Optical excitation of chemicals provides reactive excited states, which can initiate chemical reactions and can also relax via radiative photophysical processes, providing the basis for fluorescence diagnostics. The best-known example of the former is Photodynamic Therapy (PDT), which is now approved for the treatment of a number of neoplastic and non-neoplastic pathologies. Although the concept of the use of photodynamic agents in diagnostics is as old as their use in therapy, the focused development of this aspect has been relatively recent. Typically, photodynamic agents have high triplet yields and relatively long triplet lifetimes (microsecond range), which allows them to interact and destroy molecular targets near them either directly or indirectly by producing other toxic molecular species. Associated with a high triplet yield is the fortunate attribute of most PDT agents in having low but finite fluorescence quantum yields. Fluorescence from these molecules may be used not only for diagnostics of disease de novo but also for guided surgery, PDT dosimetry and therapeutic monitoring. Other uses of fluorescence in PDT (not necessarily from the PDT agents) include the development of technologies that allow tracking of cells during treatment in vivo, studies of sub-cellular localization of molecules for mechanistic studies and photosensitizer tracking for specific targeting. An overview of studies on these aspects from different laboratories will be presented.

Fluorescence-enhanced optical imaging measurements and conventional gamma camera images on human M21 melanoma xenografts were acquired for a "dual-modality" molecular imaging study. The avb3 integrin cell surface receptors were imaged using a cyclic peptide, cyclopentapeptide cyclo(lys-Arg-Gly-Asp-phe) [c(KRGDf)] probe which is known to target the membrane receptor. The probe, dual-labeled with a radiotracer, 111Indium, for gamma scintigraphy as well as with a near-infrared dye, IRDye800, was injected into six nude mice at a dose equivalent to 90mCi of 111In and 5 nanomoles of near-infrared (NIR) dye. A 15 min gamma scan and 800 millisecond NIR-sensitive ICCD optical photograph were collected 24 hours after injection of the dual-labeled probe. The image quality between the nuclear and optical data was investigated with the results showing similar target-to-background ratios (TBR) based on the origin of fluorescence and gamma emissions at the targeted tumor site. Furthermore, an analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for the subcutaneous tumor targets measured by surface regions of interest.

Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.

The design and synthesis of an environmentally sensitive long wavelength fluorescing squaraine dye for conjugation to proteins is dsecribed. Environmentally sensitive dyes are valuable for probing environmental changes that occur when labeled proteins bind their corresponding ligands and can be used to construct flyorescent sensors. Long wavelength (>650 nm) dyes would enable through-skin wireless sensing with minimum interference from the background. While several environmentally sensitive dyes are known in the visible spectrum, only a few are available in the long wavelength region, and none of them are available with reactive groups suitable for protein conjugation. Several derivatives of squarain dyes are known to be environmentally sensitive and fluorescent in the long wavelength region, but none of them are available with linkers for protein conjugation. In order to achieve this goal, we developed a synthetic scheme to introduce a reactive linker onto an anilinic squaraine that is highly sensitive to its environment. The synthesis involves the preparation of the dye with an iodoacetyl ester linker that readily reacts with a thiol on a cysteine residue of the binding protein. The squaraine dye was conjugated to known binding proteins that were evaluated as optical sensors. Ultimately, we expect these systems to measure analytes in the body and transmit information through the skin to an external monitor.

Here we present a novel class of self-quenching, double-labeled DNA probes based on the formation of non fluorescent H-type dye dimers. We therefore investigated the aggregation behavior of the red-absorbing oxazine derivative MR121 and found a dimerization constant of about 3000 M-1. This dye was successfully used to develop hairpin-structured as well as linear self-quenching DNA probes that report the presence of the target DNA by an increase of the fluorescence intensity by a factor of 3 to 12. Generally fluorescence quenching of the hairpin-structure probes is more efficient compared to the linear probes, whereas the kinetic of the fluorescence increase is significantly slower. The new probes were used for the identification of different mycobacteria and their antibiotic resistant species. As a test system a probe for the identification of a DNA sequence specific for the Mycobacterium xenopi was synthesized differing from the sequence of the Mycobacterium fortuitum by 6 nucleotides. Furthermore we developed a method for the discrimination between the sequences of the wild type and an antibiotic resistant species of Mycobacterium tuberculosis. Both sequences differ by just 2 nucleotides and were detected specifically by the use of competing olignonucleotides.

