CC BY
20B-I-7
Live cell optical microscopy from the millimeter to the nanometer range
H. Schneckenburger1, V. Richter1, P. Weber1, M. Wagner1 1Aalen University, Institute of Applied Research, Aalen, Germany
Optical microscopy is closely related to the Abbe or Rayleigh criterion, giving resolutions around 1 |im for low numeric apertures (e.g. 10' /0.30 objective lenses) and about 200 nm for high numeric apertures (e.g. 100' /1.40 lenses). Furthermore, optical microscopy provides a depth of focus L = n X/AN of more than 5 |im for low and around 400 nm for high aperture lenses. This implies that for low aperture and low magnification samples of about 1mm in diameter and 510 |im thickness, e.g. cell monolayers, can be easily imaged with a resolution around 1 |im.
If samples are thicker, some sectioning is required, and confocal as well as light sheet techniques have proven to be good standards. Both methods allow for sequential recording of individual layers and for combination in a 3D image. Only in the last 25 years methods have been described with a resolution below the Abbe criterion. They are summarized under the term "superresolution microscopy", and include (1) Stimulated Emission Depletion (STED) Microscopy where upon optical excitation in a laser scanning microscope the fluorescent spot is confined to 70 nm or less. However, the irradiance exceeds that of a conventional fluorescence microscope considerably, making live-cell microscopy very difficult and requiring essential modifications; (2) Super-localization microscopy of single molecules located within a thin illuminated layer of a sample. PALM, STORM and related techniques permit a precision of localization below 20 nm, but again require very high irradiance and exposure times; (3) Airy Scan and Structured Illumination Microscopy (SIM), both permitting an increase in resolution by a factor 1.7-2.0 compared to the Abbe criterion. They can be used at moderate light exposure, and appear ideal for live-cell fluorescence microscopy.
The present paper is focused on light sheet microscopy, SIM and their combination [1,2] as well as on Axial Tomography [3] permitting a deeper view into 3D cell assemblies and an isotropic resolution around 100 nm. For probing intermolecular distances of 10 nm or less Förster Resonance Energy Transfer (FRET) [4] is used in combination with Total Internal Reflection (TIR), e.g. in molecular test systems of pharmaceutical agents.
References
[1] H. Schneckenburger, V. Richter, M. Wagner: "Live-cell optical microscopy with limited light doses", SPIE Spotlight Series, Vol. SL 42, 2018, 38 pages.
[2] V. Richter, M. Piper, M. Wagner, H. Schneckenburger: "Increasing Resolution in Live Cell Microscopy by Structured Illumination (SIM)", Appl. Sci. 9 (6) (2019) 1188.
[3] V. Richter, S. Bruns, T. Bruns, P. Weber, M. Wagner, C. Cremer, H. Schneckenburger: "Axial Tomography in Live Cell Laser Microscopy", J. Biomed. Opt. 22(9) (2017) 91505.
[4] H. Schneckenburger, P. Weber, M. Wagner, S. Enderle, B. Kalthof, L. Schneider, C. Herzog, J. Weghuber, P. Lanzerstorfer: "Combining TIR and FRET in molecular test systems," Int. J. Mol. Sci. 20 (2019) 648.
