Multiphoton microscopy (MPM) has been the subject of major development efforts for about 25 years for imaging biological specimens at micron scale and presented as an elegant alternative to classical fluorescence methods such as confocal microscopy. In this topical review, the main interests and technical requirements of MPM are addressed with a focus on the crucial role of excitation source for optimization of multiphoton processes. Then, an overview of the different sources successfully demonstrated in literature for MPM is presented, and their physical parameters are inventoried. A classification of these sources in function with their ability to optimize multiphoton processes is proposed, following a protocol found in literature. Starting from these considerations, a suggestion of a possible identikit of the ideal laser source for MPM concludes this topical review.

The temperature-dependent fluorescence property of Rhodamine B was used to measure changes in temperature at the cellular level induced by either infrared laser light exposure or high intensity, ultrashort pulsed electric fields. The thermal impact of these stimuli were demonstrated at the cellular level in time and contrasted with the change in temperature observed in the extracellular bath. The method takes advantage of the temperature sensitivity of the fluorescent dye Rhodamine B which has a quantum yield linearly dependent on temperature. The thermal effects of different temporal pulse applications of infrared laser light exposure and of nanosecond pulsed electric fields were investigated. The temperature increase due to the application of nanosecond pulsed electric fields was demonstrated at the cellular level.

Fitting Gaussian functions to empirical data is a crucial task in a variety of scientific applications, especially in image processing. However, most of the existing approaches for performing such fitting are restricted to two dimensions and they cannot be easily extended to higher dimensions. Moreover, they are usually based on alternating minimization schemes which benefit from few theoretical guarantees in the underlying nonconvex setting. In this paper, we provide a novel variational formulation of the multivariate Gaussian fitting problem, which is applicable to any dimension and accounts for possible non-zero background and noise in the input data. The block multiconvexity of our objective function leads us to propose a proximal alternating method to minimize it in order to estimate the Gaussian shape parameters. The resulting FIGARO algorithm is shown to converge to a critical point under mild assumptions. The algorithm shows a good robustness when tested on synthetic datasets. To demonstrate the versatility of FI-GARO, we also illustrate its excellent performance in the fitting of the Point Spread Functions of experimental raw data from a two-photon fluorescence microscope.

A specific scheme is used for fiber delivery of ultrashort pulses using conventional elements. Starting from a standard femtosecond Ti:Al(2)O(3) oscillator (150 fs @ 830 nm), perfectly compressed ultrashort pulses with a duration of 45 fs are produced at the output of a standard two meter long single-mode fiber. The setup allows compensating independently and simultaneously second and third orders of chromatic dispersion as well as management of self-phase modulation in the fiber. It includes an optimized dispersion compensation line made of the assembly of diffraction gratings and prisms. The unsurpassed performances of the device are experimentally and numerically highlighted. Fiber delivery of sub-30 fs multinanojoule pulses is discussed.

The influence of infrared laser pulses on intracellular Ca signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo-4 was used to measure Ca in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo-4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca transient with similar dose-response characteristics in hippocampal neurons and glioblastoma cells (half-maximal effective energy density EC of around 58 J.cm ). For both type of cells, the source of the infrared-induced Ca transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP -induced Ca release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP cell signaling cascade has many potential applications including the development of optoceutical therapeutics.

International audienceMulticolor multiphoton microscopy is experimentally demonstrated for the first time on a spectral bandwidth of excitation of 300 nm (full width half maximum) thanks to the implementation a nanosecond supercontinuum (SC) source compact and simple with a low repetition rate. The interest of such a wide spectral bandwidth, never demonstrated until now, is highlighted in vivo: images of glioma tumor cells stably expressing eGFP grafted on the brain of a mouse and its blood vessels network labelled with Texas Red® are obtained. These two fluorophores have a spectral bandwidth covering the whole 300 nm available. In parallel, a similar image quality is obtained on a sample of mouse muscle in vitro when excited with this nanosecond SC source or with a classical high rate, femtosecond and quasi monochromatic laser. This opens the way for (i) a simple and very complete biological characterization never performed to date with multiphoton processes, (ii) multiple means of contrast in nonlinear imaging allowed by the use of numerous fluorophores and (iii) other multiphoton processes like three-photon ones

We present a new instrumental and computational pipeline named FAMOUS: Fast algorithm for 3D multiphoton microscopy of biomedical structures. This pipeline rests on a multiphoton microscopy (MPM) strategy combined with an original 3D post-processing computational approach. In the present work, FAMOUS approach is devoted to the 3D imaging of the myosin assembly of the ultrastructure of a whole striated skeletal muscle unsliced. Raw recordings of second harmonic generation (SHG) from myosin and instrumental point-spread functions (PSF) are led simultaneously all along the unsliced muscle depth. This procedure highlights a space-variant distortion of the PSF and the SHG signals and an optical degradation of the axial resolution increasing with imaging depth resulting from the optical heterogeneity of the muscle structure. A 3D mathematical modelling of the PSF, relying on the recent FIGARO method, evaluates and models the depth-variant evolution of the optical distortions. Then, the fast image deblurring algorithm BD3MG is employed to correct those non-stationary distortions all along the sample, thanks to a sounded regularized inverse problem methodology. This leads to the pipeline called FAMOUS, whose performance are highlighted for the optimization of the axial information of myosin structure, whose dimensions are close to the axial resolution limit. For the first time, the 3D organization of the myosin in skeletal muscle is visually shown from an unsliced whole muscle, starting with a solution of optical microscopy. The axial visualization of this organization presently disclosed were never shown until now without a preliminary procedure of sample slicing and labelling. Our original solution FAMOUS delivers a new point of view of this biological structure in the 3 dimensions and especially in the optical axis. Image information theoretically expected are now revealed visually in the optical axis for the first time in a whole organ unsliced and label free.

We experimentally demonstrate a compact and efficient arrangement for fiber delivery of sub-30 fs energetic light pulses at 800 nm. Pulses coming from a broadband Ti:Sapphire oscillator are negatively prechirped by a grism-pair stretcher that allows for the control of second and third orders of dispersion. At the direct exit of a 2.7-m long large mode area (LMA) photonic crystal fiber 1-nJ pulses are temporally compressed to 29 fs producing close to 30 kW of peak power. The tunability of the device is studied. Comparison between LMA fibers and standard SMF fibers is also discussed. ©2012 Optical Society of America