Victor B. Loschenov

Near-infrared fluorescent proteins

Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.

Evaluation of blood oxygen saturation in vivo from diffuse reflectance spectra

A simple method to evaluate the hemoglobin oxygen saturation and relative hemoglobin concentration in a tissue from diffuse reflectance spectra in the visible wavelength range is put forward in this paper. It was assumed that while oxygenated and deoxygenated hemoglobin contributions to light attenuation are strongly variable functions of wavelength, all other contributions to the attenuation including scattering are smooth wavelength functions and can be approximated by Taylor series expansion. Based on this assumption, a simple, robust algorithm suitable for real time monitoring of the hemoglobin oxygen saturation in the tissue has been derived. This algorithm can be used with different fiber probe configurations for delivering and collecting light passed through the tissue. An experimental technique using this algorithm has been developed for in vivo monitoring during artery occlusion and in vitro monitoring of blood samples. The experimental results obtained are presented in the paper.

Absorption spectroscopy as a tool to control blood oxygen saturation during photodynamic therapy

The photodynamic therapy (PDT) is an effective method to treat cancer and other nononcological lesions by means of light action on photosensitizer in tissue. It is considered that destroying effect is mainly due to the formation of singlet oxygen resulting from the interaction of light excited photosensitizer with molecular oxygen (triplet state in the ground state). So the destroying effect will be proportional to the rate of singlet oxygen formation which is in turn depends on light intensity, photosensitizer concentration and molecular oxygen concentration. The present work deals with the investigation of blood oxygen saturation in microcircular vessels (SO2) during light irradiation in the PDT process. It has been observed that SO2 behavior strongly correlates with the light power density applied for PDT. The high power density resulted in sharp SO2 decrease. The connection of SO2 decrease with enhanced oxygen consumption rate and vessel destruction due to PDT is discussed.

Application of aluminum phthalocyanine nanoparticles for fluorescent diagnostics in dentistry and skin autotransplantology

This paper deals with the possibility of application of aluminum phthalocyanine (AlPc) nanoparticles in clinical practice. AlPc fluoresces in the molecular form but in the form of nanoparticles it does not. Separation of molecules from an AlPc nanoparticle and therefore the appearance of fluorescence occurs under the effect of a number of biochemo-physical factors. Owing to this feature the application of AlPc nanoparticles followed by the measurement of fluorescence spectra is proposed as a diagnostics method. It was shown that after AlPc nanoparticle application on a tooth surface the fluorescence intensity in the enamel microdamage area is 2-3 times higher than that in the normal enamel area. The appearance of fluorescence after application of AlPc nanoparticles on skin autografts testifies to the presence of inflammation.

Control of photosensitizer in tissue during photodynamic therapy by means of absorption spectroscopy

The data about absorption spectra properties of the photosensitized tumor tissues in vivo are very important for the evaluation photosensitizer concentration and influence of the chemical environment on photosensitizer properties in tissue. This information will help one in the appropriate choosing of irradiation light dose and wavelength in the photodynamic therapy (PDT) treatment. The interaction of photosensitizer with tumor tissue may change its absorption spectrum. Moreover the light irradiation during PDT treatment can also affect the photosensitizer chemical structure and hence change its absorption spectrum. The simple technique based on the measurement of reflection spectra by means of fiber optic spectrometer has been developed. This method allows one to evaluate the contribution to the tissue absorption properties due to the presence of photosensitizer in it. The absorption spectra of photosensitized normal and tumor tissues presented in this paper has been obtained during PDT sessions of head and neck tumors. The photosensitizer -- sulphonated aluminum phthalocyanine (Photosence) (NIOPIC, Russia) -- has been injected intravenously in doses 0.5 - 2 mg per kg of body weight. It has been observed that absorption properties of this photosensitizer are not changed significantly in tissue as compared to that of in solution. The absorption spectra widening and red shift (4 nm) has been noticed. The qualitative pharmocokinetic of photosensitizer based on absorption spectra measurements is presented.

Photosensitizer for PDT based on phosphonate phthalocyanine derivative

It is very important to develop new sensitizer which could be efficiently excited at wavelengths exceeding 685 nm where own tissue absorption can be neglected. We have synthesize water soluble phthalocyanine derivative having phosphonate groups as substituents in macrocycle. New sensitizer efficiently absorbs in spectral range 685 - 710 nm. It exhibits intense fluorescence in range 690 -720 nm. The photodynamic activity of new sensitizer is higher than in the case of sulfonated aluminum phthalocyanine.

