The two-photon excitation (TPE) microscopy has become an important tool of noninvasive imaging due to the better penetration and relative harmlessness of the longer wavelength. However, the high photon flux in two-photon excitation can potentially lead to higher-order photobleaching within the focal volume. This paper measured the relationship between the photobleaching rate and the excitation power for Chemical dyes and green fluorescence proteins (GFPs) in vivo at biological imaging level. As expected, the photobleaching rate increased near-linearly with the excitation power for one-photon excitation. However, the two-photon photobleaching rate increased with high-order power (≥3.5) of excitation power, indicating the presence of high-order photon interaction in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study may be limited by increased photobleaching.

Specific, surface cysteine sites have been introduced into Green Fluorescent Protein (GFP) to allow site-specific chemical modification by thiol-directed reagents. These sites have been labelled using BODIPY/eosin/rhodamine reagents as chemical FRET partners for the native GFP chromophore. When they were excited at 488 nm these engineered GFP: conjugated-fluorophore constructs, showed quenching of the native GFP fluorophore emission at 511 nm. New emission bands appeared correponding to each chemical fluorophore emission. Thus the new GFP chimeras exhibited strong intramolecular FRET. GFP mutants were then engineered with trypsin-sensitive sequences located close to the chemical fluorophore-bearing cysteine site. Trypsinolysis caused major changes in the FRET fluorescence spectra. On trypsinolysis the FRET was destroyed, as the FRET partners were now on separate molecules in the cleaved products. T Consequently, the emission wavelength altered from that of the chemically conjugated FRET partner back to that of the native fluorophore of the GFP (511 nm). This provides the possibility of efficient, ratio-based detection. Thus, protein re-engineering has led to novel probes capable of enzymatic triggering based on intramolecular FRET between GFP and specifically sited chemical labels.

Adopted for the live cell, the methods of fluorescence resonance energy transfer (FRET) together with GFP-technology have taken us beyond the limits of optical resolution and allow to resolve proteinÐprotein interactions in 3-7 nm proximity in real time in a live cell. To reliably study cellular proteins involved in trafficking of cholera toxin we first analysed the maturation time of each chimeric protein. Cytosolic partners of proteins responsible for transport were fused to cyan fluorescent protein (CFP) and co-expressed with yellow fluorescent protein (YFP)-fused transmembrane Golgi proteins. Transiently transfected cells (4 - 10 h after transfection) were used in experiments since expression from inducible systems gave high background fluorescence of residual GFPs depending on different turnover of the investigated proteins. Cholera toxin induced interactions were visualised upon binding of cytosolic partners-CFP to the transmembrane proteins by an increase in sensitised emission of the acceptor (e.g. KDEL-receptor-YFP) and were confirmed by acceptor bleach. Recently we have combined two cellular systems: retrograde transport (PM -Golgi-ER, using cholera toxin as a cargo) and anterograde transport - (trafficking of newly expressed connexins from the ER-via Golgi-to the PM) and analysed them simultaneously in the same cell. Four-colour resolution of GFP, YFP, Cy3, and Cy5 was achieved by a combination of multiphoton and single photon laser techniques and by selective filter sets. The data obtained reveal a limited capacity of the Golgi to perform both types of transport, i.e. retrograde and anterograde, simultaneously.

