Strategy to Lengthen the On-Time of Photochromic Rhodamine Spirolactam for Super-resolution Photoactivated Localization Microscopy

Abstract

Rhodamine derivatives and analogues have been widely used for different super-resolution imaging techniques, including photoactivated localization microscopy (PALM). Among them, rhodamine spirolactams exhibit great superiority for PALM imaging due to a desirable bright–dark contrast during the photochromic switching process. Although considerable attention has been paid to the chemical modifications on rhodamine spirolactams, the on-time of photochromic switching, one of the key characteristics for PALM imaging, has never been optimized in previous developments. In this study, we proposed that simply installing a carboxyl group close to the lactam site could impose an intramolecular acidic environment, stabilize the photoactivated zwitterionic structure, and thus effectively increase the on-time. On the basis of this idea, we have synthesized a new rhodamine spirolactam, Rh-Gly, that demonstrated considerably longer on-time than the other tested analogues, as well as an enhancement of single-molecule brightness, an improvement on signal-to-noise ratio and an enlargement of total collected photons of a single molecule before photobleaching. Finally, super-resolution images of live cell mitochondria stained with Rh-Gly have been obtained with a good temporal resolution of 10 s, as well as a satisfactory localization precision of ∼25 nm. Through self-labeling protein tags, Rh-Gly modified with a HaloTag ligand enabled super-resolution imaging of histone H2B proteins in live HeLa cells; through immunostaining antibodies labeled with an isothiocyanate-substituted Rh-Gly, super-resolution imaging of microtubules was achieved in fixed cells. Therefore, our simple and effective strategy provides novel insight for developing further enhanced rhodamine spirolactams recommendable for PALM imaging.

Imaging the ultrastructures and dynamics of live erythrocyte membranes at the single-molecule level with a far-red probe on a microfluidic platform

“…The copyright holder for this preprint this version posted April 23, 2021. ; https://doi.org/10.1101/2021.04.22.441044 doi: bioRxiv preprint Methods Super-resolution imaging: Super-resolution imaging experiments were performed on an Olympus IX71 inverted microscope with a ×100 objective (UAPON 100×OTIRF; 1.49 numerical aperture) as described earlier. 16,26 Light from continuous 640 nm laser (100 W, Coherent) was controlled by an acousto-optic tunable filter (AOTF) and focused on the back focal plane of the objective. Evanescent lighting was achieved…”

“…Super-resolution imaging experiments were performed on an Olympus IX71 inverted microscope with a ×100 objective (UAPON 100×OTIRF; 1.49 numerical aperture) as described earlier. 16,26 Light from continuous 640 nm laser (100 W, Coherent) was controlled by an acousto-optic tunable filter (AOTF) and focused on the back focal plane of the objective. Evanescent lighting was achieved through a motorized TIRFM illuminator (IX2-RFAEVA-2, Olympus).…”

“…The raw stack images were analyzed by a home-written Matlab software as described previously. 16,26 During the analysis, the software automatically searched for single-molecule candidates, extracted fluorescent trajectories and identified the state-transforming events. On the basis of these data, the photophysics of single molecules were calculated following the methods in section 3 of Supplementary Information.…”

A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk

“…[32][33][34][35][36] We envisage that this phenomenon can be judiciously harnessed to design ratiometric probes for cellular oxidative species, and developed totalROX. We note that the bridging-group modification is a novel probe design principle for the xanthenoid chromophores, complementing such existing principles as push-pull modification, [37][38][39] conjugation disruption, 40,41 covalent-assembly, [42][43][44][45] and photo-induced electron transfer (PET) [46][47][48] (Figure 2b).…”

The cross-talk modulation of excited state electron transfer to reduce the false negative background for high fidelity imaging in vivo

“…Owing to their reactive specicity, timely reporting and biological affinity, molecular-probe-based uorescence imaging techniques have been rapidly developed for biological monitoring, molecular medicine and even surgical navigation. [1][2][3][4][5][6][7][8][9][10] However, in the actual imaging, there are several problems that are usually neglected: (1) inherent false negative background (FNB): although the background seems to be dark, the real background uorescence will be exposed when the intensity scale of microscopy is compressed (Scheme 1A). Obviously, the real background is covered by the wide intensity scale of microscopy.…”