Miron Kaliszewski

Fluorescence excitation-emission matrices of selected biological materials

EX-EM fluorescence matrices from suspensions of 11 vegetative bacteria, 5 spores, and 14 interferents (fungi, pollens) were measured and cross-sections of the selected bacteria were calculated. The changes of fluorescence characteristics of vegetative cells during their sporulation and starvation to death as well as spores during germination are shown. Influence of culture media on emission spectra and rate of spores formation under starving conditions was examined. Analysis of the measured fluorescence characteristics shows that double- or multi-wavelenghts excitation can make it possible to distinguish between particular groups of biological material, i.e., spores, vegetative cells, proteins, and interferents.

Improved laser-induced fluorescence method for bio-attack early warning detection system

Laser Induced Fluorescence (LIF) could permit fast early warning systems either for point or stand-off detection if a reliable classification of warfare biological agents versus biological or non-biological fluorescing background can be achieved. In order to improve LIF discrimination capability, a new system is described in which the fluorescence pattern is enriched by the use of multiple wavelength delayed excitation while usual spectral fluorescence analysis is extended to time domain to use both aspects as criteria for classification. General considerations and guidelines for the system design are given as well as results showing good discrimination between background and simulants.

Formation of protoporphyrin IX from carboxylic- and amino-derivatives of 5-aminolevulinic acid

BACKGROUNDnStability of ALA is an important factor for photodynamic therapy (PDT). The dimerization of ALA to pyrazines takes place via the amine group. It is, therefore, to be expected that blocking this group by addition of a formyl group should result in a more stable compound.nnnMETHODSnThe ability of a new N-formyl derivative of ALA (N-f-ALA) to form protoporphyrin IX (PPIX) was compared with that of ALA and three of its ester (methyl, butyl and hexyl) derivatives. Dark toxicity of the compounds was measured using MTT assay. Formation of PPIX was measured by fluorescence spectroscopy.nnnRESULTS AND CONCLUSIONSnN-f-ALA showed an outstanding stability in water solutions even at pH 7. However, it induced no PPIX neither in WiDr cells in vitro, nor in mouse skin in vivo. A probable reason is lack of an enzyme that can cleave the bond between the formyl group and ALA. Thus, steric hindrance may prevent processing of the compound into porphobilinogen. N-f-ALA did not inhibit PpIX formation from ALA and is unable to enter the heme cycle. Generation of ALA from its derivatives, therefore, seems to be an essential step in PPIX synthesis.

Photodynamic therapy with 5‐aminolevulinic acid and diamino acid derivatives of protoporphyrin IX reduces papillomas in mice without eliminating transformation into squamous cell carcinoma of the skin

Photodynamic therapy (PDT) is used to treat malignant and nonmalignant diseases. It is also used for cosmetological skin treatment. PDT is generally considered to have a low risk of carcinogenicity. However, instances of nonmalignant human tumors turning malignant have been linked to PDT. In this study, we used 5‐aminolevulinic (ALA) acid and 3 water soluble photosensitizers‐PP(Arg)2, PP(Ser)2Arg2, PP(Ala)2Arg2, all diamino acid derivatives of protoporphyrin IX‐to treat benign papillomas in FVB/N mice induced by 7,12‐dimethylbenz(a)anthracene (DMBA)‐12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Of these drugs, ALA and PP(Arg)2 were found the most efficient. PDT reduced the number of papillomas, but with increasing effectiveness of the drugs, the risk of malignant transformation of the papillomas into squamous cell carcinomas increased. The underlying mechanisms are not clear and further investigations are needed.