We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

Fluorescence is a tool widely employed in biological assays. Fluorescent semiconducting nanocrystals, quantum dots (QDs), are beginning to find their way into the tool box of many biologist, chemist and biochemist. These quantum dots are an attractive alternative to the traditional organic dyes due to their broad excitation spectra, narrow emission spectra and photostability. Non-specific binding is a frequently encountered problem with fluorescent labeling in biological assays. In these studies various cell lines were examined for non-specific binding to quantum dots. Evidence suggests that non-specific binding is related to cell type and, may be significantly reduced by functionalizing quantum dots with polyethyleneglycol ligands (PEG). In addition quantum dots were used to detect and monitor the progession of the viral glycoproteins ,F (fusion) and G (attachment), from Respiratory Syncytial Virus (RSV) in HEp-2 cells. RSV is the most common cause of lower respiratory tract infection in children worldwide and the most common cause of hospitalization of infants in the US. Antiviral therapy is available for treatment of RSV but is only effective if given within the first 48 hours of infection. Existing test methods require a virus level of at least 1000-fold of the amount needed for infection of most children and require several days to weeks to obtain results. The use of quantum dots may provide an early, rapid method for detection and provide insight into the trafficking of viral proteins during the course of infection.

We have developed a new functionalization approach for semiconductor nanocrystals based on a single-step exchange of surface ligands with custom-designed peptides. This peptide-coating technique yield small, monodisperse and very stable water-soluble NCs that remain bright and photostable. We have used this approach on several types of core and core-shell NCs in the visible and near-infrared spectrum range and used fluorescence correlation spectroscopy for rapid assessment of the colloidal and photophysical properties of the resulting particles. This peptide coating strategy has several advantages: it yields probes that are immediately biocompatible; it is amenable to improvements of the different properties (solubilization, functionalization, etc) via rational design, parallel synthesis, or molecular evolution; it permits the combination of several functions on individual NCs. These functionalized NCs have been used for diverse biomedical applications. Two are discussed here: single-particle tracking of membrane receptor in live cells and combined fluorescence and PET imaging of targeted delivery in live animals.

Colloidal semiconductor quantum dots (QDs) have narrow photoemission bandwidths and broad absorption spectra that are ideal for multiplexing applications. In contrast to organic dyes, which require a complex arrangement of excitation sources and filters to generate multiple signals, many populations of QDs can be simultaneously excited with a single excitation source. In a mixed sample, the narrow and symmetric emission profile of QDs allows simple deconvolution of the composite signal to generate individual QD photoluminescence (PL) contributions. We have shown that CdSe-ZnS core-shell QDs function as efficient energy donors in fluorescence resonance energy transfer (FRET) systems. In this study, we tested several QD-protein bioconjugates, each having a unique PL spectrum (or "color") functioning as independent signal channels, to assess the feasibility of a QD FRET-based multiplexing system. Several populations of QDs were self-assembled with labeled and unlabeled proteins, mixed in solution and excited at single wavelength. The resulting spectra were deconvoluted using the known QD emission profiles to reveal individual contributions of each QD population. QDs coated with dye-labeled protein acceptors showed distinct FRET-induced PL quenching due to the presence of proximal dye acceptors. Steady-state fluorescence results were verified by time-resolved spectroscopic data from the mixed samples where a reduced QD lifetime indicated the presence of proximal dye quencher on one or more QD populations. We will discuss how these findings are used to develop QD-based FRET multiplexed biosensors using a similar strategy where each QD population has surface-bound proteins that are sensitive to a unique molecular target.

