Riccardo Cicchi

Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy

We have used nonlinear imaging to evaluate collagen organization in connective tissue ex-vivo samples. Image analysis methods were tested on healthy dermis, normal scars, and keloids. The evaluation of the second harmonic to autofluorescence aging index of dermis (SAAID) has allowed a first characterization of tissues by scoring the collagen/elastin content. Further analyses on collagen morphology in healthy dermis and keloids were performed by image-pattern analysis of SHG images. The gray-level co-occurrence matrix (GLCM) analysis method has allowed classification of different tissues based on the evaluation of geometrical arrangement of collagen fibrillar bundles, whereas a pattern analysis of the FFT images has allowed the discrimination of different tissues based on the anisotropy of collagen fibers distribution. This multiple scoring method represents a promising tool to be extended to other collagen disorders, as well as to be used in in-vivo skin-imaging applications.

Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging.

The loss of organization of the corneal collagen lattice induced by photothermal effects was analyzed by using second-harmonic generation (SHG) imaging. Porcine cornea samples were treated with low-power laser irradiation in order to get localized areas of tissue disorganization. The disorder induced within the irradiated area of corneal stroma was quantified by means of Discrete Fourier Transform, auto-correlation and entropy analyses of the SHG images. Polarization modulated SHG measurements allowed to probe the changes in the structural anisotropy of sub-micron hierarchical levels of the stromal collagen. Our results emphasize the great potential of the SHG imaging to detect subtle modifications in the collagen assembly. The proposed analytical methods may be used to track several genetic, pathologic, accidental or surgical-induced disorder states of biological tissues.

Contrast and depth enhancement in two-photon microscopy of human skin ex vivo by use of optical clearing agents

We investigate the application of hyperosmotic optical clearing agents to improve the image contrast and penetration depth in two-photon microscopy of human dermis ex vivo. We show that the agents glycerol, propylene glycol, and glucose all convey significant improvements and we provide results on their dynamic behaviour and the reversibility of the effect. At suitable concentrations, such agents have the potential to be compatible with living tissue and may possibly enhance in-vivo deep-tissue imaging.

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

Second-harmonic generation (SHG) microscopy is a fantastic tool for imaging collagen and probing its hierarchical organization from molecular scale up to tissue architectural level. In fact, SHG combines the advantages of a non-linear microscopy approach with a coherent modality able to probe molecular organization. In this manuscript we review the physical concepts describing SHG from collagen, highlighting how this optical process allows to probe structures ranging from molecular sizes to tissue architecture, through image pattern analysis and scoring methods. Starting from the description of the most relevant approaches employing SHG polarization anisotropy and forward - backward SHG detection, we then focus on the most relevant methods for imaging and characterizing collagen organization in tissues through image pattern analysis methods, highlighting advantages and limitations of the methods applied to tissue imaging and to potential clinical applications.

Multidimensional non-linear laser imaging of Basal Cell Carcinoma

We have used a multidimensional non-linear laser imaging approach to visualize ex-vivo samples of basal cell carcinoma (BCC). A combination of several non-linear laser imaging techniques involving fluorescence lifetime, multispectral two-photon and second-harmonic generation imaging has been used to image different skin layers. This approach has elucidated some morphological (supported by histopathological images), biochemical, and physiochemical differences of the healthy samples with respect to BCC ones. In particular, in comparison with normal skin, BCC showed a blue-shifted fluorescence emission, a higher fluorescence response at 800 nm excitation wavelength and a slightly longer mean fluorescence lifetime. Finally, the use of aminolevulinic acid as a contrast agent has been demonstrated to increase the constrast in tumor border detection. The results obtained provide further support for in-vivo non-invasive imaging of Basal Cell Carcinoma.

Nonlinear laser imaging of skin lesions.

We investigated different kinds of human ex-vivo skin samples by combined two-photon intrinsic fluorescence (TPE), second-harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two-photon emission detection (MTPE). Morphological and spectroscopic differences were found between healthy and pathological skin samples, including tumors. In particular, we examined tissue samples from normal and pathological scar tissue (keloid), and skin tumors, including basal cell carcinoma (BCC), and malignant melanoma (MM). By using combined TPE-SHG microscopy we investigated morphological features of different skin regions. Further comparative analysis of healthy skin and neoplastic samples was performed using FLIM, and MTPE. Finally, we demonstrated the use of methyl-aminolevulinate as a contrast agent to increase the contrast in BCC border detection. The results obtained represent further support for in-vivo noninvasive imaging of diseased skin.

Time- and Spectral-resolved two-photon imaging of healthy bladder mucosa and carcinoma in situ.

