing a cryo-MIET microscope that uses the FLIM camera for the lifetime‐resolved flu‐ orescence wide‐field imaging. A combination of the polarization‐modulated excitation and the polarization‐ resolved detection will allow for disentangling the emission of several emitters in one diffraction‐limited area. The corresponding set-up for polarization microscopy investigations at cryogenic temperatures was assembled and verification experiments are in progress. In this context, we introduced and experimentally verified a simple-to-implement method for correcting orientation-induced localization errors in SMLM. It does not sacrifice collected fluorescence light, and yields additional polarization information for the identified and localized single molecules. One current restriction of our experimental implementation is that it will not work well for molecules with in-plane orientations close to one of the two detection-polarization axes. A potential solution to this problem is to alternatively record polarization-resolved images with polarization axes that switch alternatively between an orientation parallel to the horizontal/vertical image axes and an orientation diagonal to these axes, by using a Pockels cell into the detection path. We hope that the simplicity of our method will make it broadly applicable, especially for cryo-SMLM, where the restricted orientation flexibility of the dye labels cannot be neglected. We implement a novel FLIM camera (LINCam25), which is suitable for wide-field fluorescence lifetime measurements. We applied the LINCam25 for metal-induced energy transfer (MIET) imaging, which allows us to determine the axial position of individual molecules. It is the first step of applying the wide-field FLIM camera for three-dimensional single-molecule localization microscopy (SMLM) at cryogenic temperatures. Finally, we have developed a confocal laser-scanning FL-SMLM which combines sectioning and lifetime information with super-resolution imaging. The technique is straightforward to implement on a commercial CLSM with TCSPC capability and fast laser scanning. As light exposure is limited to the scanning focus, it is possible to sequentially image different regions of interest without photobleaching other regions. The lifetime resolution enables lifetime-based multiplexing within the same spectral window, distinguishing different fluorescent labels solely by their lifetimes. In combination with the optical sectioning of a CLSM, this allows for chromatic aberration-free super-resolution imaging of multiple cellular structures. The next step is to use the lifetime information for combining the lateral super-resolution of SMLM with the superior axial superresolution of Metal-Induced Energy Transfer (MIET) imaging. This will also enable 3D super-resolution imaging with exceptionally high isotropic resolution for applications in structural biology.
Projektbezogene Publikationen (Auswahl)
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"Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy" ACS Nano 14 (2020): 14190-14200
Jan Christoph Thiele, Dominic A. Helmerich, Nazar Oleksiievets, Roman Tsukanov, Eugenia Butkevich, Markus Sauer, Oleksii Nevskyi and Jörg Enderlein
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"Fluorescence polarization filtering for accurate single molecule localization" APL Photonics 5 (2020): 061302
Oleksii Nevskyi, Roman Tsukanov, Ingo Gregor, Narain Karedla and Jörg Enderlein
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"Wide-Field Fluorescence Lifetime Imaging of Single Molecules" J. Phys. Chem. A 124 (2020): 3494-3500
Nazar Oleksiievets, Jan Christoph Thiele, André Weber, Ingo Gregor, Oleksii Nevskyi, Sebastian Isbaner, Roman Tsukanov and Jörg Enderlein
