Adapting a compact confocal microscope system to a two‐photon excitation fluorescence imaging architecture

Abstract

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.

Two-Photon Analysis of Lead Accumulation in Rat Cerebellar Granule Neurons

“…Each coverslip was prepared immediately before the recording and subjected to a single treatment with Pb 2+ . Two-photon microscopy was performed on a set up that we previously described (11). The recording chamber was mounted on the stage of an inverted microscope Eclipse TE300 (Nikon Instruments).…”

“…Indo-1 has been successfully employed to measure Pb 2+ uptake in several studies with conventional (one photon) microscopy (6,10). In the present study, we have investigated if a similar approach is feasible by two-photon excitation fluorescence microscopy (TPE; (11)), a technique that allows confocal imaging with reduced phototoxicity and photobleaching (12). We demonstrate that indo-1 can be used to image Pb 2+ influx by TPE microscopy and that the lower affinity of indo-1 for Pb 2+ with respect to fura-2 can overcome the problem of dye saturation.…”

<title>Suitable microscope objectives for multiphoton digital imaging</title>

“…However, only the fluorophores from the excited plane of focus are collected. For detailed reviews of 2 photon and multiphoton (3 photon) imaging, refer to Denk et al (1995), Diaspro et al (1999b), Denk (2000), Potter (2000), Wise (2000), Xu (2000), Lemasters (2000). Even though multi photon and non-linear optical excitation have been shown to be new and powerful tools for the investigation of dynamic cell processes, the technology requires further development before it finds wide spread acceptance from plug-and-play instrumentation (Wise, 2000).…”

“…Denk (2000) elaborates on the value of option (3); and references several successes. Examples of converted systems have been described in detail by Diaspro et al (1999b), Potter (2000, and Mohler and Squirrell (2000).…”

A custom-made two-photon microscope and deconvolution system

“…Although commercial systems for two-photon microscopy are available, there are several advantages to converting an existing confocal system into a two-photon microscope [7,13,14,16,17,18]. An important advantage is the flexibility to choose all the components of the system including the microscope, confocal unit and laser.…”