Franco Fusi

The F685/F730 Chlorophyll Fluorescence Ratio as a Tool in Plant Physiology: Response to Physiological and Environmental Factors**

Summary The effect of chlorophyll concentration, light intensity and leaf temperature on the chlorophyll fluorescence ratio F685/F730 of intact leaves was evaluated. Fluorescence reabsorption that affects mainly the F685 band increases with chlorophyll concentration. This phenomenon was studied on an aurea mutant of tomato and its wild type, with very different chlorophyll content. Fluorescence spectra of the two genotypes were corrected for reabsorption using their transmittance and reflectance properties. The correction removes most of the differences in the two fluorescence spectra. The F685/F730 decreases during the declining phase of the fluorescence induction kinetics. We demonstrated that when red light is used to induce the fluorescence kinetics the variation of F685/F730 is not due to a change in the leaf absorption, as proved by the simultaneous measurement of leaf transmittance. This evidence suggests that the F685/F730 ratio is sensitive to changes in the photosynthetic activity of the leaf. Under natural conditions, the F685/F730 ratio markedly decreases as light intensity and leaf temperature increase during a daily cycle. This behaviour can be due to photoinhibitory and heat stresses. In controlled laboratory conditions, the F685/F730 ratio was seen to decrease under high light intensity (> 1000 μmol m-2 s-1) at constant leaf temperature. It decreases also when leaf temperature was decreased from 25°C to 14°C at low light intensity (150 μmol m-2 s-1). A possible interpretation of these experimental data relies on a non-negligible contribution of PSI to the total fluorescence at physiological temperatures with respect to - PSII fluorescence. Changes in the photosynthetic activity of the two photosystems may induce variation in the F685/F730 ratio. Our results indicate that light intensity and leaf temperature are important parameters to take into account when the F685/F730 ratio is used as stress indicator.

A simple approach to the evaluation of the reabsorption of chlorophyll fluorescence spectra in intact leaves

Abstract A simple approach to the estimation of the effect of reabsorption on chlorophyll fluorescence spectra in intact leaves is reported. It permits one to calculate the chlorophyll fluorescence spectrum effectively emitted inside the leaf using the measured fluorescence spectrum and the total transmittance and reflectance of the leaf. Fluorescence spectra, excited by various laser wavelengths, were measured on leaves of an aurea mutant of tomato and its wild type which differ very much in chlorophyll content and chloroplast ultrastructure. Measured chlorophyll fluorescence spectra for the aurea mutant have higher values of the peak fluorescence ratio F 685 / F 735 ratio of the two fluorescence bands than do the wild type. It is the result of a lower reabsorption effect which affects mainly the shorter-wavelength (685 nm) fluorescence band. The correction for reabsorption removes almost completely the differences between the fluorescence spectra of the two genotypes, confirming the validity of the model. Our work suggests that the evaluation of fluorescence reabsorption must be considered in the widely used methods of vegetation monitoring by chlorophyll fluorescence spectral measurements. It can improve the correlation between fluorescence properties and the physiological state of the plant.

QUANTUM YIELDS FOR LASER PHOTOCYCLIZATION OF BILIRUBIN IN THE PRESENCE OF HUMAN SERUM ALBUMIN. DEPENDENCE OF QUANTUM YIELD ON EXCITATION WAVELENGTH

