Super resolution techniques are gaining more and more importance in order to enlighten biological processes. Although, state of the art super methods such as dSTORM or STED microscopy enable a resolution down to the 10 nm range, their imaging rate is limited to less than one frame per second (fps), because those methods require the subsequent recording of several thousand images (dSTORM) or are based on laser scanning approaches (STED).
In contrast, SIM as wide field technique allows a fast image acquisition, since a super resolved image can be reconstructed out of only 9 (two-beam SIM) or 15 (three-beam SIM) raw images, respectively. Consequently, SIM is perfectly suited for video rate acquisition of super resolved images. The drawback of this technique is, however, its basic limitation of the resolution to the quarter wavelength (half of the Abbe limit).
In our lab we are currently developing a structured illumination microscope (SIM) that enables video rate acquisition of super resolved images at several excitation wavelengths. The basic setup is sketched in figure 1.

Fig.1: Schematic setup of the fast multi-color structured illumination microscope

We use a spatial light modulator for fast projection of the illumination pattern onto the sample [1]. The sCMOS camera, as limiting component for the acquisition speed, allows a raw image rate of 100 fps, corresponding to a super resolution rate of 11 fps. Exploiting the rolling shutter of the sCMOS camera we managed to boost the acquisition rates up to 714 fps (raw images) corresponding to a 79 fps of super resolved images [2].
The fiber coupled multi line laser source allows luminescence excitation at 405 nm, 488 nm, 561 nm and 638 nm. The emitted signal is split up into three color channels (500 nm < λEm­ ­< 525 nm), (575 nm < λEm­ ­< 625 nm) and (λEm­ ­< 650 nm). Thus we are able to provide a functional analysis of biological tissue and can estimate crosstalk between different excitation wavelengths and fluorophores.

Currently we are establishing our structured illumination reconstruction code on graphical processing units (GPU) in order to integrate “on-the-fly” image reconstruction into our setup.

Besides pushing the acquisition rate of the SIM we are as well working on extending its resolution by exploiting nonlinear emission properties of selected fluorophores. The nonlinearity is achieved by using photo switchable dyes. These can be pumped into a dark state (deactivation) by one wavelength and brought back to the bright state (activation) by a different wavelength [3].

Currently we are investigating Kohinoor, a photo switchable derivate of the green fluorescent protein (GFP) which can be deactivated by 405 nm and activated as well as excited by 488 nm [4, 5].


[1] R. Förster, H.-W. Lu-Walther, A. Jost, M. Kielhorn, K. Wicker, and R. Heintzmann, „Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulater“, Opt. Express, 22 (17), 2014, 20664

[2] L. Song, H-W. Lu-Walther, R. Förster, A. Jost, M. Kielhorn, J. Zhou, and R. Heintzmann „Fast structured illumination microscopy using rolling shutter cameras“, Meas. Sci. Technol., 27 (5), 2016, 055401

[3] D. Li, L. Shao, Bi-Chang Chen, x. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. Hammer III, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, „Extended-resoluiton structured illumination imaging of endocytic and cytoskeletal dynamics”, Science, 349 (6251), 2016, 944

[4] H.-W. Lu-Walter, W. Hou, M. Kielhorn, Y. Arai, T. Nagai, M. M. Kessels, B. Qualmann, and R. Heintzmann, „Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength“, PLoS ONE, 11(10), 2016, e0165148

[5] D. K. Tiwari, Y. Arai, M. Yamanaka, T. Matsuda, M. Agetsuma, M. Nakano, K. Fujita, and T. Nagai, “A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy”, Nat. Meth. 12(6) 2015, 515

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