15th European Molecular Imaging Meeting
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Frontiers in Microscopy | joint session with COMULIS

Session chair: Giannis Zacharakis (Heraklion, Greece); Andreas Walter (Vienna, Austria)
 
Shortcut: FS 03
Date: Thursday, 27 August, 2020, 5:30 p.m. - 7:00 p.m.
Session type: Focus Session

Focus Sessions are organized in co-operation with related societies or projects. COMULIS is an EU funded COST Action dedicated to "Correlated Multimodal Imaging in Life Sciences"

Contents

Abstract/Video opens by clicking at the talk title.

5:30 p.m. FS 03-01

MINFLUX: Super Resolution at the Molecular Scale

Francisco Balzarotti1

1 Research Institute for Molecular Pathology, Vienna, Austria

Content

Superresolution microscopy methods such as stimulated emission depletion STED and SMLM have revolutionized far-field optical fluorescence microscopy by manipulating state transitions of the emitters, offering potentially unlimited resolution. In practice, however, the resolution of an image is limited by the finite photon budget of fluorescent probes. The recently introduced localization concept, termed MINFLUX [1,2], tackles this limitation by rendering each emitted photon more informative, achieving single digit nanometer resolution.

MINFLUX localizes an emitter by repeatedly probing its location with an excitation beam that features a zero of intensity. The emitter is localized with the knowledge of the beam shape and the number of photons collected at each probed location by the beam. The localization precision is roughly proportional to the size of the probed region, imposing a trade-off between photon-efficiency and observable range.

Here, I will present the MINFLUX concept and a strategy [3] that achieves high photon efficiency in arbitrarily large regions. thus allowing to image in fixed and living cells. This is accomplished by iteratively approaching each photo-activated emitter with a set of MINFLUX localizations, while gradually shrinking the probed region size. This allows isotropic localization precision and surpasses the typical ∝1⁄√N dependence, as photons are made increasingly informative as they are acquired. A multi-color modality for 3D-MINFLUX imaging will be also presented, together with several biological applications.

References
1 Balzarotti, F, Eilers, Y., Gwosch, K.C., Gynnå, A.H., Westphal W., Stefani, F.D., Elf J., Hell, S.W. 'Nanometer Resolution Imaging and Tracking of Fluorescent Molecules with Minimal Photon Fluxes.' Science 355, no. 6325: 606–12.
2 Eilers, Y., Ta, H., Gwosch, K.C., Balzarotti, F., Hell, S.W., 2018. 'MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution'. Proceedings of the National Academy of Sciences of the United States of America 201801672. 
3 Gwosch, K.C., Pape, J.K., Balzarotti, F., Hoess, P., Ellenberg, J., Ries, J., Hell, S.W., 2020. 'MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells'. Nature Methods 17, 217–224.
Keywords: super resolution optical microscopy, single molecule localization, single molecule tracking, MINFLUX
6:00 p.m. FS 03-02

3D correlative cryo-imaging using super resolution fluorescence microscopy and soft X-ray tomography

Maria Harkiolaki1

1 Diamond Light Source, Life Sciences, Didcot, United Kingdom

Content

Advances in the molecular understanding of biomedical systems have led to the accumulation of a wealth of biochemical information that warrants further investigation in the context of the living cell to allow the localisation of molecules of interest within cells as well as their effect on cellular structure and function.

 

B24 is the full field X-ray tomography beamline at Diamond currently delivering X-ray absorption contrast imaging of biological material, such as cells and tissues, to 25nm resolution. The resulting cryo-Soft X-ray Tomography (cryoSXT) 3D data allows the unambiguous delineation of cellular ultrastructure and is employed in the interpretation of the effects of biological chemical and mechanical cues depending on the subject matter. B24 is currently fully operational and available to the wider user community.

CryoSXT also provides context for further investigation on the molecular level. The latter is gained via super resolution fluorescence microscopy methods that provide invaluable information as to the molecular localisation of key parameters relevant to the system under study within the context of cellular maps defined via cryoSXT. To that effect, a bespoke cryo-fluorescence super resolution module has been developed offering both cryo-Structured Illumination Microscopy (cryoSIM) and dSTORM at the B24 beamline.