The quantum yield for laser photocyclization of bilirubin to lumirubin in the presence of human serum albumin (tHLR) was measured at five monochromatic excitation wavelengths in the range 450–530 nm. Solutions used were optically thin throughout the wavelength range and precautions were taken to exclude contributions from photocyclization of bilirubin XHIa impurities. The values obtained (7.2‐18 10‐4) were lower than those previously reported and showed the following wavelength dependence: 457.9 < 488.0 < 501.7 < 514.5 528.7. However, the rate of lumirubin formation, normalized to constant fluence, decreased with wavelength over the same wavelength range and no evidence was found that photoisomerization of bilirubin to lumirubin is faster with green (514.5 or 528.7 nm) than with blue (457.9 or 488.0 nm) light. The stereoselectivity of the configurational isomerization of bilirubin to 4Z,15E and 4E,15Z isomers also was studied. This reaction became less regioselective for the 4Z,15E isomer with increasing wavelength. The observed wavelength dependence of tHLR and of the [4Z,15E]: [4Z,15E] ratio at photoequilibrium are consistent with an exciton coupling model in which intramolecular energy transfer can occur between the two pyrromethenone chromophores of the bilirubin molecule in the excited state. Relative rates of lumirubin formation in vivo at different excitation wavelengths and constant fluence were estimated for different optical thicknesses and for different skin thicknesses. These estimates suggest that the recently reported clinical equivalence of blue and green phototherapy lights probably reflects the marked variation of skin transmittance with wavelength more than wavelength‐dependent photochemistry. The calculations also indicated that the optimal wavelength for phototherapy is probably on the long wavelength side of the bilirubin absorption maximum.

The F685/F730 Chlorophyll Fluorescence Ratio as Indicator of Chilling Stress in Plants

Summary The response of chlorophyll fluorescence to chilling temperatures was evaluated by two different experiments. In the first, the F685/F730 and the F v /F m chlorophyll fluorescence ratios were measured in Phaseolus vulgaris L., cv. Mondragone plants under chilling stress at 4 °C and moderate light (100 μmol m -2 s -1 ) up to 72 hours. F v /F m decreased linearly with chilling time indicating a photoinhibitory effect (no change was observed in the dark under the same conditions). F685/F730 underwent a rapid exponential decay followed by a linear slow decline. In a second experiment, the F685/F730 ratio, the total chlorophyll fluorescence, F685 + F730, and the leaf temperature were monitored on a single leaf in a climate chamber as the temperature was decreased from 20 to 4 °C. The experiment was run simultaneously on the chilling-sensitive Phaseolus vulgaris and on the chilling-tolerant Pisum sativum L. (cv. Shuttle) plants. For both species two phases related to the leaf temperature can be distinguished: the first 4-hour period during which the leaf temperature decreased from 24 to 4 °C, and a second period during which the leaf temperature slightly oscillated around 4°C. The behaviour of F685/F730 for the bean was quite different from that of the pea plant. During the first phase, it decreased markedly for the chilling-sensitive bean while a slight increase was observed for the chilling-resistant pea. In the following period, the F685/F730 values for the pea remained constant while those for the bean were found still to decrease. On the basis of our results, the use of the chlorophyll fluorescence ratio as indicator of plant chilling sensitivity can be envisaged.

ANALYSIS OF LASER-INDUCED FLUORESCENCE LINE SHAPE OF INTACT LEAVES : APPLICATION TO UV STRESS DETECTION

Abstract— In vivo laser‐induced fluorescence spectra of intact leaves of healthy and UV‐irradiated Salvia splendens plants excited at 337 nm by a nitrogen laser were recorded using an optical multichannel analyzer system. The spectra showed the typical fluorescence bands centered around 450, 530, 685 and 730 nm. Exposure to UV radiation changed the relative intensity values of these bands and their peak positions. The analysis of the acquired spectra in terms of a linear combination of Gaussian bands was carried out to determine accurately the peak positions and the relative intensity contribution of the various bands to the laser‐induced fluorescence spectra on healthy and UV‐treated plants of different age.

Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues.

Abstract Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red–green–blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.

WAVELENGTH-DEPENDENT QUANTUM YIELD FOR Z E ISOMERIZATION OF BILIRUBIN COMPLEXED WITH HUMAN SERUM ALBUMIN

Abstract— The quantum yield, φZE, for configurational photoisomerization (4Z,15Z→ 4Z,15E) of bilirubin bound non‐covalently to human serum albumin was determined (at 23 ± 2°C) by laser excitation and chromatographic analysis of products. Values obtained for photoexcitation at 465 nm were about one‐half those previously reported. The quantum yield was dependent on excitation wavelength, decreasing from a value of 0.109 ± 0.010 for excitation at 457.9 nm to a value of 0.054 ± 0.005 for excitation at 514.5 nm. The wavelength dependence is consistent with rapid transfer of excitation energy between the two non‐identical pyrromethenone chromophores of bilirubin in the singlet excited state. Since the quantum yields for photoisomerization and luminescence of bilirubin bound to serum albumin at room temperature are both low, internal conversion processes, rather than Z→E configurational isomerizations, are probably the major pathways for deactivation of photo‐excited bilirubin.

