Green fluorescent protein variants have been developed that report real-time change in pH and redox potential in living cells. The variants involve cysteine substitutions near the chromophore, which greatly alter the sensitivity of the protein to changes in its environment. Measurements can be made on single living cells in the fluorescence microscope or in cell suspension with an ordinary fluorimeter. The indicators are ratiometric by emission and/or excitation, which means that measurements at two different wavelengths are sufficient to determine both the quantity being measured and the indicator GFP concentration. The photophysics of a novel blue/green dual emission GFP variant will be presented. The design principles, crystal structures and ultrafast spectroscopic analysis of probe response will be discussed in terms of atomic models involving excited state proton transfer. Some applications in living cells will be presented.

Photoactivation, the rapid conversion of photoactivatable molecules to a fluorescent state by intense irradiation, can be used to mark and monitor selected molecules within cells¹. We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) based on a mutation at position 203 that upon intense irradiation with 413 nm light exhibits a stable 60-100 fluorescence increase under 488 nm excitation. The photoactivated form of this mutant named photoactivatable GFP (PA-GFP), is stable under a number of conditions. PA-GFP can be used to analyze protein dynamics in living cells, offering enormous potential for addressing outstanding questions in protein trafficking and turnover, organelle dynamics, and cell lineage patterns.

The red fluorescent protein eqFP611 shows favorable properties for applications as molecular marker. Its usefulness is, however, limited by its tendency to form tetramers at physiological concentrations. To provide a basis for the rational design of monomeric variants, we examined the monomer interfaces in the x-ray structure of eqFP611. The arrangement of the four ß cans is very similar to that of other GFP-like proteins such as DsRed and RTMS5. In eqFP611, the monomers are linked by comparatively weak interactions, as inferred from the dissociation into monomers in the presence of SDS or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. Some structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. Functional dimeric variants could be generated by altering the A/B interface by single point mutations (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface resulted as yet in essentially complete loss of fluorescence. Presumably, the folding of eqFP611 into its functional form relies on A/C interfacial interactions.

Since 1982, phycobiliproteins have served as fluorescent labels in a wide variety of cell and molecule analyses. The exceptional spectroscopic properties of these labels include very high absorbance coefficients and quantum yields, and large Stokes shifts. The spectroscopic diversity of these reagents is restricted to a subset of naturally occurring phycobiliproteins with stable assembly states in vitro, whose target specificity is generated by chemical conjugation to proteins or small molecules. The latter step generates heterogeneity. These limitations have been overcome by expressing various recombinant phycobiliprotein constructs in the cyanobacterium Anabaena sp. PCC7120. Modular recombinant phycobiliprotein-based labels were constructed with some or all of the following features (a) an affinity purification tag; (b) a stable oligomerization domain (to maintain stable higher order assemblies of the phycobiliprotein monomers at very low protein concentration); (c) a biospecific recognition domain. Such phycobiliprotein constructs are readily purified from crude cell extracts by affinity chromatography and used directly as fluorescent labels. To generate constructs for intracellular in vivo labeling, the entire pathways for the biosynthesis of the His-tagged holo- α (phycocyanobilin-bearing) subunit of phycocyanin (emission max. 641 nm) and of the His-tagged holo-α (phycobiliviolin-bearing) subunit of phycoerythrocyanin (emission max. 582 nm) were reconstituted in Escherichia coli.

Phytochromes are biliprotein photoreceptors which exist in two photointerconvertible forms - a red light absorbing Pr form and far-red light absorbing Pfr form. Substitution of their native linear tetrapyrrole (bilin) prosthetic group with an unnatural bilin analog was shown to yield strongly fluorescent adducts that can be reconstituted in living cells⁽¹⁾. These self-assembling fluorescent adducts, phytofluors, hold great promise for in vivo cell biological applications; however, unlike the green fluorescent protein (GFP), exogenous bilins are needed for phytofluor formation in cells. In the present study, a directed evolution approach was undertaken with the goal of creating mutant phytochromes with novel spectroscopic properties. Error-prone PCR was employed to generate point mutations at random positions within the PHY domain of the cyanobacterial phytochrome Cph1. We hypothesize that alterations in this domain will result in spectrally shifted and red/far-red fluorescent holophytochrome mutants 'locked' in either the Pr or Pfr form. Apophytochrome mutant libraries, expressed in strains of Escherichia coli engineered to synthesize different bilin precursors, were screened using digital imaging spectroscopy (DIS) and fluorimaging methodologies^(2;3). A variety of mutants with altered absorption properties were identified. In vitro DNA shuffling is in progress to enrich the diversity of mutant phenotypes.