The use of quantum dots to study and solve biological issues is bringing scientists and engineers into the realm of pure biology. Yet quantum dots are still a young technology that has not fulfilled all its immense promises. The prospects of using quantum dots in biology motivates a wealth of different studies that include the targeting of organisms in live cells and the characterization of the optical properties of the bioconjugates at the single molecule level. Here, I want to review some experiments using silanized quantum dots. In particular, I want to focus on the versatility of silanized quantum dots for bioconjugation and show how they can be programmed to target the nucleus of live cells. In parallel, silanized qdots are remarkable chromophores that can be used as FRET sensors. FRET efficiencies up to 100% can be reached by tuning the spectral properties of the donor and acceptor pairs.

We present a simple and efficient method for marking living human red blood cells using CdS (Cadmium Sulfide) quantum dots (QDs). The nanocrystals were obtained via colloidal synthesis in aqueous medium with final pH=7 using sodium polyphosphate as the stabilizing agent. The methodology implementation is simple, do not requires additional capping layers nor narrow size QDs distribution. The synthesized nanoparticles were conjugated to monoclonal A anti-body. The resulting conjugates QDs/anti-A were incubated with human erythrocytes of blood groups A and O for 30 min at 37°C. The living cells in contact with the quantum dots maintained their properties for several days showing the low level of citotoxicity of the quantum dots. The conjugation of CdS QDs/anti-A show simultaneous red and green fluorescence when excited with 543 and 488 nm respectively. The efficiency of the conjugation QDs/anti-body to the erythrocytes, for each system, was monitored by confocal microscopy. The comparative analysis of the micrographs was done with the luminescence intensity maps of the samples obtained under constant capture conditions, such as, pinhole, filters, beam splitters and photomultiplier gain. The conjugates QDs/anti-A intensely marked group A erythrocytes and did not show any luminescence for group O erythrocytes, showing the sensitivity of the labeling procedure. In conclusion, we show the viability of the use of high luminescent and stable quantum dots as fluorescent labels for human erythrocytes with a methodology of simple implementation and the possibility to use them to distinguish different blood groups.

The detection of the waterborne pathogens Giardia lamblia and Cryptosporidium parvum in environmental water bodies requires concentration of large volumes of water due to the low dose required for infection. The highly concentrated (10,000-fold) water sample is often rich in strongly autofluorescent algae, organic debris and mineral particles that can obscure immunofluorescently labeled (oo)cysts during analysis. Time-resolved fluorescence techniques exploit the long fluorescence lifetimes of lanthanide chelates (ms) to differentiate target fluorescence from background autofluorescence (ns). Relatively simple instrumentation can be used to enhance the signal-to-noise ratio (S/N) of labelled target. Time-resolved fluorescence techniques exploit the large difference in lifetime by briefly exciting fluorescence from the sample using a pulsed excitation source. Capture of the resulting fluorescence emission is delayed until the more rapidly decaying autofluorescence has faded beyond detection, whereon the much stronger and slower fading emission from labelled target is collected. BHHCT is a tetradentate beta-diketone chelate that is activated to bind with protein (antibody) as the chlorosulfonate. The high activity of this residue makes conjugations difficult to control and can lead to the formation of unstable immunoconjugates. To overcome these limitations a 5-atom hydrophylic molecular tether was attached to BHHCT via the chlorosulfonate and the BHHCT derivative was then activated to bind to proteins as the succinimide. The new compound (BHHST) could be prepared in high purity and was far more stable than the chlorosulfonate on storage. A high activity immunocojugate was prepared against Cryptosporidium that yielded an 8-fold increase in SNR using a lab-built time-resolved fluorescence microscope.

In this paper we applied the efficient fluorescence quenching of the red-absorbing oxazine derivative MR121 by the amino acid tryptophan to develop a new fluorescence based enzyme assay that can be used for detection of exopeptidases and endopeptidases. Therefore, we developed peptide substrates labeled with only one chromophore, which is quenched by a neighbored tryptophan residue via photoinduced electron transfer. The specific cleavage site for the target enzyme is located between the chromophore and the tryptophan residue. After digestion of the substrate the contact formation between tryptophan and fluorescent dye is precluded and a significant increase in fluorescence intensity occurs. To demonstrate the new assay technique for exopeptidases, a substrate for the Carboxypeptidase A was designed and a detection limit below the picomolar range (~10^-13 M) was achieved with standard fluorescence spectrometry. The primary objective was the detection of the HIV-protease, which is an endopeptidase digesting substrates containing seven specific amino acids in the cleavage site. We designed a substrate, which enables the detection of 10^-9 M HIV-protease, whereas the continuous monitoring of the fluorescence signal also allows kinetic studies.

