Two-Photon Probes for Intracellular Free Metal Ions, Acidic Vesicles, And Lipid Rafts in Live Tissues

Abstract

Optical imaging with fluorescence microscopy is a vital tool in the study of living systems. The most common method for cell imaging, one-photon microscopy (OPM), uses a single photon of higher energy to excite the fluorophore. However, two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is growing in popularity among biologists because of several distinct advantages. Using TPM, researchers can image intact tissue for a long period of time with minimum interference from tissue preparation artifacts, self-absorption, autofluorescence, photo-bleaching and photodamage. However, to make TPM a more versatile tool in biology, researchers need a wider variety of two-photon probes for specific applications. In this Account, we describe a series of two-photon probes that we developed that can visualize the distribution of intracellular metal ions, acidic vesicles, and lipid rafts in living cells and tissues. The development of these probes requires a significant two-photon cross section for the bright image and receptors (sensing moieties) that triggers the emission of the two-photon excited fluorescence upon binding with the ions or membrane in the living system. These probes also must be sensitive to the polarity of the environment to allow selective detection of cytosolic and membrane-bound probes. In addition, they need to be cell-permeable, water-soluble for the staining of cells and tissues, and highly photostable for long-term imaging. The resulting probes-AMg1 (Mg2+), ACa1-Aca3 (Ca2+), AZn1 and AZn2 (Zn2+), AH1, AH2, and AL1 (acidic vesicles), and CL2 (membrane)-use 2-acetyl-6-aminonaphthalene as the fluorophore and receptors for the target ions or membrane, All of these two-photon turn-on probes can detect the intracellular free metal ions, acidic vesicles, and lipid rafts at 100-300 mu m depth in live tissues. Moreover, with AlCall-AM, we could simultaneously visualize the spontaneous Ca2+ waves in the somas of neurons and astrocytes at similar to 120 mu m depth in fresh hypothalamic slices for more than 1000 s without appreciable decay. Furthermore, AL1 could visualize the transport of the acidic vesicles between cell body and axon terminal along the axon in fresh rat hippocampal slices at similar to 120 mu m depth.