Glucose is the major source of carbon, and glutamine is the major source of nitrogen in cell culture media. Thus, glucose and glutamine monitoring are important in maintaining optimal conditions in industrial bioprocesses. Here we report reagentless glucose and glutamine sensors using the E. coli glucose binding protein (GBP) and the glutamine binding protein (GlnBP). Both of these proteins are derived from the permease system of the gram-negative bacteria. The Q26C variant of GBP was labeled at the 26-position with anilino-naphthalene sulfonate (ANS), while the S179C variant of GlnBP was labeled at the 179-position with acrylodan. The ANS and acrylodan emissions are quenched in the presence of glucose and glutamine, respectively. The acrylodan-labeled GlnBP was labeled at the N-terminal with ruthenium bis-(2,2’-bipyridyl)-1,10-phenanthroline-9-isothiocyanate. The ruthenium acts as a non-responsive long-lived reference. The apparent binding constant, Kd’, of 8.0 μM glucose was obtained from the decrease in intensity of ANS in GBP. The reliability of the method in monitoring glucose during yeast fermentation was determined by comparison with the YSI Biochemistry Analyzer. The apparent binding constant, Kd’, of 0.72 μM glutamine was calculated from the ratio of emission intensities of acrylodan and ruthenium (I₅₁₅/I₆₁₀) in GlnBP. The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of ± 0.02 μM. The conversion of the GBP into a similar ratiometric sensor was described.

The food industry is increasingly adopting food safety and quality management systems that are more proactive and preventive than those used in the past which have tended to rely on end product testing and visual inspection. The regulatory agencies in many countries are promoting one such management tool, Hazard Analysis Critical Control Point (HACCP), as a way to achieve a safer food supply and as a basis for harmonization of trading standards. Verification that the process is safe must involve microbiological testing but the results need not be generated in real-time. Of all the rapid microbiological tests currently available, the only ones that come close to offering real-time results are bioluminescence-based methods. Recent developments in application of bioluminescence for food safety issues are presented in the paper. These include the use of genetically engineered microorganisms with bioluminescent and fluorescent phenotypes as a real time indicator of physiological state and survival of food-borne pathogens in food and food processing environments as well as novel bioluminescent-based methods for rapid detection of pathogens in food and environmental samples. Advantages and pitfalls of the methods are discussed.

A spectral imaging technique applied to in vivo bioluminescent imaging is presented that provides an estimate of the depth of bioluminescent reporters inside living animals. The model, based on the standard diffusion approximation of light propagating in a slab sample, is described in this paper. Validation experiments performed on phantom and tissue models, as well as preliminary in vivo mouse images, demonstrate the ability of spectral imaging to provide a correct estimate of depth based upon a single view imaging system.

Bioluminescence resonance energy transfer (BRET) is a naturally occurring phenomenon taking place in some marine coelenterates. Emission of light in these organisms involves the energy transfer between chromophores of donor luciferase and acceptor fluorescent protein. Due to the strict dependence of BRET efficiency on the inter-chromophore distance, the phenomenon has been applied to study protein-protein interactions by fusing interacting partners with either donor or acceptor proteins. Here we describe a BRET-based homogeneous protein-protein interaction assay exploiting novel donor-acceptor pair formed by photoproteins of jellyfish Aequorea victoria bioluminescent system, aequorin and green fluorescent protein enhanced variant (EGFP). Two known interacting proteins, streptavidin (SAV) and biotin carboxyl carrier protein (BCCP) were fused, respectively, with aequorin and EGFP. The fusions were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-Aequorin and BCCP-EGFP was followed by BRET between aequorin (donor) and EGFP (acceptor) resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-Aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

Luciferases are used as the reporter gene for promoter activity, whereas a green fluorescent protein (GFP) is used as marker for cellular function and localization. Recently, bioluminescence resonance energy transfer (BRET) between luciferase and YFP is used for analysis of inter-molecular reaction such as ligand-receptor in the living cells. The neuropeptides nocistatin (NST) and nociceptin/orphanin FQ (Noc/OFQ) are derived from the same precursor protein, while NST exhibits antagonism against Noc/OFQ-actions. In this study, we attempt an intra-molecular BRET system for monitoring dynamic biological process of the production of NST and Noc/OFQ in the living cells. At first, we constructed a fusion protein (Rluc-GFP) covalently linking luciferase (Renilla luciferase; Rluc) to Aequorea GFP as an intra-molecular BRET partner. Furthermore, we inserted constructs of mouse NST and Noc/OFQ (Rluc-m-GFP) or bovine NST and Noc/OFQ (Rluc-b-GFP) containing a proteolytic cleavage motif (Lys-Arg) within Rluc-GFP. When these constructions were transfected into Cos7 cells, all fusion proteins had luciferase activity and specific fluorescence. Luminescence spectra of Rluc-GFP, Rluc-m-GFP and Rluc-b-GFP fusion proteins with DeepBlueC as a substrate showed two peaks centered at 400 nm and 510 nm, whereas Rluc showed one peak centered at 400 nm. These results indicate that the proteolytic cleavage motif inserted fusion proteins between luciferase and GFP are available for intra-molecular BRET systems at first step.