Photodynamic therapy (PDT) is a cancer therapy that utilizes optical energy to activate a photosensitizer drug in a target tissue. Reactive oxygen species (ROS), such as ¹O₂ and superoxide, are believed to be the major cytotoxic agents involved in PDT. Although current PDT dosimetry mostly involves measurements of light and photosensitizer doses delivered to a patient, the quantification of ROS production during a treatment would be the ultimate dosimetry of PDT. Technically, it is very difficult and expensive to directly measure the fluorescence from ¹O₂, due to its extreme short lifetime and weak signal strength. In this paper, Photofrin(R) and 635nm laser were used to generate ¹O₂ and superoxide in a PDT in solution. Compound 3,7- dihydro-6-{4-[2-(N’-(5-fluoresceinyl) thioureido) ethoxy] phenyl}-2- methylimidazo{1,2-a} pyrazin-3-one sodium salt,an Cyp- ridina luciferin analog commonly referred as FCLA, was used as a chemical reporter of ROS. The 532nm chemiluminescence (CL) from the reaction of the FCLA and ROS was detected with a photon multiplier tube (PMT) system operating at single photon counting mode. With the setup, we have made detections of ROS generated by PDT in real time. By varying the amount of conventional PDT dosage (photosensitizer concentration, light irradiation fluence and its delivery rate) and the amount of FCLA, the intensity of CL and its consumption rate were investigated. The results show that the intensity and temporal profile of CL are highly related to the PDT treatment parameters. This suggests that FCLA CL may provide a highly potential alternative for ROS detection during PDT.

BSA (Bovine Serum Albumin) can enlarge the CL intensity of FCLA(3,7-dihydro-6-{4-{2-(N'-(5-fluoresceinyl) thioureido)ethoxy}phenyl}-2-methylimi-dazo{1,2-a}pyrazin-3-one dosium salt) to 763%. This report presents novel methods for determination of Aflatoxin B1 (AfB1) mediated by FCLA+BSA. The concentration of AFB1 showed an obvious positive correlation with the chemiluminescence (CL) intensity mediated by FCLA+BSA, correlative coefficient R@0.94. This method could measure accurately ng/ml of AfB1 concentration. 365nm as excitated wavelength, 440nm and 520nm-two fluorescence peaks of FCLA+BSA+AfB1 were found. The fluorescence intensity of peak at 440nm showed an obvious positive correlation with the concentration of AFB1, R@0.97; the fluorescence intensity of peak at 520nm showed a positive correlation with the concentration of AFB1, R@0.90. Comparing the peak of FCLA, FCLA+BSA and FCLA+BSA+AfB1 had a 6nm Einstein shift (red shift). The study suggested that CL and fluorescence spectrum methods mediated by FCLA+BSA might be applicable to the determination of AfB1 concentration.

A new method for identification of point mutations was proposed. Polymerase chain reaction (PCR) amplification of a sequence from genomic DNA was followed by digestion with a kind of restriction enzyme, which only cut the wild-type amplicon containing its recognition site. Reaction products were detected by electrochemiluminescence (ECL) assay after adsorption of the resulting DNA duplexes to the solid phase. One strand of PCR products carries biotin to be bound on a streptavidin-coated microbead for sample selection. Another strand carries Ru(bpy)₃²⁺ (TBR) to react with tripropylamine (TPA) to emit light for ECL detection. The method was applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than 3 orders of magnitude, thus, make quantitative analysis possible. The genotype can be clearly discriminated. Results of the study suggest that ECL-PCR is a feasible quantitative method for safe, sensitive and rapid detection of point mutation in human genes.

