A Two‐Photon Turn‐On Probe for Lipid Rafts with Minimum Internalization

Abstract

The lo domains, also called lipid rafts, are believed to serve as a platform to support various cellular processes such as signal transduction, membrane trafficking, pathogen invasion, cholesterol homeostasis, neurodegenerative diseases, and angiogenesis. The existence of lipid rafts has been demonstrated in mammalian cells and in model membranes by using various techniques such as atomic force microscopy, fluorescence microscopy, and coherent anti-Stokes Raman scattering microscopy. To understand their roles in cell biology, it is crucial to visualize such domains in the live cells and tissues. An ideal tool for this purpose is two-photon microscopy (TPM) that utilizes two near-infrared photons for excitation. The advantages of TPM include the capability of visualizing the real-time activities of the biological targets in live cells and intact tissues at >100 mm depth with minimum interference due to the tissue preparation artifacts that can extend ~70 mm from the tissue surface. For maximum utilization of TPM, many TP probes for specific applications are needed. However, TP probes for such applications are rare. Earlier, we reported a membrane TP polarity probe, CL, which showed several advantages over laurdan including greater sensitivity to the solvent polarity, brighter TPM image, and more precise reflection of the cell environment. We also developed a TP turn-on probe, CL2, which showed larger TP action cross-section and higher sensitivity to the polarity of the environment than CL. This allowed selective detection of the lipid rafts in live cells and tissues by TPM. Unfortunately, CL2 had one shortcoming; it internalized into the cytoplasm upon prolonged incubation with cells, causing a blurred TPM image. To overcome this problem, we have designed a new TP turn-on (off-on) probe (SL2) that has sodium sulfonate in the head group and exclusively stains the plasma membrane. Herein, we report that SL2 can detect the lipid rafts in the live cells and intact tissues without internalization problems by simply collecting the TP excited fluorescence (TPEF) by using TPM. The preparation of SL2 is given in the Supporting Information. The solubility of SL2 in water was 7 mm, which is sufficient to stain the cells (Figure S1 in the Supporting Information). The change of the head group from a carboxylic acid (CL2, 4 mm) to a sulfonate increased the water solubility by ~ twofold. The absorption and emission spectra of SL2 showed gradual bathochromic shifts with increasing solvent polarity (Figure 1A and Table S1) indicating the charge-transfer character of the emitting state. The shifts were larger for the emission than for the absorption spectra (21 vs. 126 nm). Moreover, the fluorescence quantum yield (F) increased by 22-fold as the solvent was changed from EtOH/H2O (F=0.04) to THF (F=0.88). CL2 showed similar behavior except that fluorescence enhancement factor (FEF) was lower [FEF(CL2)=16 versus FEF(SL2)= 22] (Table S1). Hence, the fluorescence of SL2 is more sensitive to the polarity of the environment than that of CL2. The fluorescence intensity of SL2 showed similar decrease with the hydrophilicity of the large unilamellar vesicles (LUVs) in the order, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (DPPC/CHL (40 mol%), lo)>DOPC/sphingomyelin/CHL (1:1:1, raft mixture)>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, ld ; Figure S2). The relative fluorescence intensities of SL2 in DPPC/CHL/raft mixture/DOPC in the 420–520 nm range are 7.0/4.8/1.0, respectively; SL2 emits sevenfold stronger fluorescence in the lo than in the ld domain. The slightly weaker fluorescence in the raft mixture than in DPPC/CHL can be explained if SL2 exists in 63/37 ratio in the lo/ld domains of the raft mixture with 7/1 intensity ratio (vide supra). A similar result was reported for CL2 (lo/ld=86:14). [12] This indicates high preference of these probes to reside in the lo domain, which can be attributed to the more favorable hydrophobic interactions with sphingomyelin when compared to DOPC. The sensitivity of the laurdan derivatives on the polarity of the environment originates from the intramolecular charge [a] C. S. Lim, J. H. Lee, Dr. Y. S. Tian, Prof. B. R. Cho Department of Chemistry, Korea University 1-Anamdong, Seoul, 136-701 (Korea) Fax: (+82)2-3290-3544 E-mail : chobr@korea.ac.kr [b] H. J. Kim, Prof. H. M. Kim Division of Energy Systems Research, Ajou University Suwon, 443-749 (Korea) E-mail : kimhm@ajou.ac.kr [c] Dr. C. H. Kim, Prof. T. Joo Department of Chemistry, Pohang University of Science and Technology Pohang, 790-784 (Korea) E-mail : thjoo@postech.ac.kr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201000609.