The spectral properties and the subunit components of an R-phycoerythrin that was stable at 37°C in phosphate buffer (pH 7.0) with sodium dodecyl sulfate (SDS) were investigated. The R-phycoerythrin was obtained from the phycobilisome that was prepared from the marine red algae Polysiphonia urceolata by step-gradient sucrose centrifugation. By Sephadex G-150 column chromatography and polyacrylamide gel electrophoresis the R-phycoerythrin was prepared from the phycobilisome disassociatin that was incubated at 37¡C for 6 hr in 0.05M phosphate buffer (pH 7.0) containing 5% (w/v) SDS, 2% (w/v) mercaptoethanol and 10% (v/v) glycerol. The absorption spectrum of the R-phycoerythrin in 0.05M phosphate buffer (pH 7.0) showed that it has three absorption peaks at 498 nm, 537 nm and 566 nm, respectively; and therefore, it belongs to three-peak R-phycoerythrin. At room temperature, its fluorescence emission spectrum showed that the emission peak occurs at 578nm. The component analysis by SDS-polyacrylamide gel electrophoresis showed that the R-phycoerythrin is composed of 17.8 KD, 21 KD and 31KD of three colored polypeptides. Linker peptides existed in the R-phycoerythrin may account for its stability in SDS Solution at 37°C. The stable feature, together with its high fluorescence emission efficiency, like most other phycobiliproteins, may let the obtained R-phycoerythrin be a promising agent of fluorescence label for diagnostic uses of various purposes.

The green fluorescent protein (GFP) from jellyfish Aequorea victoria is a useful tool in biochemistry and cell biology due to its ability to develop the fluorescence in wide variety of organisms. This indicates that maturation of GFP does not require any specific enzymes from jellyfish and chromophore formation may be spontaneous or dependent on ubiquitous factors. There is a great progress in elucidation of mechanism of this process since the GFP was discovered. Nevertheless, the last step of chromophore formation, the oxygen dependent dehydration of Ä„-Äñ bond of Tyr66, still remains unclear. We proposed that this dehydration require a reactive oxygen species (ROS) and arise through the hydroxylation of CÄñ atom of Tyr66 followed by the water elimination and formation of double bond. Recently six novel fluorescent proteins (FPs) have been cloned. We have found that maturation of these FPs in E.coli can be dramatically accelerate by addition of paraquat (superoxide generator).

The red fluorescent protein DsRed (drFP583), has become an excellent addition to Green Fluorescent Protein (GFP) and its mutants in biological and biotechnical applications due to the significantly red-shifted emission maximum at 583 nm. However, the use of DsRed is complicated by its oligomerization. According to the data from dynamic light scattering, steady-state fluorescence polarization, DsRed and its no-aggregated mutants are organized as tetramers in concentrated solutions. But at the concentration of 10^-8-10^-9 M fluorescence polarization shows the existence of the monomeric form of DsRed. Neither dimer nor trimer forms of DsRed were detected. Upon dilution of concentrated solutions of DsRed, the intensity of fluorescence decreased with time. Observed fluorescence kinetics fits well to the tetramer - monomer transition. The brightness of the monomeric form of DsRed was suggested to be significantly less compared to the tetrameric form.

