Fabio Cannone

Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels

Many of the effects exerted on protein structure, stability, and dynamics by molecular crowding and confinement in the cellular environment can be mimicked by encapsulation in polymeric matrices. We have compared the stability and unfolding kinetics of a highly fluorescent mutant of Green Fluorescent Protein, GFPmut2, in solution and in wet, nanoporous silica gels. In the absence of denaturant, encapsulation does not induce any observable change in the circular dichroism and fluorescence emission spectra of GFPmut2. In solution, the unfolding induced by guanidinium chloride is well described by a thermodynamic and kinetic two‐state process. In the gel, biphasic unfolding kinetics reveal that at least two alternative conformations of the native protein are significantly populated. The relative rates for the unfolding of each conformer differ by almost two orders of magnitude. The slower rate, once extrapolated to native solvent conditions, superimposes to that of the single unfolding phase observed in solution. Differences in the dependence on denaturant concentration are consistent with restrictions opposed by the gel to possibly expanded transition states and to the conformational entropy of the denatured ensemble. The observed behavior highlights the significance of investigating protein function and stability in different environments to uncover structural and dynamic properties that can escape detection in dilute solution, but might be relevant for proteins in vivo.

Two-photon thermal bleaching of single fluorescent molecules.

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.

Two-photon microscopy and spectroscopy based on a compact confocal scanning head

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two-photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method

We report on the construction details of a compact autocorrelator set‐up for the measurement of the width of infrared laser pulses at the focal plane of a microscope for two‐photon excitation fluorescence imaging. One of the novelties of the set‐up, which leads to an improved measurement accuracy, is the use of a modulation technique that is achieved by mounting one of the interferometer mirrors on a loudspeaker driven by a sinusoidal bias at low frequency. A non‐linear least‐square routine selects only that part of the fluorescence signal that is modulated at the same frequency as the loudspeaker bias. To further increase the accuracy, the laser pulse width is obtained from a series of measurements at different values of the modulation bias. The autocorrelator is a compact single bread‐board (10 × 20 cm); it is PC‐controlled both for the acquisition and the analysis of the data and can be coupled to different ports of the microscope. The increase in the pulse width measured for three different ports of the microscope is well accounted for by the group velocity dispersion and the glass thickness of the optics found along these paths.

High sensitivity optical microscope for single molecule spectroscopy studies

We describe the setup and apply two algorithms for fast imaging in a sample raster scanning two photon microscope. Imaging can be performed at a rate of 1–100 lines per second with a closed loop piezo actuator, and the detection is performed via avalanche photodiodes. This allows to investigate single molecule emission with 50 ms time resolution. In a slow scanning algorithm we have implemented fluorescence fluctuation analysis by computing the photon counting histogram (PCH) on each pixel of the image. In a fast-scan acquistion method the image acquistion rate is 5 lines per second on a large field of view and high resolution(50 nm scanning step, 100×100 μm2 field of view) and ≅100 lines per second on smaller field of views with optically limited resolution (200 nm scanning step, 20×20 μm2 field of view). This figure, which is lower than the typical value for normal confocal scanning imaging (≅500 lines per second), allows nevertheless to perform imaging studies of extended samples in reasonable times fo...

Single molecule photodynamics by means of one- and two-photon approach

Single molecule spectroscopy allows to investigate heterogeneous behaviours on photochemical and structural grounds. We report on studies of the effect of the excitation intensity on the internal photodynamics of simple dyes immobilized on chemically etched glass slides. The use of the excitation intensity needed for two-photon excitation induces local heating, structural changes and transitions to dark states. Similar behaviour is found on single green fluorescent proteins immobilized on glass slides or embedded in silica gels upon single-photon excitation. However, by sampling the images with sufficiently low frequency, we are able to follow relevant biological events, such as the unfolding kinetics. We find that the glass slides are preferable in terms of the signal-to-noise ratio but the protein is not preserved in its native state, while evidence for the native conformation of the single proteins in the silica gels is found in the uniformity of the fluorescence emission.

