Super-resolution fluorescence microscopy has revolutionized biological imaging. However, resolution improvement is only one side of the story. Equally important is structural preservation of the sample. We are combining fast-freezing techniques (vitrification) with the concept of super-resolution imaging to achieve both.
Super-resolution fluorescence microscopy under cryo-conditions (super-resolution cryo-FM) is a new field of microscopy that is aiming to combine super-resolution concepts with the benefits of cryo-immobilized samples. Structural preservation of the sample is critical for imaging with high resolution to prevent artefacts and misinterpretation of the data. Making use of the near-native state of the sample when immobilized by fast freezing methods leads to two major application areas of super-resolution cryo-FM:
1) Provide an alternative to conventional (ambient temperature) super-resolution FM methods, where currently the common practice for immobilization is chemical fixation.
2) Bridge the resolution gap in cryo-CLEM (correlative light and electron cryo-microscopy) to take full advantage of the complementary features of both imaging modalities.
Correlative super-resolution cryo-FM and cryo-EM. IMAGE: Moser & Pražák et al. (2019) PNAS
The key for super-resolution imaging in general is photo-switching of the fluorophores. As this is only poorly studied and understood under cryo-conditions, one of our major aims is to gain a deeper insight into the photo-physical mechanisms in different molecules at a temperature range suitable for amorphous ice (devitrification point of water: -135°C).
Prospective applications of super-resolution cryo-FM in the context of cryo-CLEM. IMAGE: Wolff et al. (2016) Biol. Cell.
We are focusing on developing new techniques and methodology in the area of super-resolution cryo-FM with a particular interest on super-resolution cryo-CLEM (imaging the same sample with super-resolution cryo-FM and subsequent cryo-EM). Minimizing the resolution gap in cryo-CLEM is opening up a broad spectrum of applications in the field of structural biology.
Microscope setup on optical bench. IMAGE: R. Kaufmann.
In close collaboration with other groups in the CSSB we are using super-resolution cryo-CLEM to tackle current biological questions in the context of infectious diseases. Integrating cryo super-resolution fluorescence microscopy into the cryo-EM workflow enables for example identification and localization of rare biological events in host-pathogen interactions.
Future projects and goals
Super-resolution cryo-FM/cryo-CLEM is currently at a very early and experimental stage. Overcoming technical challenges and a better understanding of the underlying photo-physics will help us to push the boundaries of this method and turn it into a powerful tool for structural biology.
Listed projects are aimed at Physics or Nano Science students, but open to other disciplines too. If you are interested, simply contact us for more information.
Gartenmann L, Wainman A, Qurashi M, Kaufmann R, Schubert S, Raff JW, Dobbie IM (2017) A combined 3D-SIM/SMLM approach allows centriole proteins to be localized with a precision of∼ 4–5 nm. Current Biology 27: R1054-R1055 doi: 10.1016/j.cub.2017.08.009
Johnson E, Kaufmann R (2017) Correlative In-Resin Super-Resolution Fluorescence and Electron Microscopy of Cultured Cells. Super-Resolution Microscopy. Springer, 163-177 doi: /10.1007/978-1-4939-7265-4_14
2016
Wolff G, Hagen C, Grünewald K, Kaufmann R (2016) Towards correlative super‐resolution fluorescence and electron cryo‐microscopy. Biology of the Cell 108: 245-258 doi: 10.1111/boc.201600008
Kong Y, Janssen BJ, Malinauskas T, Vangoor VR, Coles CH, Kaufmann R, Ni T, Gilbert RJ, Padilla-Parra S, Pasterkamp RJ (2016) Structural Basis for Plexin Activation and Regulation. Neuron 91: 548-560 doi: 10.1016/j.neuron.2016.06.018
Wegel E, Göhler A, Lagerholm BC, Wainman A, Uphoff S, Kaufmann R, Dobbie IM (2016) Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison. Scientific Reports 6: 27290 doi: 10.1038/srep27290
2015
Ball G, Demmerle J, Kaufmann R, Davis I, Dobbie IM, Schermelleh L (2015) SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy. Scientific reports 5: 15915 doi: 10.1038/srep15915
Zhang Y, Máté G, Müller P, Hillebrandt S, Krufczik M, Bach M, Kaufmann R, Hausmann M, Heermann DW (2015) Radiation Induced Chromatin Conformation Changes Analysed by Fluorescent Localization Microscopy, Statistical Physics, and Graph Theory. PloS one 10: e0128555 doi: 10.1371/journal.pone.0128555
Johnson E, Seiradake E, Jones EY, Davis I, Grünewald K, Kaufmann R (2015) Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Scientific reports 5: 9583 doi: 10.1038/srep09583
2014
Schellenberger P, Kaufmann R, Siebert CA, Hagen C, Wodrich H, Grünewald K (2014) High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers. Ultramicroscopy 143: 41-51 doi: 10.1016/j.ultramic.2013.10.011