Fluorescence of the yellow fluorescent protein zFP538 strongly depends on concentration and starting pH form which pH profile is recorded. pH transitions typical for chromopeptides isolated from zFP538 can be observed for whole protein in diluted solutions. The quenching fluorescence of zFP538 is irreversible upon acidification or alkalization of the low concentrated soultions. In concentrated solutions, according to the data of dynamic light scattering, the protein zFP538 is strongly aggretaed (or oligomerized) and become more stable against acid denaturation. Spectral changes on pH are almost reversible both for fluorescence and absorbance. Two major chromopeptides obtained from zFP538 have different spectral properties and no similarity to the spectral properties of the chromopeptide obtained from GFP.

A baculovirus expression system was used to produce DsRed fusion protein in insect larvae. As the baculovirus/insect larvae system requires precise harvest timing to achieve high yield of protein, a low-cost miniature all-solid state optical probe was used for detection of the protein concentrations in the frozen larvae. Three batches of infected larvae were monitored at different post-infection times. The calibration curve of the probe was obtained by simultaneous measurements both in laboratory fluorimeter and using gel electrophoresis analysis. The results show good correlation between the optical measurements and the standard laboratory technique.

The specificity of a novel EGF-Cy5.5 fluorescent optical probe was assessed using CW fluorescence imaging accomplished via an ICCD camera. Imaging was performed on mice with MDA-MB-468 cancer, known to overexpress EGFr, and contrasted against an analogous cell line, MDA-MB-435, that does not express EGFr. Fluorescence images on mice bearing s.c. inoculated tumors were obtained every 6 seconds for a period of 20 minutes following i.v. injection of ICG, Cy5.5, or EGF-Cy5.5 and every 24 hrs thereafter for up to 192 hrs. In addition, mice with MDA-MB-468 tumors were injected i.v. with anti-EGFr antibody C225 24 hrs prior to injection of EGF-Cy5.5. Monitoring the time-sensitive fluorescence intensity confirms that ICG and Cy5.5 show no favorable binding to tumor, regardless of EGFr expression level. In contrast, EGF-Cy5.5 exhibits selective accumulation only in the MDA-MB-468 tumor. Moreover, tumor uptake of EGF-Cy5.5 was blocked by pre-injection of C225 antibody, demonstrating specificity of the targeted contrast agent. Data further demonstrate that ICG and Cy5.5 fluorescence is completely absent from the tumor site, regardless of EGFr expression level, 24-hrs post injection. Similarly, little EGF-Cy5.5 fluorescence was detected in the EGFr-negative tumor after 24 hrs, however, for the MDA-MB-468 tumor, EGF-Cy5.5 fluorescence did not reach undetectable levels until 192 hrs.

The predominant long-chain carotenoids found in the human skin are lycopene and β-carotene. They are powerful antioxidants and thought to act as scavengers for free radicals and single oxygen that are formed by excessive exposure of skin to sunlight. However the role of the particular representatives of the carotenoid antioxidants family in the skin defense mechanism is still unclear and has to be clarified. We demonstrate the opportunity for fast non-invasive selective quantitative detection of β-carotene and lycopene in human skin employing Raman spectroscopy. Analyzing Raman signals originating from the carbon-carbon double bond stretch vibrations of the molecules under blue and green laser excitation we were able to characterize quantitativly the concentrations of each carotenoid in alive human skin. In this method we take an advantage of different Raman cross-section spectral profile for β-carotene and lycopene molecules. This novel technique allows the quantitative assessment of individual carotenoid species in the skin rather then the cumulative level of long-chain carotenoids mixture as we could measure in our previous works. The required laser light exposure levels are well within safety standards. Prelimininary dichoromatic Raman measurements reveal significant differences in the carotenoid composition of different volunteer's skin: even in statistically small group of seven subjects the ratio of β-carotene-to-lycopene in their skin vary from 0.5 to 1.6. This technique holds promise as a method of rapid screening of carotenoids composition of human skin in large populations and suitable in clinical studies for assessing the risk for cutaneous diseases.

