On the Road Toward More Efficient Biocompatible Two‐Photon Excitable Fluorophores

Abstract

Red-to-NIR absorption and emission wavelengths are key requirements for intravital bioimaging. One of the way to reach such excitation wavelengths is to use twophoton excitation. Unfortunately, there is still a lack of twophoton excitable fluorophores that are both efficient and biocompatible. Thus, we design a series of biocompatible quadrupolar dyes in order to study their ability to be used for live-cell imaging, and in particular for two-photon microscopy. Hence, we report the synthesis of 5 probes based on different donor cores (phenoxazine, acridane, phenazasiline and phenothiazine) and the study of their linear and nonlinear photophysical properties. TD-DFT calculations were performed and were able to highlight the structure-property relationship of this series. All these studies highlight the great potential of three of these biocompatible dyes for twophoton microscopy, as they both exhibit high two-photon cross-sections (up to 3650 GM) and emit orange to red light. This potential was confirmed through live-cell two-photon microscopy experiments, leading to images with very high brightness and contrast.

Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labeling in live cells

“…We based our design on the recently published Acri-Py (Figure 1). 38 The nitrogen atom of the acridan is an ideal anchor point for tetrazine attachment. We focused on the through-bond energy transfer (TBET) and photoinduced electron transfer (PeT) quenching mechanisms as we wanted to compare these designs on our two-photon excitable fluorophore.…”

“…All had high absorption cross sections (>2,400 GM), like Acri-Py. 38 These values are particularly impressive for two-photon excitable redemissive fluorogenic probes, as the highest values reported to date are below 65 GM. 37 We also compared the performances of the in terms of two-photon brightness (δ×ΦF), the most relevant property for imaging.…”

“…All dyes are very weakly fluorescent in native forms. 38 As they became fluorescent upon immobilization, their photophysical properties and those of their click adducts were determined in a buffer containing Bovine Serum Albumin (BSA) to mimic a constrained environment. We first showed that 100 equivalents of BSA were sufficient to reach the fluorescence plateau for each fluorogenic probe and its clicked product (Supporting Figure S3).…”

“…Our recently reported optimized probe, Acri-Py, meets all the criteria for use in live-cell imaging, including high twophoton brightness and red emission (Figure 1). 38 Finally, it also has a high cross-section relative to its molecular weight (/M=7.9) -an essential feature of biocompatible fluorophores. We then envisaged different tetrazine attachments to this scaffold to compare different quenching mechanisms (Figure 1).…”

Ultrabright two-photon excitable red-emissive fluorogenic probes for fast and wash-free bioorthogonal labeling in live cells

“…All had high absorption cross sections (>2,400 GM), like Acri-Py. 38 These values are particularly impressive for two-photon excitable red-emissive fluorogenic probes, as the highest values reported to date are below 65 GM. 37 We also compared the performances of the two dyes in terms of two-photon brightness (δ×ΦF), the most relevant property for imaging.…”