Size dependent biological profiles of PEGylated gold nanorods

The perspective of introducing plasmonic particles for applications in biomedical optics is receiving much interest. However, their translation into clinical practices is delayed by various factors, which include a poor definition of their biological interactions. Here, we describe the preparation and the biological profiles of gold nanorods belonging to five different size classes with average effective radii between ∼5 and 20 nm and coated with polyethylene glycol (PEG). All these particles exhibit decent stability in the presence of representative proteins, low cytotoxicity and satisfactory compatibility with intravenous administration, in terms of their interference with blood tissue. However, the suspension begins to become unstable after a few days of exposure to blood proteins. Moreover, the cytotoxicity is a little worse for smaller particles, probably because their purification is more critical, while undesirable interactions with the mononuclear phagocyte system are minimal in the intermediate size range. Overall, these findings hold implications of practical relevance and suggest that PEGylated gold nanorods may be a versatile platform for a variety of biomedical applications.

Modeled gravitational unloading triggers differentiation and apoptosis in preosteoclastic cells

Gravity acts permanently on organisms as either static or dynamic stimulation. Understanding the influence of gravitational and mechanical stimuli on biological systems is an intriguing scientific problem. More than two decades of life science studies in low g, either real or modeled by clinostats, as well as experimentation with devices simulating different types of controlled mechanical stimuli, have shown that important biological functions are altered at the single cell level. Here, we show that the human leukemic line FLG 29.1, characterized as an osteoclastic precursor model, is directly sensitive to gravitational unloading, modeled by a random positioning machine (RPM). The phenotypic expression of cytoskeletal proteins, osteoclastic markers, and factors regulating apoptosis was investigated using histochemical and immunohistochemical methods, while the expression of the corresponding genes was analyzed using RT‐PCR. A quantitative bone resorption assay was performed. Autofluorescence spectroscopy and imaging were applied to gain information on cell metabolism. The results show that modeled hypogravity may trigger both differentiation and apoptosis in FLG 29.1 cells. Indeed, when comparing RPM versus 1 × g cultures, in the former we found cytoskeletal alterations and a marked increase in apoptosis, but the surviving cells showed an osteoclastic‐like morphology, overexpression of osteoclastic markers and the ability to resorb bone. In particular, the overexpression of both RANK and its ligand RANKL, maintained even after return to 1 × g conditions, is consistent with the firing of a differentiation process via a paracrine/autocrine mechanism. J. Cell. Biochem. 98: 65–80, 2006.

Irradiation of amelanotic melanoma cells with 532 nm high peak power pulsed laser radiation in the presence of the photothermal sensitizer Cu(II)-hematoporphyrin: a new approach to cell photoinactivation.

Abstract— Cu(II)‐hematoporphyrin (CuHp) was efficiently accumulated by B78H1 amelanotic melanoma cells upon incubation with porphyrin concentrations up to 52 μM When the cells incubated for 18 h with 13 μM CuHp were irradiated with 532 nm light from a Q‐switched Nd: YAG laser operated in a pulsed mode (10 ns pulses, 10 Hz) a significant decrease in cell survival was observed. The cell photoinactivation was not the consequence of a photodynamic process, as CuHp gave no detectable triplet signal upon laser flash photolysis excitation and no decrease in cell survival was observed upon continuous wave irradiation. Thus, it is likely that CuHp sensitization takes place by photothermal pathways. The efficiency of the photoprocess was modulated by different parameters; thus, while varying the amount of added CuHp in the 3.25–26 μM range had little effect, pulse energies larger than 50 mJ and irradiation times of at least 120 s were necessary to induce a cell inactivation of about 50%. The porphyrin‐cell incubation time prior to irradiation had a major influence on cell survival, suggesting that the nature of the CuHp microenvironment can control the efficiency of photothermal sensitization.