Acidic pH-Activatable Visible to Near-Infrared Switchable Ratiometric Fluorescent Probe for Live-Cell Lysosome Targeted Imaging

Abstract

Here, we have designed and synthesized acidic pH-activatable visible to NIR switchable ratiometric pH-sensitive fluorescent dye. The design consists of a cell-permeable organic probe containing a lysosome targeting morpholine functionality and an acidic pH-activatable oxazolidine moiety. The visible closed oxazolidine form (λ abs 418 nm) can be switched to the highly conjugated NIR Cy-7 form (λ abs 780 nm) through ring opening of the oxazolidine moiety at acidic pH. This switching of the ratiometric fluorescent probe is highly reversible and can be controlled by pH. NMR, UV/vis, and fluorescence spectroscopies allowed monitoring of pH switching behavior of the probe. This bioresponsive in situ acidic organelle activatable fluorophore showed reversible pH-switchable ratiometric optical properties, high photostability, huge bathochromic emission shift of 320 nm from basic to acidic pH, off-to-on narrow NIR absorption and emission bands with enhanced molar extinction coefficient at lysosomal pH, good quantum yield, low cytotoxicity, and targeted imaging ability of live cell lysosomes with ideal pK a . The report demonstrated ratiometric imaging with improved specificity of the acidic lysosome while minimizing signals at the NIR region from nontargeted neutral or basic organelles in human carcinoma HeLa and A549 as well as rat healthy H9c2(2-1) live cells, which is monitored by confocal laser scanning microscopy.

Real-Time and High-Fidelity Tracking of Lysosomal Dynamics with a Dicyanoisophorone-Based Fluorescent Probe

“…Initially, these four lysosomes were independent (5.28 s), and lysosomes 2 and 3 approached and finally merged (6.16 s). This process used only 0.88 s, which is less than the reported time gap. ,− After that, these four lysosomes continued to touch and communicate with each other, and their mutual merging, dissociating, hiding, and appearing all could be recorded clearly. These results suggest that DCIP has the ability for real-time and high-fidelity imaging of lysosomal movements through a common CLSM.…”

“…Due to the rapid movement of lysosomes, it is very challenging to obtain fast and high-fidelity imaging in a short time, which requires high-quality imaging in a very short exposure time. However, a short exposure time will reduce the fluorescence brightness and degrade the image quality, which may be the reason why the time interval of most lysosomal movements recorded by fluorescent probes was >1 s, even 5 s or longer. ,− Long-time interval imaging using a long exposure time may mask important information. With the excellent imaging performance of DCIP for lysosomes, we further studied whether it can track lysosomal movement in a shorter exposure mode.…”

“…Several lysosomal trackers have been developed and commercialized, such as LysoTracker Green (LTG), LysoTracker Red (LTR), and other LysoSensor dyes, − which greatly promoted the study of lysosomes. However, these LysoTrackers/Sensors have some obvious limitations, such as narrow Stokes shift (LTG, 7 nm; LTR, 13 nm), poor photostability, high background fluorescence staining, and high sensitivity to pH within the normal lysosomal pH range, which restricts them for long-time and high-fidelity lysosomal tracking. − To solve these problems, recently some new lysosomal probes based on nanoparticles, , polymers, metal complexes, aggregation-induced emission (AIE) molecules, − and others − have been reported one after another (some of them are shown in Table S1). However, some shortcomings also exist in these new probes, such as poor cell permeability, , difficult synthesis, poor biocompatibility, , and low water solubility. − With the deepening of lysosomal research, it is still very necessary to further develop new lysosomal probes, especially those with easy preparation, high photostability, long emission wavelength, high quantum yield, large Stokes shift, high-resolution imaging, and best use in a very low dose.…”

Fluorescent Benzothiadiazole Derivatives as Fluorescence Imaging Dyes: A Decade of New Generation Probes

“…Fluorescent small organic molecules are of paramount importance in the study of lysosomes and their dynamic processes. In this sense, several organic dyes have been used to stain lysosomes selectively [184–191] . Although the large number of fluorescent dyes applied to visualize lysosomes and their cellular events, up to now, only four fluorescent BTD derivatives (Figure 22) have been described as selective bioimaging probes to stain this specific organelle and all of them published very recently.…”

Lighting up Individual Organelles With Fluorescent Carbon Dots

“…Studies on the use of morpholine as a functional group for lysosome-targeting probes have been reported (Wan et al, 2014;Wu X. et al, 2017;He et al, 2017;Biswas et al, 2021;Mukherjee et al, 2021). For example, Wu et al synthesized CDs functionalized with a morpholine derivative (CDs-PEI-ML) to serve as a lysosomal probe (Wu L. et al, 2017).…”