Cremer C, Kaufmann R, Gunkel M, Polanski F, Müller P, Dierkes R, Degenhard S, Wege C, Hausmann M, Birk U (2014) Application perspectives of localization microscopy in virology. Histochemistry and cell biology 142: 43-59 doi: 10.1007/s00418-014-1203-4
Müller P, Lemmermann NA, Kaufmann R, Gunkel M, Paech D, Hildenbrand G, Holtappels R, Cremer C, Hausmann M (2014) Spatial distribution and structural arrangement of a murine cytomegalovirus glycoprotein detected by SPDM localization microscopy. Histochemistry and cell biology 142: 61-67 doi: 10.1007/s00418-014-1185-2
Kaufmann R, Hagen C, Grünewald K (2014) Fluorescence cryo-microscopy: current challenges and prospects. Current Opinion in Chemical Biology 20: 86-91 doi: 10.1016/j.cbpa.2014.05.007
Kaufmann R, Schellenberger P, Seiradake E, Dobbie IM, Jones EY, Davis I, Hagen C, Grünewald K (2014) Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions. Nano Letters 14: 4171-4175 doi: 10.1021/nl501870p
Wang Q, Dierkes R, Kaufmann R, Cremer C (2014) Quantitative analysis of individual hepatocyte growth factor receptor clusters in influenza A virus infected human epithelial cells using localization microscopy. Biochimica et Biophysica Acta (BBA)-Biomembranes 1838: 1191-1198 doi: 10.1016/j.bbamem.2013.12.014
2013
Seiradake E, Schaupp A, del Toro Ruiz D, Kaufmann R, Mitakidis N, Harlos K, Aricescu AR, Klein R, Jones EY.et al. (2013) Structurally encoded intraclass differences in EphA clusters drive distinct cell responses. Nature Structural & Molecular Biology 20 (8): 958-964
2012
Kaufmann R, Gall JG, Cremer C (2012) Superresolution imaging of transcription units on newt lampbrush chromosomes. Chromosome Research 20 (8): 1009-1015
Huber O, Brunner A, MaierP, Kaufmann R, Couraud PO, Cremer C, Fricker G (2012) Localization microscopy (SPDM) reveals clustered formations of P-glycoprotein in a human blood-brain barrier model. Plos One. 7 (9): e44776
Kaufmann R, Piontek J, Grüll F, Kirchgessner M, Rossa J, Blasig I, Cremer C (2012) Visualization and quantitative analysis of reconstituted tight junctions using localization microscopy. Plos One 7 (2): e31128
2011
Cremer C, Kaufmann R, Gunkel M, Pres S, Weiland Y, Ruckeslshausen T, Lemmer P, Geiger F, Degenhard S, Wege C, Lemmermann N, Holtappels R, Strickfaden H, Hausmann M (2011) Superresolution imaging of biological nanostructures by spectral precision distance microscopy. Biotechnology Journal 6 (9): 1037-1051
Grüll F, Kirchgessner M, Kaufmann R, Hausmann M, Kepschull U (2011) Accelerating image analysis for localization microscopy with FPGAs. Field Programmable Logic and Applications 1-5
Kaufmann R, Müller P, Hausmann M, Cremer C (2011) Imaging label-free intracellular structures by localisation microscopy. Micron 42 (4): 348-352
Kaufmann R, Müller P, Hildenbrand G, Hausmann M, Cremer C (2011) Analysis of Her2/neu membrane protein clusters in different types of breast cancer cells using localization microscopy. Journal of Microscopy 242 (1): 46-54
2010
Müller P, Schmitt E, Jacob A, Hoheisel J, Kaufmann R, Cremer C, Hausmann M (2010) COMBO-FISH enables high precision localization microscopy as a prerequisite for nanostructure analysis of genome loci. International Journal of Molecular Sciences 11 (10): 4094-4105
Bohn M, Diesinger P, Kaufmann R, Weiland Y, Müller P, Gunkel M, von Ketteler A, Lemmer P, Hausmann M, Heermann DW, Cremer C (2010) Localization microscopy reveals expression-dependent parameters of chromatin nanostructure. Biophysical Journal 99 (5): 1358-1367
Cremer C, von Ketteler A, Lemmer P, Kaufmann R, Weiland Y, Müller P, Hausmann M, Gunkel M, Ruckelshausen T, Baddeley D, Amberger R (2010) Far-field fluorescence microscopy of cellular structures at molecular optical resolution. Nanoscopy and Multidimensional Optical Fluorescence Microscopy 3.1‑3.35
2009
Lemme, P, Gunkel M, Weiland Y., Müller P, Baddeley D, Kaufmann R, Urich A, Eipel H, Amberger R, Hausmann M, Cremer C (2009) Using conventional fluorescent markers for far‐field fluorescence localization nanoscopy allows resolution in the 10‐nm range. Journal of Microscopy 235 (2): 163-171
Gunkel M, Erdel F, Rippe K, Lemmer P, Kaufmann R, Hörmann C, Amberger R, Cremer C (2009) Dual color localization microscopy of cellular nanostructures. Biotechnology Journal 4 (6): 927-938
Kaufmann R, Lemmer P, Gunkel M, Weiland Y, Müller P, Hausmann M, Baddely D, Amberger R, Cremer C (2009) SPDM: single molecule superresolution of cellular nanostructures. Proceedings of SPIE 7185: 71850J
2008
Lemmer P, Gunkel M, Baddeley D, Kaufmann R, Urich A, Weiland Y, Reymann J, Müller P, Hausmann M, Cremer C (2008) SPDM: light microscopy with single-molecule resolution at the nanoscale. Applied Physics B: Lasers and Optics 93 (1): 1-12
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