Combined non-linear imaging techniques were used to deeply image human ex-vivo fresh biopsies of bladder as well as to discriminate between healthy bladder mucosa and carcinoma in situ. Morphological examination by two-photon excited fluorescence and second-harmonic generation has shown a good agreement with corresponding common routine histology performed on the same samples. Tumor cells appeared slightly different in shape and with a smaller cellular-to-nuclear dimension ratio with respect to corresponding normal cells. Further differences between the two tissue types were found in both spectral emission and fluorescence lifetime distribution by performing temporal- and spectral- resolved analysis of fluorescence. This method may represent a promising tool to be used in a multi-photon endoscope, in a confocal endoscope or in a spectroscopic probe for in-vivo optical diagnosis of bladder cancer.

Combined non-linear laser imaging (two-photon excitation fluorescence microscopy, fluorescence lifetime imaging microscopy, multispectral multiphoton microscopy) in cutaneous tumours : first experiences

Background  Two‐photon excitation (TPE) fluorescence microscopy is a high‐resolution laser‐scanning imaging technique enabling deep imaging inside biological tissues. TPE microscopy has the triple advantage of offering high spatial resolution (250 nm radially, 800 nm axially), high penetration depth inside skin (200mm ), and low photodamage effects. Further, cells and extracellular matrix intrinsically contain a variety of fluorescent molecules (NADH, tryptophan, keratins, melanin, elastin, cholecalciferol and others), so that biological tissues can be imaged by TPE microscopy without any exogenous probe.

Convergence of integrins and EGF receptor signaling via PI3K/Akt/FoxO pathway in early gene Egr-1 expression†

The early gene early growth response (Egr‐1), a broadly expressed member of the zing‐finger family of transcription factors, is induced in many cell types by a variety of growth and differentiation stimuli, including epidermal growth factor (EGF). Here we demonstrate that Egr‐1 expression is mainly regulated by integrin‐mediated adhesion. Integrin‐dependent adhesion plays a dual role in Egr‐1 regulation, either being sufficient “per se” to induce Egr‐1, or required for EGF‐dependent expression of Egr‐1, which occurs only in adherent cells and not in cells in suspension. To dissect the molecular basis of integrin‐dependent Egr‐1 regulation, we show by FLIM‐based FRET that in living cells beta1‐integrin associates with the EGF receptor (EGFR) and that EGF further increases the extent complex formation. Interestingly, Egr‐1 induction depends on integrin‐dependent PI3K/Akt activation, as indicated by the decrease in Egr‐1 levels in presence of the pharmacological inhibitor LY294002, the kinase‐defective Akt mutant and Akt1/2 shRNAs. Indeed, upon adhesion activated Akt translocates into the nucleus and phosphorylates FoxO1, a Forkhead transcription factors. Consistently, FoxO1silencing results in Egr‐1‐increased levels, indicating that FoxO1 behaves as a negative regulator of Egr‐1 expression. These data demonstrate that integrin/EGFR cross‐talk is required for expression of Egr‐1 through a novel regulatory cascade involving the activation of the PI3K/Akt/Forkhead pathway. J. Cell. Physiol. 218: 294–303, 2009.

Optical methods in the study of protein-protein interactions.

Förster (or Fluorescence) resonance energy transfer (FRET) is a physical process in which energy is transferred nonradiatively from an excited fluorophore, serving as a donor, to another chromophore (acceptor). Among the techniques related to fluorescence microscopy, FRET is unique in providing signals sensitive to intra- and intermolecular distances in the 1-10 nm range. Because of its potency, FRET is increasingly used to visualize and quantify the dynamics of protein-protein interaction in living cells, with high spatio-temporal resolution. Here we describe the physical bases of FRET, detailing the principal methods applied: (1) measurement of signal intensity and (2) analysis of fluorescence lifetime (FLIM). Although several technical complications must be carefully considered, both methods can be applied fruitfully to specific fields. For example, FRET based on intensity detection is more suitable to follow biological phenomena at a finely tuned spatial and temporal scale. Furthermore, a specific fluorescence signal occurring close to the plasma membrane (< or = 100 nm) can be obtained using a total internal reflection fluorescence (TIRF) microscopy system. When performing FRET experiments, care must be also taken to the method chosen for labeling interacting proteins. Two principal tools can be applied: (1) fluorophore tagged antibodies; (2) recombinant fluorescent fusion proteins. The latter method essentially takes advantage of the discovery and use of spontaneously fluorescent proteins, like the green fluorescent protein (GFP). Until now, FRET has been widely used to analyze the structural characteristics of several proteins, including integrins and ion channels. More recently, this method has been applied to clarify the interaction dynamics of these classes of membrane proteins with cytosolic signaling proteins. We report two examples in which the interaction dynamics between integrins and ion channels have been studied with FRET methods. Using fluorescent antibodies and applying FRET-FLIM, the direct interaction of beta1 integrin with the receptor for Epidermal Growth Factor (EGF-R) has been proved in living endothelial cells. A different approach, based on TIRFM measurement of the FRET intensity of fluorescently labeled recombinant proteins, suggests that a direct interaction also occurs between integrins and the ether-a-go-go-related-gene 1 (hERG1) K+ channel.