Photobleaching of endogenous fluorochroms in tissues in vivo during laser irradiation

The photobleaching phenomenon has been previously first of all widely studied for exogenous photosensitizers applied in photodynamic therapy (PDT). The present paper deals with detailed investigation of photobleaching of endogenous fluorochroms in tissues in vivo during its laser irradiation at 532, 633 and 670 nm at different powers. The fluorescence decay curves during skin irradiation in vivo at these wavelengths have been obtained and analyzed. The similarity in bleaching behavior of endogenous fluorochroms and exogenous photosensitizers used in PDT gave us the reasons to imply the possible connection between native fluorochroms and low intensity laser therapy effects. The results obtained may be applied for tissue diagnostics and therapy control.

Photobleaching of photosensitizers applied for photodynamic therapy

It is well known that photosensitizers used for PDT are liable to photobleaching. This phenomenon should be taken into account when developing the appropriate tactics of treatment. The present paper deals with detailed investigation of photobleaching of two photosensitizers: ALA induced protoporphyrin IX (PPIX) and sulphonated aluminum phthalocyanines. The fluorescence decay curves during light irradiation in vivo and in vitro have been obtained and analyzed. The mathematical model taking into account both first and second order photobleaching as well as spatial inhomogeneity of light distribution in tissue is presented. The experimental data for fluorescence decay of ALA induced PPIX during light irradiation are fitted to this mode. As opposed to PPIX the photobleaching behavior for sulphonated aluminum phthalocyanines is rather complicated to be fitted by proposed mathematical mode. The fluorescence outburning and residual fluorescence for sulphonated aluminum phthalocyanines have been observe.d It has been shown that fluorescence maximum correlates with blood oxygen saturation decrease induced by PDT effect.

Spectroscopic research of upconversion nanomaterials based on complex oxide compounds doped with rare-earth ion pairs: Benefit for cancer diagnostics by upconversion fluorescence and radio sensitive methods/Spektroskopische Untersuchung von mit Ionenpaaren Seltener Erden dotierten Upconversion-Nanokompositen: Nutzen für die Krebsdiagnostik durch Upconversion-Fluoreszenz und strahlungssensitive Methoden

