Multimodal two-photon imaging using a second harmonic generation-specific dye

Abstract

Second harmonic generation (SHG) imaging can be used to visualize unique biological phenomena, but currently available dyes limit its application owing to the strong fluorescent signals that they generate together with SHG. Here we report the first non-fluorescent and membrane potential-sensitive SHG-active organic dye Ap3. Ap3 is photostable and generates SH signals at the plasma membrane with virtually no fluorescent signals, in sharp contrast to the previously used fluorescent dye FM4-64. When tested in neurons, Ap3-SHG shows linear membrane potential sensitivity and fast responses to action potentials, and also shows significantly reduced photodamage compared with FM4-64. The SHG-specific nature of Ap3 allows simultaneous and completely independent imaging of SHG signals and fluorescent signals from various reporter molecules, including markers of cellular organelles and intracellular calcium. Therefore, this SHG-specific dye enables true multimodal two-photon imaging in biological samples.

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

“…For the specific experiment of FM 4-64 with S. aureus, we attribute the decrease of SHG signal at longer times to the flip-flop of the probe molecule from the outer to inner leaflet. The expected rate of flip-flop is not well known as a large number of different experiments on different systems has produced a wide range of results (21,28,(56)(57)(58)(59)(60)(61)(62)(63)(64). It should be first noted that FM probes have been reported to be permanently bound to the outer leaflet of a bilayer membrane due to their double positive charge (65), incapable of translocating to the inner leaflet without pore or endocytosis mechanisms (66).…”

“…Two other prominent mechanisms of SHG signal decrease that have been observed in living cells and do not require changing the number of molecules in the membrane are ion flux (11) and flip-flop (21,28,37,45). A reduction in the SHG signal due to ion flux would arise from a loss of membrane potential; however, such a loss would also be expected to affect cell viability (46).…”

“…Second harmonic generation (SHG) is a nonlinear optical technique that is sensitive to molecular species at interfaces and has previously been been applied to the study of small molecules interacting with both model (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) and living cell membranes (22)(23)(24)(25)(26)(27)(28)(29). In SHG, incoming light at a frequency, ω, with sufficient intensity induces a second order polarization in the sample.…”

Molecule‐Membrane Interactions in Biological Cells Studied with Second Harmonic Light Scattering

“…There has been an extensive body of work on the application of SHG as an imaging modality following the first SH imaging study on the microstructure of polycrystalline ZnSe . More recent applications include visualizing cell membranes stained with SH‐active dyes, monitoring drug binding interactions at planar membrane surfaces, mapping water surface potentials in glass micro‐capillaries and on lipid membranes, and development of super‐resolution techniques for SH imaging …”

Multimodal optical contrast agents as new tools for monitoring and tuning nanoemulsion internalisation into cancer cells. From live cell imaging to in vivo imaging of tumours

“…20 Exogenous SHG membrane probes, including push-pull amphiphilic dyes, have also been developed as membrane dyes to probe membrane potentials. [21][22][23][24][25][26] Hence amphiphilic push-pull dyes D1 and D2 were meant to act as multimodal optical contrast agents allowing (i) in vitro multiphoton imaging by simultaneous two-photon excited uorescence (TPEF) and SHG microscopy, [23][24][25][26][27][28][29][30] (ii) monitoring the fate of the encapsulated dyes in cancer cells at the subcellular level and (iii) in vivo imaging of tumours in small animals using NIRF.…”