A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

Abstract

The development of stimulated emission depletion (STED) microscopyr epresented am ajor breakthrough in cellular and molecular biology.H owever,t he intense laser beams required for both excitation and STED usually provoke rapid photobleaching of fluorescent molecular probes,w hich significantly limits the performance and practical utility of STED microscopy. We herein developed ap hotoresistant fluorescent dye C-Naphoxa sapractical tool for STED imaging.Withexcitation using either a l = 405 or 488 nm laser in protic solvents,C -Naphox exhibited an intense red/orange fluorescence (quantum yield F F > 0.7) with alarge Stokes shift (circa 5900 cm À1 ). Even after irradiation with aX elamp (300 W, l ex = 460 nm, full width at half maximum (FWHM) = 11 nm) for 12 hours,99.5 %ofC-Naphoxremained intact. The high photoresistance of C-Naphoxa llowed repeated STED imaging of HeLa cells.Even after recording 50 STED images, 83 %o fthe initial fluorescence intensity persisted.Super-resolution nanoscopic techniques,s uch as stimulated emission depletion (STED) microscopy,h ave evolved continuously over the past decade and have allowed scientists to visualize structural details in biological systems. [1][2][3][4][5][6][7][8][9] In STED imaging,afluorescent dye is exposed to irradiation from two different laser sources,namely an excitation laser and aSTED beam, where the STED beam is adonut-shaped beam for the depletion of excited molecules.T he spatial resolution of STED imaging increases with the I STED /I sat ratio,where I STED is the STED beam intensity and I sat represents the saturation intensity at 50 %depletion.[10] An increase of the I STED value, however, simultaneously accelerates the photobleaching of the dye,mainly through multiphoton absorption, [11] and even the most advanced photostable fluorophores currently available suffer from this limitation. Substantially enhancing the photostability of af luorescent dye has therefore been ap rimary concern in order to employ STED microscopy for live imaging.H erein, we report promising fluorescent small molecules which exhibit outstanding photostability,e ven in living cells and under continuous STED imaging conditions, relative to currently available photostable fluorophores.T his compound class is thus ap owerful tool for the advancement of STED microscopy,with potential applications,for instance, in repeated time-lapse STED imaging and live video imaging. Our molecular design was based on ac ore scaffold of (N,N-diphenylamino)phenyl-substituted benzophosphole Poxide (Bphox;F igure 1), [12] which offers several distinct advantages as af luorescent probe.F irst, the combination of the electron-donating properties of the amino moiety with the electron-accepting properties of the benzophosphole P-oxide leads to compounds with intense fluorescence emissions, which can retain high fluorescence quantum yields (F F )even in polar and protic solvents.S econd, this donor-acceptor combination gives rise to an intramolecular charge transfer (ICT) character of the excited s...

A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae

“…contains an electron-withdrawing phosphine oxide (P = O) moiety. We have previously reported that the donor-π-acceptor-type dye C-Naphox, which consists of a naphthophosphole P-oxide skeleton and an electron-donating triphenylamine moiety, exhibits sufficient photoresistance to allow successive STED scans (11). While this dye showed significantly decreased fluorescence quantum yields (Φ F ) in aqueous media due to its intramolecular charge transfer (ICT) character in the excited state, intense fluorescence was revived in nonpolar environments.…”

“…To solve this issue, we have recently proposed a structural reinforcement strategy of electron-deficient π-conjugated molecular skeletons, which significantly improves the photostability of fluorophores (11). Based on this strategy, superphotostable phospholebased fluorescent molecules have been developed, and these allow multiple acquisition of STED images under physiological conditions (12).…”

Negative fluorescence anisotropy of phosphole oxide-based dyes in nematic liquid crystals

“…The components of S TDM also suggest that CBF-S has lower anisotropy in its shape (low λ S and S LX~0 .23), which is the main reason for the low S TDM , similarly to the cases found on CP2 and CBM. Originally, the phenyl group attached to the phosphorus has been designed and required because of the synthetic reason, ie, availability of the starting materials and the stability toward the reaction conditions 39,47,48 . Here it is theoretically suggested that the structural nature, ie, >P(=O)Ph, also plays an important role in realising negative fluorescence anisotropy of phosphole oxide-based dyes in the present NLCs.…”

“…Recently the negative anisotropy of a phosphole oxide-based dye, C-Naphox 39 , in NLC was reported 40 . Since this dye has a high photobleaching resistance, the detailed FPOM study of NLC defects has become possible, eg, uncovering their three-dimensional structures being based on alignment information complementary to that given by the dyes with positive anisotropy.…”

“…Here, with the aid of such theoretical analysis, we investigate steady-state fluorescence anisotropy of a series of phosphole oxidebased dyes 39,[46][47][48] in NLCs to clarify the relation between the experimentally observed negative anisotropy and the molecular structures. In addition, the theoretically derived general principle of the molecular design of dyes for controlling the fluorescence anisotropy and molecular alignment in NLCs, as well as conditions for realising strong negative anisotropy, is discussed.…”