Abstract Background: Highly photochemically stable nanoparticles, in which upconversion luminescence can be excited – so-called upconversion nanocrystals (UC-NCs) – exhibit widely separated (up to 500 nm) narrow luminescence bands in the visible (VIS) region located far from the excitation near-infrared (NIR) laser radiation, and thus can be more easily identified compared to organic luminophores and semiconductor nanoparticles. Due to a deep penetration of exciting infrared (IR) radiation, the absence of parasitic fluorescence of biomolecules and the absence of phototoxicity and photobleaching upon near IR excitation, UC-NCs can be efficiently used as fluorescent probes in biological studies and fluorescence diagnostics (FD). The doping of such nanoparticles with Gd3+ ions provides the additional possibility of combining fluorescence visualization with magnetic resonance imaging, which will considerably improve the sensitivity of diagnostics of cancer tumors even in the early stages. Materials and methods: We studied the upconversion characteristics of inorganic nanoparticles made of different materials doped with rare-earth ion (REI) pairs Yb3+-Er3+ and Yb3+-Tm3+ as functions of the concentration and composition of a dopant at different excitation intensities. Matrices chosen for doping were complex polycrystalline oxide rare earth compounds Gd2GeMoO8, La4Gd10B6Ge2O34, and Gd11SiP3O26 which permit the introduction of significant concentrations of the activator luminescence ions (Yb3+, Er3+ and Tm3+), synthesized by solid-phase reaction methods from corresponding oxides. The final product was obtained by combined precipitation of initial components from aqueous solutions followed by the annealing of hydroxide mixtures and grinding. The redistribution of the intensity of the 550 nm 2H11/2, 4S3/2→4I15/2 and 650 nm 4F9/2→4I15/2 upconversion luminescence bands in Er3+ was investigated depending on matrices, dopants, and the laser power density. The quantum yield and lifetime of upconversion luminescence were determined for individual electronic transitions, which were used to optimize the composition of dopants in matrices. Results: Based on the results obtained, the matrix La4Gd10B6Ge2O34 is most effective for the upconversion process in the VIS spectrum. Doping nanoparticles by REI pairs Yb3+-Er3+ and Yb3+-Tm3+, each has its advantages. REI pair Yb3+-Er3+ is good for energy transfer in the green and/or red part of the spectrum as well as for the FD and can be used for a further energy transfer to the photosensitizers at photodynamic therapy (PDT). REI pair Yb3+-Tm3+ transform the infrared radiation in the blue region of the spectrum, which is also suitable for FD and PDT and additional intensive energy conversion in NIR will allow for deep tissue imaging. Conclusion: The investigated complex polycrystalline oxide compounds are promising as diagnostic agents for biological tissues visualization by fluorescence, light scattering, and nuclear magnetic resonance imaging. Zusammenfassung Hintergrund: Photochemisch hochstabile Nanopartikel, in denen Upconversion-Lumineszenz angeregt werden kann – die sogenannten Upconversion-Nanokomposite (UC-NCs) – weisen eng begrenzte, schmale Lumineszenz-Banden (bis 500 nm) im sichtbaren Wellenlängenbereich auf, die weit genug von der Anregungs-Laserstrahlung im Nahinfrarot (NIR)-Bereich entfernt sind und somit leichter gegenüber organischen Luminophoren und Halbleiternanopartikeln zu identifizieren sind. Durch die hohe Eindringtiefe des infraroten (IR) Anregungslichtes und das Fehlen parasitärer Fluoreszenz durch Biomoleküle sowie Phototoxizitäts- und Photobleechingeffekte im Bereich der IR-Anregung können UC-NCs effizient als Fluoreszenzsonden für biologische Untersuchungen und die Fluoreszenzdiagnostik (FD) eingesetzt werden. Die Dotierung solcher Nanopartikel mit Gd3+-Ionen bietet zusätzlich die Möglichkeit der Kombination von Fluoreszenz-Visualisierung und Magnetresonanz-Bildgebung, was die Empfindlichkeit der Diagnostik von Tumoren auch in frühen Stadien deutlich verbessert. Material und Methoden: Wir untersuchten die Upconversion-Eigenschaften von anorganischen Nanopartikeln aus unterschiedlichen Materialien, die mit Ionenpaaren Seltener Erden dotiert wurden (Yb3+-Er3+ und Yb3+-Tm3+), und zwar in Abhängigkeit von der Konzentration und Zusammensetzung des Dotands bei unterschiedlichen Anregungsenergien. Als Grundsubstanzen für eine Dotierung wurden komplexe polykristalline Oxidverbindungen Seltener Erden ausgewählt (Gd2GeMoO8, La4Gd10 B6Ge2O34, und Gd11SiP3O26), die die Einführung signifikanter Konzentrationen der Aktivator-Lumineszenz-Ionen (Yb3+, Er3+ und Tm3+) gestatten – synthetisiert durch Festphasenreaktionsmethoden aus korrespondierenden Oxiden. Das Endprodukt wurde durch die kombinierte Ausfällung der anfänglichen Komponenten aus wässriger Lösung und anschließendes Glühen und Mahlen der Hydroxidgemische erzeugt. Die Umverteilung der Intensität der 550 nm 2H11/2, 4S3/2→4I15/2 und 650 nm 4F9/2→4I15/2 Upconversion-Lumineszenz-Banden wurde in Abhängigkeit von den Grundstoffen, Dotanden und der Laserleistungsdichte untersucht. Die Quantenausbeute und die Lebensdauer der Upconversion-Lumineszenz wurden für einzelne elektronische Übergänge bestimmt, die zur Optimierung der Dotanden-Zusammensetzung verwendet wurden. Ergebnisse: Wie sich zeigte, ist die La4Gd10B6Ge2O34-Matrix im sichtbaren Spektralbereich am effektivsten für den Upconversion-Prozess. Beide dotierenden Ionenpaare Seltener Erden (Yb3+-Er3+ und Yb3+-Tm3+) haben ihre Vorteile. Das Yb3+-Er3+-Ionenpaar ist gut für die Energieübertragung im grünen und/oder roten Spektralbereich als auch für die FD und kann für eine weitere Energieübertragung auf den Photosensibilisator während der photodynamischen Therapie (PDT) verwendet werden. Das Yb3+-Tm3+-Ionenpaar verwandelt die IR-Strahlung im blauen Bereich des Spektrums, was ebenfalls für die FD und PDT geeignet ist und eine zusätzliche intensive Energieumwandlung in NIR bewirkt, was ein Imaging tiefer Gewebeschichten erlaubt. Fazit: Die untersuchten komplexen polykristallinen Oxidverbindungen sind als diagnostische Mittel zur Darstellung biologischen Gewebes mittels Fluoreszenz, Lichtstreuung und Magnetresonanz-Bildgebung geeignet.

Simultaneous measurement of photosensitizer absorption and fluorescence in patients undergoing photodynamic therapy

This paper deals with photosensitizer quantification in patients undergoing photodynamic therapy. Both fluorescence and diffuse reflectance spectroscopy were applied to evaluate concentration of sulphonated aluminum phthalocyanine in different tissues. The mixtures of Intralipid, blood and photosensitizer with different concentrations were used as standard samples to solve the problem in question. While fluorescence method is more sensitive and more convenient to apply in clinics, the absorption technique may be applied to non fluorescent dyes and used to evaluate the shifts in adsorption peak position due to interaction of dye with tissues. Finally, the concentration dynamics of non fluorescent dye (cobalt phthalocyanine) in patients was obtained with the use of absorption method alone.