Fluorescence imaging techniques have been central to much biomedical science research over the past two decades. In particular, functional imaging has provided important new information about processes that occur at cellular and sub-cellular levels. With this approach, living tissues are stained with dyes whose emission is modulated by changes in the environment to which the dye is exposed. The fluorescence imaging systems used within this context typically incorporate relatively complex free space optical assemblies and a stable platform is necessary to maintain appropriate alignment of their components. Because of the poor efficiency of these systems, it is necessary to use powerful light sources and sensitive photo-detectors. We have developed a novel fluorescence imaging system in which free-space optics are replaced by optical fibers, passive optical splitters and associated components. Solid state lasers are used as the excitation light source. A variety of detection systems have been utilized including a spectrometer. The feasibility of the approach has been established using a rat heart preparation stained with the membrane potential-sensitive dye, di-4-ANEPPS. Detailed emission spectra for this dye, at different levels of resting membrane potential, are presented here for 532 nm and 488 nm excitation. Cardiac action potentials obtained with the modular fiber optic system correspond closely to intracellular potentials acquired at adjacent sites in the isolated rat heart preparation. Our modular fiber optic system is cheaper, more efficient, more flexible and more robust than conventional fluorescence imaging systems. Using a high-speed spectrometer for photodetection, it is possible to implement the signal processing required for multi-line or ratiometric imaging in software, which further enhances the efficiency and flexibility of the system. We believe that this approach has wide potential applications for biomedical fluorescence imaging.

The new field of in vivo cell biology is being developed with multi-colored fluorescent proteins. With the use of fluorescent proteins, the behavior of individual cells can be visualized in the living animal. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of the tumor-stroma cell-cell interaction especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. Another example is the color-coding of cells with RFP or GFP such that both cell types and their interaction can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo with fluorescent proteins. Mice, in which the regulatory elements of the stem-cell marker nestin drive GFP expression, can be used to visualize hair follicle stem cells including their ability to form hair follicles as well as blood vessels. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Dual-color cells also enable the in vivo imaging of cell and nuclear deformation as well as trafficking in capillaries in living animals. Multiple-color labeling of cells will enable multiple events to be simultaneously visualized in vivo including cell-cell interaction, gene expression, ion fluxes, protein and organelle trafficking, chromosome dynamics and numerous other processes currently still studied in vitro.

The rat represents a perfect animal for broadening medical experiments, because its physiology has been well understood in the history of experimental animals. In addition, its larger body size takes enough advantage for surgical manipulation, compared to the mouse. Many rat models mimicking human diseases, therefore, have been used in a variety of biomedical studies including physiology, pharmacology, transplantation, and immunology. In an effort to create the specifically designed rats for biomedical research and regenerative medicine, we have developed the engineered rat system on the basis of transgenic technology and succeeded in establishing various transgenic rat strains. The transgenic rats with green fluorescent protein (GFP) were generated in the two different strains (Wistar and Lewis), in which GFP is driven under the chicken beta-actin promoter and cytomegalovirus enhancer (CAG promoter). Their GFP expression levels were different in each organ, but the Lewis line expressed GFP strongly and ubiquitously in most of the organs compared with that of Wistar. For red fluorescence, DsRed2 was transduced to the Wistar rats: one line specifically expresses DsRed2 in the liver under the mouse albumin promoter, another is designed for the Cre/LoxP system as the double reporter rat (the initial DsRed2 expression turns on GFP in the presence of Cre recombinase). LacZ-transgenic rats represent blue color, and LacZ is driven the CAG (DA) or ROSA26 promoter (Lewis). Our unique transgenic rats’ system highlights the powerful performance for the elucidation of many cellular processes in regenerative medicine, leading to innovative medical treatments.

