Abstract
With the growing popularity of cryogenic correlative light and electron microscopy, it is becoming increasingly important to bridge the resolution gap between these two modalities. At cryogenic temperatures, the photon yield of fluorophores is a few orders of magnitude higher than at room temperature, enabling localization precisions on the \AA{}ngstr\"om scale. The current challenge is to induce sparsity at cryogenic temperatures such that individual fluorescent molecules can be localized. In this paper, we demonstrate the progress of using polarized stimulated-emission depletion (STED) to induce sparsity at cryogenic temperatures and in vacuum. We generate linear polarization of arbitrary in-plane orientations to achieve polarized STED with a sparsity of 3.3:1. Furthermore, we have probed the dark-state lifetime of ATTO 647N at cryogenic temperatures and in vacuum at room temperature. This dark state in vacuum is long-lived ($\ensuremath{\tau}=38$ ms) and could be the cause for reduced photostability of fluorophores under STED illumination in vacuum. The experiments were done on an in-house designed and built liquid nitrogen cryostat, enabling 30 hours of stable cryogenic fluorescence microscopy.
Highlights
The diffraction limited resolution in fluorescence microscopy can be circumvented with localization microscopy [1,2,3]
The FWHM achieved with significant stimulated-emission depletion (STED) powers does not induce enough sparsity to perform localization microscopy on dense biological samples
This would approximately square the amount of sparsity but would be excessively complicated to realize in a low numerical a√perture (NA) cryogenic microscope
Summary
The diffraction limited resolution in fluorescence microscopy can be circumvented with localization microscopy [1,2,3]. To induce sparsity at cryogenic temperatures, we have proposed the use of polarized stimulated-emission depletion (STED) in our earlier work [9] This idea is based on the work of Hafi et al where they used mutually orthogonal linearly polarized excitation and depletion beams to modulate the fluorescence to achieve super-resolution [17]. Their implementation, was later shown to owe its resolution enhancement primarily to the sparsity enhancing deconvolution and not the polarization modulation [18]. The method is assessed by the degree of sparsity that can potentially be achieved and by the number of emitted photons per single molecule It appears that longer lived triple states [19] limit the achievable photon yield. Several aspects of the experimental methods used (optical setup, cryostat, sample preparation) are described in the Appendices
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