Francesca Cella

A New FRAP/FRAPa Method for Three-Dimensional Diffusion Measurements Based on Multiphoton Excitation Microscopy

We present a new convenient method for quantitative three-dimensionally resolved diffusion measurements based on the photobleaching (FRAP) or photoactivation (FRAPa) of a disk-shaped area by the scanning laser beam of a multiphoton microscope. Contrary to previously reported spot-photobleaching protocols, this method has the advantage of full scalability of the size of the photobleached area and thus the range of diffusion coefficients, which can be measured conveniently. The method is compatible with low as well as high numerical aperture objective lenses, allowing us to perform quantitative diffusion measurements in three-dimensional extended samples as well as in very small volumes, such as cell nuclei. Furthermore, by photobleaching/photoactivating a large area, diffusion along the optical axis can be measured separately, which is convenient when studying anisotropic diffusion. First, we show the rigorous mathematical derivation of the model, leading to a closed-form formula describing the fluorescence recovery/redistribution phase. Next, the ability of the multiphoton FRAP method to correctly measure absolute diffusion coefficients is tested thoroughly on many test solutions of FITC-dextrans covering a wide range of diffusion coefficients. The same is done for the FRAPa method on a series of photoactivatable green fluorescent protein solutions with different viscosities. Finally, we apply the method to photoactivatable green fluorescent protein diffusing freely in the nucleus of living NIH-3T3 mouse embryo fibroblasts.

Role of three-dimensional bleach distribution in confocal and two-photon fluorescence recovery after photobleaching experiments

The quantitative analysis of fluorescence perturbation experiments such as fluorescence recovery after photobleaching (FRAP) requires suitable analytical models to be developed. When diffusion in 3D environments is considered, the description of the initial condition produced by the perturbation (i.e., the photobleaching of a selected region in FRAP) represents a crucial aspect. Though it is widely known that bleaching profiles approximations can lead to errors in quantitative FRAP measurements, a detailed analysis of the sources and the effects of these approximations has never been conducted until now. In this study, we measured the experimental 3D bleaching distributions obtained in conventional and two-photon excitation schemes and analyzed the deviations from the ideal cases usually adopted in FRAP experiments. In addition, we considered the non-first-order effects generated by the high energy pulses usually delivered in FRAP experiments. These data have been used for finite-element simulations mimicking FRAP experiments on free diffusing molecules and compared with FRAP model curves based on the ideal bleach distributions. The results show that two-photon excitation more closely fits ideal bleaching patterns even in the event of fluorescence saturation, achieving estimations of diffusion coefficients within 20% accuracy of the correct value.

Studying the illumination puzzle towards an isotropic increase of optical resolution

The aim of this work is to propose and analyze optical schemes to obtain an improvement of resolution in optical fluorescence microscopy. This goal can be achieved by implementing interfering illumination beams. We start from the insertion, on the illumination arm of the confocal microscope, of appropriately phase plates inducing laterally interfering beams, and then we propose to exploit two-photon excitation, too. We plan to implement solutions for shaping also the axial component of the point spread function by use of phase-only pupil filters and binary filters. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach coupled to experimental procedures utilizing ultra-thin fluorescent layers and thick gels containing immobile fluorescent molecules as 2D and 3D phantoms, respectively. As well, image processing and successive views can be recombined to get a final isotropic improvement of resolution.

Non-linear effects and role of scattering in multiphoton imaging of thick biological samples

Non linear optical scanning microscopy has became a useful tool for living tissue imaging. Biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. While performing imaging of biological scattering tissues in non linear excitation regime, the localization of the maximum 2PE intensity was found to shift closer to the surface1 and the 2PE imaging depth limit appears strongly limited by near surface fluorescence.2 In this work we computed the illumination and the photobleaching distribution3 in order to characterize the effects induced by scattering. The simulations have been performed for different scattering coefficients and different focus depth. An experimental test has been carried out by imaging, with 0.9 numerical aperture objective, thick scattering fluorescent immobile sample (polyelectrolyte gel). Results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface.

Improving image formation from the illumination side: linear and non-linear excitation cases

Diffraction imposes for each optical system a resolution limit which could be described by using the vectorial theory of Richards and Wolf. This theory defines the intensity distribution of a point like source imaged by a lens assuming ideal imaging conditions. Unfortunately, these conditions can not be completely achieved in practical situations as a recorded microscope image is always affected by noise which makes the resolution limit worse. In this work we propose and analyze optical set-up schemes towards an image quality improvement in terms of Signal to Noise Ratio (SNR) in linear and non-linear fluorescence microscopy. In order to reach this purpose we insert, on the illumination arm of the microscope, a proper amplitude ring filter inducing laterally interfering beams. The effect induced by the filter results in a shape engineering of the 3D-PSF and in a redistribution of the spatial frequencies of the OTF. In particular, the high frequencies information are collected at improved SNR. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach analyzing the results in both space and frequency domain to characterize the optical system response. Analysis reveals that, although the theoretical resolution of the system is unchanged, when we impose a certain noise level the practical imaging quality could be improved in the ring filtering scheme. The results suggest that further improvement can be reached by the usage of the proposed annular filers in combination with image restoration. A comparison between linear and non-linear excitation cases is presented.

SIPcharts using uniform ultra-thin and thin layers for Z-response measurements in two-photon excitation fluorescence microscopy

Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy. Layers can be realized as ultra-thin (<< 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart), we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the z-response of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be effciently used for real time alignment of the microscope.