The genetic modification for organ transplantation is one of the most promising strategies to regulate allogeneic immune response. Organ-selective gene transfer has especially benefit to control local immune responses. Based on the catheter technique, we tested to deliver naked plasmid DNA to target graft organs of rats (liver and limbs) by a rapid injection (hydrodynamics-based transfection). Recent advances in transplantation have been achieved by visualization of cellular process and delivered gene expression during the inflammatory process by using non-invasive in vivo imaging. Herein, we examined the fate of genetically modified grafts using a firefly luciferase expression plasmid. For liver modification before transplantation, 6.25% of body weight PBS containing plasmid DNA was injected into the liver through the inferior vena cava using a catheter, and the liver was subsequently transplanted to the recipient rat. For limb modification, the femoral caudal epigastric vein was used. In the rat liver transplantation model, substantial luciferase expression was visualized and sustained for only a few days in the grafted liver. We also addressed stress responses by this hydrodynamics procedure using reporter plasmids containing cis-acting enhancer binding site such as NF-kappa B, cAMP, or heat shock response element. In contrast to hepatic transduction, this genetic limb targeting achieved long lasting luciferase expression in the muscle for 2 months or more. Thus, our results suggest that this catheter-based in vivo transfection technique provides an effective strategy for organ-selective gene modification in transplantation, and the bioluminescent imaging is broadening its potential for evaluation to various preclinical studies.

Apoptosis is one of the important modes in PDT-induced cell death. Activation of caspase-3 is considered to be the final step in many apoptosis pathways. In this study, we used SCAT3, a fluorescence resonance energy transfer (FRET) probe containing caspase-3 substrate, to study the dynamics of caspase-3 activation in living ASTC-a-1 cells expressing stably SCAT3. The FRET analysis results indicated that caspase-3 activation in response to tumor necrosis factor-α or PDT resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The SCAT3 was cleaved immediately after PDT treatment, but that for TNF-a treatment was delayed two hours. Our experimental results suggested that the different apoptotic pathways induced by TNF-α or PDT caused different cleavage kinetics of SCAT3. This study shows that FRET technique based on GFPs could be used to study the mechanism of PDT-induced apoptosis in living cells.

Biocompatible organic dyes, in general, exhibit poor photostability in aqueous solution. This is especially true at the high concentrations needed for bolus administration in a clinical application. Several years ago, we showed that by using carefully chosen macromolecular additives, the stability of aqueous dye solutions may be enhanced significantly. We now report the observation of an unexpected long-term stability of a near infrared dye-peptide conjugate. The Mallinckrodt developed dye-peptide conjugate known as Cytate is an indocyanine type dye attached to the peptide octreotate. We have previously shown that this compound targets somatostatin receptor rich tumors and provides unambiguous contrast for optical imaging. A freshly prepared aqueous solution of Cytate was mixed with blood (from a rat) in a small glass bottle. An optical image was taken soon after, along with several control samples. The Cytate-blood bottle fluoresced quite nicely. The sample bottles were then left in a lab for three plus years, at room temperature with ordinary exposure to lab lights. The sample bottles were imaged after the three year period, and the Cytate-blood bottle fluoresces still.

Semiconductor nanocrystals composed by few hundred to a few thousand atoms also known as quantum dots have received substantial attention due to their size tunable narrow-emission spectra and several other advantages over organic molecules as fluorescent labels for biological applications, including resistence to photodegradation, improved brightness and only one laser excitation that enable the monitoring of several processes simultaneously. In this work we have synthesized and characterized thiol-capped CdTe and bioconjugated them to macrophages. We have mapped the fluoroscence intensity along the macrophage's body in our set up consisting of an optical tweezer plus a non-linear micro-spectroscopy system to perform scanning microscopy and observe spectra using two photon excited luminescence.

We had previously reported about high aggregation number of the yellow fluorescent protein zFP538, as was shown by gel-filtration and dynamic light scattering. In this study we used atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) to image zFP538 in solid state. Height and phase measurements of the sample were taken using NT-MDT AFM operated in tapping mode. Height data provides three-dimensional topographical information on the sample while the phase data, which measures the phase shift in cantilever oscillation, responds to attractive and repulsive interactions between the cantilever tip and the sample. This signal can be related to the stiffness of the sample. Unlike EGFP, which crystallizes upon drying of water solution, zFP538 forms ring-like films due to its surfactant properties. According to AFM these films are comprised of ellipsoidal protein granules, with the major axis of the granules lying in the range from 50 to 300 nm and the minor axis - in the range from 30 to 130 nm. Average volume of these granules is about 19500 times higher than volume of zFP538 monomer, calculated as in the case of GFP molecule (a cylinder with a height of 4.2 nm and 2.4 nm diameter). NSOM with a 532 nm laser emission from CW Nd:YAG shows that fluorescence of zFP538 is retained in solid state. A ratio of maximum emission to shoulder at 580 nm is 0.65 for solid zFP538 and only 0.25 for water solution.

