Anna C. Croce

Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine.

Photodynamic therapy is a moderately invasive therapeutic procedure based on the action of photosensitizers (PSs). These compounds are able to absorb light, and dissipate energy through photochemical processes leading to the production of oxidizing chemical species (singlet oxygen, free radicals or reactive oxygen species) which can damage the cell molecular structures eventually inducing cell death. To increase the entering through the plasma membrane, a PS with suitable chemical structure can be modified by addition of chemical groups (e.g., acetate or phosphate): this affects both the fluorescence emission and of the photosensitizing properties of the native PS. The modified compounds behave as fluorogenic substrates (FSs), since inside the cell the bound groups can be enzymatically removed and the fluorescence and photosensitizing properties of the native molecules are restored. With the aim to detect the subcellular localization of photoactive molecules at transmission electron microscopy, we loaded cultured HeLa cells with two different FSs, Rose Bengal acetate (RB-Ac) or Hypocrellin B acetate (HypB-Ac), and took advantage of the photophysical properties of the intracellularly restored PS molecules to obtain the photoconversion of diaminobenzidine (DAB) into an electrondense product. We demonstrated that RB-Ac and HypB-Ac are mostly internalized by endocytosis, and are converted into the native PSs already at the cell surface. Endocytosed PS molecules apparently follow the endosomes–lysosome route, being found in endosomes, lysosomes and multivescicular bodies; PS molecules were also detected in the cytosol. This ultrastructural localization of the photoactive molecules is fully consistent with the multiorganelle photodamage observed after irradiation in culture of RB-Ac- or HypB-Ac-loaded cells. Due to the very short half-life of the oxidizing chemical species and their limited mobility, DAB deposits do localize in close proximity of the very place where photoactive molecules elicited the production of reactive oxygen species upon light irradiation. Therefore, DAB photoconversion promises to be a suitable tool for directly visualizing in single cells the PS molecules at high resolution, helping to elucidate their mode of penetration into the cell as well as their dynamic intracellular redistribution and organelle targeting.

In vivo autofluorescence spectrofluorometry of central serotonin

The autofluorescence properties of serotonin (5-HT) were investigated by light spectrofluorometry in in vitro, ex vivo and in vivo experiments. Ex vivo samples were prepared from rat brain regions containing serotonin (5-HT) i.e. cortex, striatum, hippocampus. Rats were untreated (controls) or previously submitted to chronic behavioural or pharmacological treatments known to affect endogenous 5-HT levels. Autofluorescence analysis (excitation: 366 nm) on hippocampus homogenates supplied with exogenous 5-HT revealed spectral alterations attributable to changes of endogenous 5-HT levels. In vivo, real time fluorescence studies were performed via a 50 microm diameter optic fiber probe stereotaxically implanted into selected brain areas of anaesthetised rats treated with fluoxetine or 5-OH-tryptophan. All autofluorescence data were consistent with those obtained in parallel experiments performed with ex vivo or in vivo voltammetry, confirming that auto-fluorescence spectroscopy is a suitable technique for the direct assessment of fluorescent neurotransmitters. This is a reliable evidence of the in vivo application of spectroscopy together with optic fiber probe for in vivo, in situ and real time measurement of 5-HT in discrete brain areas.

Autofluorescence discrimination of metabolic fingerprint in nutritional and genetic fatty liver models

Liver tissue autofluorescence (AF) has been characterized in two models with a different potential to undergo disease progression to steatohepatitis: Wistar rats, administered with a methionine, choline deficient diet (MCD), and Zucker (fa/fa) rats, homozygous for a spontaneous mutation of leptin receptor. AF spectra were recorded from liver tissue cryostatic sections by microspectrofluorometry, under 366nm excitation. Curve fitting analysis was used to estimate the contribution of different endogenous fluorophores (EFs) to the overall AF emission: i) fluorescing fatty acids, a fraction of liver lipids up to now poorly considered and complicated to detect by conventional procedures; ii) lipofuscin-like lipopigments, biomarkers of oxidizing events; iii) NAD(P)H and flavins, biomarkers of energy metabolism and tissue redox state. AF data and biochemical correlates of hepatocellular injury resulted to depend more on rat strain than on intratissue bulk lipid or ROS levels, reflecting a different metabolic ability of the two models to counteract potentially harmful agents. AF analysis can thus be proposed for extensive applications ranging from experimental hepatology to the clinics. AF based diagnostic procedures are expected to help both the prediction of the risk of fatty liver disease progression and the prescreening of marginal organs to be recruited as donors for transplantation. A support is also foreseen in the advancement and personalization of strategies to ameliorate the donor organ preservation outcome and the follow up of therapeutic interventions.