The particular attraction of the system is that samples that are due to be used for-ray imaging can be processed there first to generate 3D fluorescence information at high resolution on identified areas of interest before taken to the transmission X-ray microscope allowing for the accumulation of directly correlated localisation data; i.e. the same sample is imaged through a variety of methods, and the results are directly correlated avoiding sample to sample variations and therefore allowing the unambiguous interpretation of data across modalities. The B24 cryoSIM capacity is fully implemented now, being available to the user community on a commissioning basis. The dSTORM methodology is implemented optically but has not been tested with samples yet. Software is being developed to handle data processing and correlation across imaging modes. The beamline workflow will be presented with examples of recent data collected along with highlights and pitfalls of the correlative scheme employed.

References
1. Kounatidis, I. et al. 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway. Cell 182, 1–16 (2020).
Correlative cryo-imaging at the UK synchrotron beamline B24
Workflow and methods development at beamline B24 including a list of our involvement with development and dissemination of new methodology with views of the microscopes available and the space that houses them. 
Keywords: Correlative cryo-imaging, X-ray tomography, Super resolution fluorescence imaging
6:30 p.m. FS03-03

Mid-infraRed Optoacoustic Microscopy (MiROM): A new tool for label-free metabolic imaging of living cells and tissues

Miguel A. Pleitez1

1 Helmholtz Zentrum München, Institute of Biological and Medical Imaging (IBMI), Neuherberg, Baden-Württemberg, Germany

Content

Imaging modalities based on vibrational spectroscopy (Raman scattering or mid-infrared absorption) have demonstrated high chemical specificity for different biomolecules in a label-free manner. Nevertheless, the sensitivity of label-free Raman imaging, especially in the fingerprint region, is limited to concentrations above 1 mM, which is inadequate for live-cell analytical imaging of biomolecules with concentrations below the µM range. On the other hand, conventional mid-infrared methods, with high sensitivity in the fingerprint region, have been limited mostly to dry tissues and fixed cells due to the strong mid-infrared absorption of water and due to the negative-contrast detection scheme of conventional mid-infrared imaging.

Here, we introduce positive-contrast Mid-infraRed Optoacoustic Microscopy (MiROM) for label-free metabolic imaging in living cells. MiROM achieves low-micromolar concentration sensitivity with negligible cell photodamage by using low laser excitation power in the range of 100’s of µW. We showcase the unique label-free biomolecular contrast capabilities of MiROM in living cells by monitoring the spatiotemporal distribution of carbohydrates, lipids, and proteins in 3T3-L1 cells during lipogenesis as well as monitoring the lipid-protein dynamics in brown and white adipocytes during lipolysis. For the first time, we visualize carbohydrate patterns in early-stage adipocytes revealing, over time, an initial spread throughout the adipocyte body, followed by a co-localization with lipid droplets upon adipocyte maturation2. We discuss how MiROM yields unique label-free metabolic imaging abilities for a broader range of bioanalytical studies in living cells, additionally, showing its potential for analytical histology in fresh/unprocessed tissues.

References
1 Cheng, J.-X. & Xie, X. S., (2015) Science, 350, aaa8870–aaa8870.
2 Pleitez, M.A., Khan, A.A., Soldà, A. et al., (2019) Nat Biotechnol . https://doi.org/10.1038/s41587-019-0359-
Figure 1: Mid-infraRed Optoacoustic Microscopy (MiROM)
a, Excitation/sensing scheme for MiROM. b, Comparison of MiROM and ATR-FTIR spectrum of glucose and 1,2-dioleoyl-3-palmitoyl-rac-glycerol (TAG). c, MiROM micrographs of differentiated 3T3-L1 cells compared to brightfield (VIS) microscopy. d, MiROM micrographs of freshly excised inguinal White Adipocyte Tissue (iWAT)2.
Keywords: mid-infrared, label-free molecular imaging, optoacoustic microscopy, live-cell