Our experiments show that fluorescence dependence of zFP538 on protein concentration is nonlinear over the range from 1.2•10^-9 M to 5.5•10^-7 M. Under pH from 5 to 9 it can be divided into two linear regions with different slope at concentration more and less than 1•10^-7 M. One may conclude, there are at least two different forms of the yellow protein observed in the high (>1•10^-7 M) and low (<1•10^-7 M) concentration regions. These forms may be different aggregation states of the protein. We have obtained acid denaturation kinetics curves for zFP538 over pH range from 3.5 to 5.5 at different protein concentrations. Under all pH values relative residual fluorescence of the most concentrated solution (1.33•10^-6 M) is higher than that for the solutions of lower protein concentration (1.36•10^-7 M and 1.36•10^-8 M). Fluorescence decrease under pH from 3.5 to 4.24 is three or two exponential. Under higher pH values kinetic curves have a plateau at first and then fluorescence drops in exponential way. The results obtained support the hypothesis that at higher protein concentrations zFP538 is more stable against acid denaturation.

In this work we studied in vivo the combined action of cyclophosphamide and the extract of mycelium of Pleurotus ostreatus on mice bearing melanoma B16-F0, expressing green fluorescent protein (GFP). This model allows to recognize small-size tumors and metastases, unrecognizable by other methods. It was found that combined administration of cyclophosphamide (300 mg/kg) and the extract of mycelium of Pleurotus ostreatus (100 mg/kg), administered for 10 days after cyclophosphamide injection, as well administration of cyclophosphamide alone, cause inhibition of tumor growth about 97%. It was shown that administration of the extract of mycelium of Pleurotus ostreatus alone leads to inhibition of tumor growth of 61%. It was found that in case of combined administration of cyclophosphamide and the extract of mycelium of Pleurotus ostreatus, leucopenia was less expressed than in case of administration of cyclophosphamide alone.

It has been proved that singlet oxygen is the major cytotoxic agent in Photodynamic therapy (PDT). Chemiluminescence(CL) mediated with Cyp- ridina luciferin analog (FCLA) was recently reported to successfully detect singlet oxygen in chemical and biological systems. The present study has focused on establishing the experiment conditions of FCLA-assisted CL method and tested the validity of the system as a dosimetric tool for PDT in vitro. HL-60 leukemia cell suspensions were sensitized with varying dose of Photofrin and irradiated with 635nm laser light at different rate. The FCLA-CL associated with singlet oxygen was measured with a band-pass filtered photon multiplier tube (PMT) system. We have observed that the CL intensity of FCLA is dependent on PDT treatment parameters. In addition, Cell survival corresponded strongly with peak CL intensity. These results suggest that the FCLA-assisted CL system can be an effective means in in vitro PDT study as an indicator of reactive oxygen species. The system may as well, with additional investigations, provide an alternative dosimetry technique for PDT.

Compared with the traditional organic fluorescent materials, the lanthanide coordination compounds have a large variety of applications, for example glucose sensing, based on the luminescence enhancement of a lanthanide-tetracycline complex due to enzymatically generated H₂O₂ at physiological glucose concentrations. It is known that the fluorescence is based on the energy-transfer from the ligand to the central lanthanide ion. The aim of this work was to study the optical properties of Europium, Erbium, Ytterbium, Holmium, Terbium and Neodymium tetracycline complexes. An increase in europium emission band was observed for the first time, with addition of urea peroxide in the solution. This method works at neutral pH and the luminescence was detected at visible lanthanides luminescence after a 10 min. incubation time of the samples.