Targeted synthetic sensors. (A) Transmission of [Ca²⁺]_c changes into the mitochondrial matrix of single hepatocytes challenged with a maximal dose of vasopressin (VP). [Ca²⁺]_m was monitored with dihydro-Rhod 2-AM, and [Ca²⁺]_c was measured with Fura 2-AM. The inset shows cell population mean responses for [Ca²⁺]_c and [Ca²⁺]_m. The figure is modified from Hajnóczky et al. (1995) with permission from Elsevier. (B) PM potential measured using the FRET-based sensor. (left) Scheme of the voltage-sensitive FRET mechanism. At resting negative membrane potential (top), the permeable oxonols have a high concentration at the extracellular surface of the PM, and energy transfer from the extracellularly bound FL-WGA (acceptor molecule) is favored. FRET is symbolized as the straight arrow from lectin to oxonol. Upon membrane depolarization (bottom), the anions diffuse inside the cell and energy transfer is greatly reduced. (right) Confocal image of a voltage-clamped astrocytoma cell at −70 (A) and 50 mV (B), stained with FLOX6 (from González and Tsien [1995] with permission from Elsevier). (C) Mitochondrial membrane potential measured using TMRM. Cerebellar granule neurons loaded with TMRM (left) were exposed to the uncoupler FCCP and monitored over 60 min using TMRM in both the quenched (100 nM, middle) and nonquenched (30 nM, right) mode (from Ward et al. [2007] with permission from the Society for Neuroscience). (D) [Ca²⁺]_ER measured with Mag-Fura2. (left) Pseudo-color ratio image of permeabilized BHK-21 cells loaded with mag-Fura2. (middle) Same cell of left panel after stimulation with InsP₃, causing release of Ca²⁺ from the ER. (right) Representative kinetic of mag-Fura2 ratio collected from selected areas of the cell (from Hofer et al. [1995] with permission from the Federation of American Societies for Experimental Biology). (E) Exocytosis from large dense core vesicles (LDCVs) measured using FFN511. (left) Chemical structure of FFN511. (middle) Multiphoton image of a chromaffin cell shows distribution of FFN511 in LDCVs. Bar, 5 µm. (right) FFN511 exocytosis from an LDCV observed with total internal reflection fluorescence microscopy (TIRFM) images. The upper row shows consecutive images of a single vesicle. Orthogonal section through this vesicle and its integrated intensity are in the middle and bottom panels. The dotted line indicates stimulation by high potassium (from Gubernator et al. [2009] with permission from The American Association for the Advancement of Science).