The use of contrast agents in almost every imaging modality has been known to enhance the sensitivity of detection and improve diagnostic capabilities by site-specifically labeling tissues or cells of interest. The imaging capabilities of Optical Coherence Tomography (OCT) need to be improved in order to detect early neoplastic changes in medicine and tumor biology. We introduce and characterize the optical properties of several types of optical contrast agents in OCT, namely encapsulating microspheres that incorporate materials including melanin, gold, and carbon. Micron-sized microspheres have been fabricated by state-of-the-art sonicating and ultrasound technology. The optical properties of optical contrast agents have been characterized according to their scattering and absorption coefficients and lifetimes using OCT and the oblique incidence reflectometry method. Finally, we demonstrate the use of these optical contrast agents in in vitro mice liver and analyze the contrast improvement from the OCT images. These optical contrast agents have the potential to improve the detection of in vivo pathologies in the future.

We have shown that a specific cytosolic pH (pH^cyt) regulatory mechanism, i.e., vacuolar type H+-ATPases at the plasma membrane (pmV-ATPases), allows angiogenic and metastatic cells to survive in an acidic and hostile environment. However, a functional evaluation of this pump's activity in situ (i.e., in living animal models) has not been attempted. We developed a mouse model of angiogenesis and metastasis based on the dorsal skin fold chamber, and implanted highly metastatic human tumor cells that have been engineered to express green fluorescent protein (GFP). GFP can be used as a pH reporter because its fluorescence is pH sensitive. Our studies in isolated single cells indicated that there are distinct pH^cyt gradients in the invadipodia versus the lamellipodia due to the preferential expression of pmV-ATPases at the leading edge. We hypothesize that in vivo, these pH gradients also exist. We employed spectral imaging and real time confocal imaging microscopy, since these approaches are complementary and exhibited unsurpassed temporal and spectral resolution, thus allowing us to study pH^cyt in discrete subcellular regions of the cells expressing GFP. We can acquire a full frame (i.e., 512 x 512 pixels) in real time confocal imaging at ca. 25-50 msec, whereas spectral imaging allow us to obtain spectral information from discrete domains of ca. 10 μm in the x-y plane and every 10 μm from leading to lagging edge within a time frame of 5 msec at 0.4 nm spectral resolution. This is possible because we employ frame transfer cooled CCD cameras and spectrographs. Studies are under way to evaluate proton gradients using multiphoton approaches since this will allow us to evaluate pH deeper into the tissue (i.e., 300-600 μm), and should allow us to follow pH^cyt and the progression of tumor metastasis.

Water is routinely monitored for environmental pathogens such a Cryptosporidium and Giardia using immunofluorescence microscopy (IFM). Autofluorescence can greatly diminish an operators capacity to resolve labeled pathogens from non-specific background. Naturally fluorescing components (autofluorophores) encountered in biological samples typically have fluorescent lifetimes (τ) of less than 100 nanoseconds and their emissions may be excluded through use of time-resolved fluorescence microscopy (TRFM). TRFM relies on the large differences in τ between autofluorescent molecules and long-lived lanthanide chelates. In TRFM, targets labeled with a time-resolvable fluorescent immunoconjugate are excited by an intense (UV) light pulse. A short delay is imposed to permit the decay of autofluorescence before capture of luminescence from the excited chelate using an image intensified CCD camera. In our experience, autofluorescence can be reduced to insignificant levels with a consequent 30-fold increase in target visibility using TRFM techniques. We report conjugation of a novel europium chelate to a monoclonal antibody specific for Giardia lamblia and use of the immunoconjugate for TRFM studies. Initial attempts to conjugate the same chelate to a monoclonal antibody directed against Cryptosporidium parvum led to poorly fluorescent constructs that were prone to denature and precipitate. We successfully conjugated BHHCT to anti-mouse polyvalent immunoglobulin and used this construct to overcome the difficulties in direct labeling of the anti-Cryptosporidium antibody. Both Giardia and Cryptosporidium were labeled using the anti-mouse protocol with a subsequent 20-fold and 6.6-fold suppression of autofluorescence respectively. A rapid protocol for conjugating and purifying the immunoconjugate was found and methods of quantifying the fluorescence to protein ratio determined. Performance of our TRFM was dependent on the quality and brightness of the immunoconjugate and optimization of the conjugation process is necessary to reap the full benefit of time-resolved techniques.