Two-photon interactions at single fluorescent molecule level

Single-molecule spectroscopy and single-molecule detection are emerging areas that have many applications when combined with scanning, imaging, and spectroscopy techniques. We have combined a commercial confocal scanning head, to a Ti:sapphire laser and to an inverted microscope, for the detection of single molecule fluorescence of varies dyes by two-photon excitation. We collected spot images of fluorescent molecules that have been deposited on a substrate considering both blinking and photobleaching behavior of fluorescent spots. Here, we report data related to two-photon interactions that occur with the following fluorescent molecules: Indo-1, Rhodamine 6G, Fluorescein, and Pyrene. The choice of these specific dyes is based upon their wide use in biological and medical applications together with the varying complexity of their chemical structure that increases from Pyrene to Indo-1. Moreover, we report some data about single molecule studies related to denaturation of an enhanced green fluorescent protein, GFPmut2, under one photon excitation regime, that show a very similar trend to that observed for the already mentioned fluorescent molecules.

Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

Discovery of Green Fluorescent Protein (GFP) constituted an important improvement for living cell studies on submicron resolution allowing in vivo fluorescence labeling. We studied the photo-physical properties of single GFP molecules incorporated in a charged polyelectrolyte environment by means of single molecule spectroscopy. The fluorescence characteristics change dramatically in terms of photo-stability,lifetime and blinking behavior so that the proteins scale up to quantum dots. The reported results highlight interesting applications in the design of fluorescent markers and in the development of optical data storage architectures.

Functional imaging of living Paramecium by means of confocal and two-photon excitation fluorescence microscopy

Confocal and Two-photon excitation laser scanning microscopy allow gathering three-dimensional and temporal information from biological systems exploiting fluorescence labeling and autofluorescence properties. In this work we study biological events linked to functionality in Paramecium primaurelia. The internalization of material in ciliated one-celled organisms (protozoa) occurs via different mechanisms, even if most of nutrients, particulate or not, is taken up by food vacuoles formed at the bottom of the oral cavity. The endocytosis of small-sized molecules occurs at the parasomal sacs, located next the ciliar basal bodies. Vital fluorescent dyes (BSA-FITC, WGA-FITC, dextran-Texas Red, cholesteryl-Bodipy) and autofluorescence were used to study formation, movement, and fusion of vesicles during endocytosis and phagocytosis of Paramecium primaurelia. By immobilizing living cells pulsed with food vacuole and endosome markers at successive times after chasing in unlabeled medium, the intracellular movement and fusion of food vacuoles and of endosomes were visualized. A temporal analysis of fluorescence images and the false-color technique were used. Starting from time series or 3D data sets composite images were generated by associating with each originally acquired image a different color corresponding to each sampling point in time and along the z-axis. Second Harmonic Generation Imaging attempts are also outlined.

Environment effects on the oscillatory unfolding kinetics of GFP

The chromophore of a green fluorescent protein (GFP) mutant engineered to enhance emission and stability is known to display erratic switchings among a few of its chemical substates and, in particular, between the anionic A and the neutral N substates, whose difference is associated with a proton exchange and a consequent conformation rearrangement. However, when close to unfolding, the A–N switchings suddenly become very regular as shown by fluorescence oscillations that have been recently observed for molecules embedded in wet silica gel. In order to establish whether the matrix hosting the protein is responsible for these oscillations, we investigated the effect of another medium (silanized surfaces), of a different denaturant (urea) and of cosolvents (D2O and glycerol). The occurrence of periodic A–N switchings, in the last milliseconds before GFP unfolding, is observed under all investigated conditions, together with three specific frequency values that characterize the pre-unfolding fluorescence. Urea and guanidinium, the denaturants employed in order to unfold GFP, do not lead to appreciable differences in the observed switching parameters, whereas the different media embedding the protein give rise only to frequency shifts that scale with the viscosity of the host. The periodicity of the GFP A–N switchings and their dependence on cosolvents suggest that they could be associated with oscillatory motions between meta-stable conformations of the β-barrel surrounding the chromophore near protein unfolding.