Clearance of protoporphyrin IX induced by 5-aminolevulinic acid from WiDr human colon carcinoma cells

5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is the most widely practiced form of PDT in dermatology. One of the advantages of ALA-PDT is that undesirable photosensitization lasts only for 24-48 h. In order to optimize ALA-PDT it is necessary to understand the mechanisms controlling intracellular PpIX clearance (efflux and transformation into heme) in order to decrease protoporphyrin IX (PpIX) clearance rates in the early stages of its production. The aim of this study was to investigate the factors controlling the clearance of intracellular PpIX. Fluorescence spectroscopy was used to study PpIX kinetics in WiDr cells initially treated with ALA. The clearance rate of PpIX in WiDr cells was faster after application of a low concentration of ALA (0.1 mM) than after application of high concentration of ALA (1 mM). PpIX was cleared faster from cells which initially were seeded at low densities than cells seeded at higher densities. The presence of the iron chelator deferoxamine reduced the clearance rate of PpIX, while the presence of ferrous sulfate acted oppositely. The decay rate of PpIX in WiDr cells was faster at higher temperature than at lower. The ferrochelatase activity at pH 7.2 was significantly greater than that at pH 6.7. ALA concentration, application time, cell density, temperature, pH, intracellular iron content, intracellular amount and localization of PpIX are factors controlling PpIX clearance.

The application of semiconductor based UV sources for the detection and classification of biological material

Fluorescence analysis of dry samples of biological origin like pollens, fungi, flours and proteins was presented. In the laboratory study presentenced here two fluorescence methods using semiconductor light sources were applied. Firstly, laser induced fluorescence emission (LIF) spectra of the samples were recorded under 266 and 375 nm excitation. The second technique covered fluorescence decay (FD) at 280 and 340 nm excitation. Hierarchical Cluster Analysis (HCA) of acquired spectra and decays was performed. Both LIF and FD showed that single wavelength excitation 266 and 280 nm, respectively allow distinguishing of pollens from other samples. Combining data of both excitation wavelengths, for LIF and FD, respectively, resulted in substantial improvement of data classification for groups according to the samples origin.

Fluorimetric techniques in analysis and classification of fuels

In presented work the analysis of liquid fuels with use of optical spectroscopy was performed. The fuels were examined with the use of two fluorescence techniques: steady-state fluorescence and time-resolved fluorescence. Recorded excitation-emission matrices (EEMs), with the use of steady-state fluorescence, allowed for estimation of the absorption and emission maxima, classification of the fuels was partially possible. Time-resolved fluorescence allowed for classification of the fuels into four groups. Among examined fuels the longest time of fluorescence decay was observed in diesel, petrol Pb98 and Pb95, in descending order.

The laser detector of bio-aerosols

In the paper we describe a new construction and preliminary laboratory tests of airborne particle flow detector. The device is designed for continuous monitoring of biological particles in ambient air. The scattering and fluorescence signals from each particle are detected simultaneously. The device uses 375 nm laser for fluorescence excitation. The luminescence is measured with three broad bands. Principal Component Analysis was applied for data analysis. It has been demonstrated proper classification between various biological aerosols.

Optoelectronic methods in potential application in monitoring of environmental conditions

Allergic rhinitis, also known as hay fever is a type of inflammation which occurs when the immune system overreacts to allergens in the air. It became the most common disease among people. It became important to monitor air content for the presence of a particular type of allergen. For the purposes of environmental monitoring there is a need to widen the group of traditional methods of identification of pollen for faster and more accurate research systems. The aim of the work was the characterization and classification of certain types of plant pollens by using laser optical methods, which were supported by the chemmometrics. Several species of pollen were examined, for which a database of spectral characteristics was created, using LIF, Raman scattering and FTIR methods. Spectral database contains characteristics of both common allergens and pollen of minor importance. Based on registered spectra, statistical analysis was made, which allows the classification of the tested pollen species. For the study of the emission spectra Nd:YAG laser was used with the fourth harmonic generation (266 nm) and GaN diode laser (375 nm). For Raman scattering spectra spectrometer Nicolet IS-50 with a excitation wavelength of 1064 nm was used. The FTIR spectra, recorded in the mid infrared1 range (4000-650 cm-1) were collected with use of transmission mode (KBr pellet), ATR and DRIFT.