Carbon 60 has been used in a functionalized form in a bioassay for a common herbicide, atrazine. It was found that the C₆₀ is a very effective quencher of fluorescence from a number of common dyes. C₆₀ was conjugated to atrazine for use in an immunoassay in which fluorescence from rhodamine was measured. Quenching of the rhodamine emission provided a detection scheme in this assay that yielded a very good limit of detection. The C₆₀ quenching scheme can be used with a wide variety of fluorescent dyes, permitting the potential use of a range of small, cheap excitation sources.

We describe the design, fabrication and performance of novel, electrically pumped, vertical compound cavity 976nm InGaAs lasers that emit at 488nm via intracavity second harmonic generation. The resulting light source is an ideal replacement for Ar-ion lasers used in a wide variety of bio-analytical instruments. We present characterization data for the laser to demonstrate its capabilities. Lastly, future directions for the technology are discussed, including a monolithic form and devices operating at 460 and 532nm.

We have recently developed a new type of reporter (upconverting chelate) for biomedical diagnostics. For this reporter, the light is absorbed and emitted by a lanthanide ion, rather than an organic molecule, as is the case for a typical fluorescent dye. These materials do not photobleach and have no autofluorescent background. We focus in this paper on neodymium ions complexed with the familiar chelating agents, EDTA, DPA, DTPA and DOTA. We have performed experimental measurements with one- and two-color laser light excitation for different chelate compounds. The samples are excited using two Nd:YAG-pumped dye laser systems that provide laser light near 587 nm and 800 nm. For one-color excitation, the emitted light depends quadratically on the incident laser power, as expected. Three strongly emitting lines are observed, located near 360 nm, 387 nm, and 417 nm. We observed more efficient upconversion in EDTA although the DPA chelates show comparable ground state absorbance. We have studied the influence of temporal delay between the two laser pulses and obtained the decay lifetime of the first intermediate state in the various chelated compounds.

The development of a rapid, inexpensive, and accurate in vivo phenotyping methodology for characterizing drug-metabolizing phenotypes with reference to the cytochrome P450 (CYP450) enzymes would be very beneficial. In terms of application, in the wake of the human genome project, considerable interest is focused on the development of new drugs whose uses will be tailored to specific genetic polymorphisms, and on the individualization of dosing regimens that are also tailored to meet individual patient needs depending upon genotype. In this investigation, chemical probes for CYP450 enzymes were characterized and identified with Raman spectroscopy. Furthermore, gold-based metal colloid clusters were utilized to generate surface enhanced Raman spectra for each of the chemical probes. Results will be presented demonstrating the ability of SERS to identify minute quantities of these probes on the order needed for in vivo application.

We proposed and analyzed the D-P (Dye-to-Photon) photobleaching mechanism, the interaction between the excitation photon(s) and the fluorophore dye molecules in excited states. At single molecule level, the D-P photobleaching probability with One- and Two-photon excitation (OPE and TPE) should always depend linearly on the probability of fluorophore molecule in excited states. However, at biological imaging level, the dependence of the D-P photobleaching probability on the probability of fluorophore molecules in excited states may depend on the excitation way: the photobleaching states, while the photobleaching rate depends on the high-order power of the probability of fluorophore molecule in the excited states, especially in the case of high excitation intensity.