Selective Plane Illumination Microscopy (SPIM) is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET). Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca2+ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca2+ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca2+ signal percolation, predicted in previous studies, has been directly visualized.

We introduce flow optical projection tomography, an imaging technique capable of visualizing the vasculature of living specimens in 3-D. The method detects the movement of cells in the bloodstream and creates flow maps using a motion-analysis procedure. Then, flow maps obtained from projection taken at several angles are used to reconstruct sections of the circulatory system of the specimen. We therefore demonstrate an in vivo, 3-D optical imaging technique that, without the use of any labeling, is able to reconstruct and visualize the vascular network of transparent and weakly scattering living specimens.

Microscopy techniques can readily visualize the finest details of embryo vasculature, but still lack to provide a complete three-dimensional representation of blood flow parameters. We present an in-vivo 3D imaging technique, able to reconstruct the blood cell velocity vector over a large volume of zebrafish embryos. This low cost and relatively simple technique is exploited to quantitatively assess blood velocity in the zebrafish tail at different stages of development.

Optical imaging through biological samples is compromised by tissue scattering and currently various approaches aim to overcome this limitation. In this paper we demonstrate that an all optical technique, based on non-linear upconversion of infrared ultrashort laser pulses and on multiple view acquisition, allows the reduction of scattering effects in tomographic imaging. This technique, namely Time-Gated Optical Projection Tomography (TGOPT), is used to reconstruct three dimensionally the internal structure of adult zebrafish without staining or clearing agents. This method extends the use of Optical Projection Tomography to optically diffusive samples yielding reconstructions with reduced artifacts, increased contrast and improved resolution with respect to those obtained with non-gated techniques. The paper shows that TGOPT is particularly suited for imaging the skeletal system and nervous structures of adult zebrafish.

Optical Projection Tomography (OPT) is a three dimensional imaging technique that is particularly suitable for studying millimeter sized biological samples and organisms. Similarly to x-ray computed tomography, OPT is based on the acquisition of a sequence of images taken through the sample at many angles (projections). Assuming the linearity of the optical absorption process, the projections are combined to reconstruct the 3-D volume of the sample, typically using a filtered back-projection algorithm. OPT has been applied to in-vivo imaging of zebrafish (Danio rerio). The instrument and the protocol for in vivo imaging of zebrafish embryos and juvenile specimens are described. Light scattering remains a challenge for in vivo OPT, especially when samples at the upper size limit, like zebrafish at the adult stage, are under study. We describe Time-Gated Optical Projection Tomography (TGOPT), a technique able to reconstruct adult zebrafish internal structures by counteracting the scattering effects through a fast time-gate. The time gating mechanism is based on non-linear optical upconversion of an infrared ultrashort laser pulse and allows the detection of quasi-ballistic photons within a 100 fs temporal gate. This results in a strong improvement in contrast and resolution with respect to conventional OPT. Artifacts in the reconstructed images are reduced as well. We show that TGOPT is suited for imaging the skeletal system and nervous structures of adult zebrafis