Fluorescing fatty acids in rat fatty liver models

The autofluorescence (AF) of NAD(P)H and flavins has been at the basis of many in-situ studies of liver energy metabolism and functionality. Conversely, few data have been so far reported on fluorescing lipids. In this work we investigated the AF of liver lipid extracts from two fatty liver models, Wistar rats fed with MCD diet for 12 days (Wi-MCD), and obese (fa/fa) Zucker rats. Among the most abundant fatty acids in the lipid extracts, indicated by mass spectrometry, arachidonic acid (AA) exhibited higher quantum yield than the other fluorescing fatty acids (FLFA), and red shifted AF spectrum. This allowed to estimate the AA contribution to the overall emission of lipid extracts by curve fitting analysis. AA prevailed in obese Zucker livers, accounting for the different AF spectral profiles between the two models. AF and mass spectrometry indicated also a different balance between the fluorescing fraction and the overall amount of AA in the two models. The ability of AF to detect directly AA and FLFA was demonstrated, suggesting its supportive role as tool in wide-ranging applications, from the control of animal origin food, to experimental investigations on liver fat accumulation, lipotoxicity and disease progression, with potential translation to the clinics.

Autofluorescence-based optical biopsy: An effective diagnostic tool in hepatology

Autofluorescence emission of liver tissue depends on the presence of endogenous biomolecules able to fluoresce under suitable light excitation. Overall autofluorescence emission contains much information of diagnostic value because it is the sum of individual autofluorescence contributions from fluorophores involved in metabolism, for example, NAD(P)H, flavins, lipofuscins, retinoids, porphyrins, bilirubin and lipids, or in structural architecture, for example, fibrous proteins, in close relationship with normal, altered or diseased conditions of the liver. Since the 1950s, hepatocytes and liver have been historical models to study NAD(P)H and flavins as in situ, real‐time autofluorescence biomarkers of energy metabolism and redox state. Later investigations designed to monitor organ responses to ischaemia/reperfusion were able to predict the risk of dysfunction in surgery and transplantation or support the development of procedures to ameliorate the liver outcome. Subsequently, fluorescent fatty acids, lipofuscin‐like lipopigments and collagen were characterized as optical biomarkers of liver steatosis, oxidative stress damage, fibrosis and disease progression. Currently, serum AF is being investigated to improve non‐invasive optical diagnosis of liver disease. Validation of endogenous fluorophores and in situ discrimination of cancerous from non‐cancerous tissue belong to the few studies on liver in human subjects. These reports along with other optical techniques and the huge work performed on animal models suggest many optically based applications in hepatology. Optical diagnosis is currently offering beneficial outcomes in clinical fields ranging from the respiratory and gastrointestinal tracts, to dermatology and ophthalmology. Accordingly, this review aims to promote an effective bench to bedside transfer in hepatology.

NAD(P)H and Flavin Autofluorescence Correlation with ATP in Rat Livers with Different Metabolic Steady State Conditions

The monitoring of NAD(P)H and flavin autofluorescence (AF) is at the basis of numerous investigations on energy metabolism. Nevertheless, the ability of these AF biomarkers to accurately represent the energy currency, ATP, is poorly explored. Here, we focused on the AF/ATP correlation in lean and fatty livers with different steady‐state metabolic conditions, achieved after organ isolation, preservation and recovery, in a likely dependence on both liver intrinsic metabolic features and externally induced perturbations. Within these eventual, various conditions, a significant correlation was detected between liver NAD(P)H and flavin AF, measured via fiber‐optic probe, and biochemical ATP data, strengthening AF as biomarker of energy metabolism in steady‐state conditions for wide‐ranging experimental and diagnostic applications.