Genetic introduction of fluorescent labels such as the Visible Fluorescent Proteins (VFP) has revolutionized the visualization and characterization of cellular proteins. Lateral diffusion measurements, most commonly accomplished through Fluorescence Photobleaching Recovery (FPR or FRAP), provide important information on such molecules’ size, environment and participation in intermolecular interactions. However, serious difficulties arise when these techniques are applied to VFP fusion proteins since cytoplasmic species contribute to the fluorescence recovery signal and thus distort measurements aimed at surface molecules. Two new methods help eliminate these difficulties through distinctly different strategies. In Total Internal Reflection Interference Fringe FPR, interfering laser beams enter a 1.65 NA Olympus objective at the periphery of the back focal plane where the NA exceeds 1.38. This creates an interference pattern totally internally reflected at the coverslip-medium interface. Fluorescence excitation occurs only where the cell contacts the coverslip so no contribution arises from cytoplasmic species. Alternatively, High Probe Intensity (HPI) FPR measurements retain the intrinsic confocality of spot measurements to eliminate interference from fluorescent cytoplasmic species. However, HPI-FPR methods lift the previous requirement that FPR procedures be performed at probe beam intensities low enough to not induce bleaching in samples during measurements. The high probe intensities now employed provide much larger fluorescence signals and thus more information on molecular diffusion from each measurement. We report successful measurements of membrane dynamics of various VFP species obtained by these techniques and compare them with results of earlier FPR methods which previously proved unsatisfactory in these instances.

Dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP) expressing transgenic mice has been shown to be a powerful technology to study tumor-host interaction. Host animals include mice which express the GFP transgene in essentially all cells as well as animals in which the regulatory elements of the stem cell marker nestin drive GFP. The general GFP-transgenic mouse is available in both the normal and athymic nude (nu/nu) background. These models show with great clarity the details of the tumor-stroma interaction especially tumor induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. GFP-expressing tumor vasculature could be visualized interacting with the RFP-expressing tumor cells transplanted to the nestin-driven GFP transgenic mice which expressed nestin-GFP in nascent blood vessels was shown as a marker of nascent tumor angiogenesis. Dual-color fluorescence imaging, which visualizes the tumor-host interaction by whole-body imaging and at the cellular level in fresh tissues, dramatically expanding previous studies in fixed and stained preparations (1).

Colored proteins are widely used as gene markers in biotechnology. Chromophores result from autocatalytic posttranslational reactions involving several amino acids. The protein asCP595 was isolated for the first time from the coral as a weakly fluorescent chromoprotein with a fluorescence maximum at 595 nm. Strong illumination in the blue wing of the low energy absorption band results in a superlinear increase of the fluorescence yield and shifts its fluorescence spectrum by about 10 nm to the red. Time resolved fluorescence measurements using excitation pulses with 10 ps duration revealed a multiexponential decay pattern with time constants in the range from 20 ps to 2.1 ns. The ratio of amplitudes related to the different time constants depends on the intensity of illumination favoring the ns component at high intensities. Transient absorption measurements using ultrashort excitation pulses (150 fs, 1 kHz repetition rate) did not reveal excited states with nanosecond lifetimes as observed in fluorescence upon excitation using 10 ps pulses. This observation leads to the notion that within 10 ps a second photon is absorbed by a state not yet populated within 150 fs. As a consequence we propose two different excited singlet states operative in asCP595, one with low fluorescence quantum yield peaking at 595 nm and one with high fluorescence quantum yield peaking at 605 nm which is populated via the consecutive absorption of two photons at high excitation intensities.

One of the most intriguing findings in single molecule spectroscopy (SMS) is the observation of Raman spectra of individual molecules, despite the small cross section of the transitions involved. The observation of the spectra can be explained by the surface enhanced Raman scattering (SERRS) effect. At the single-molecule level, the SERRS-spectra recorded as a function of time reveal inhomogeneous behaviour such as on/off blinking, spectral diffusion, intensity fluctuations of vibrational line, and even splitting of some lines within the spectrum of one molecule. Single-molecule SERRS (SM-SERRS) spectroscopy opens up exciting opportunities in the field of biophysics and biomedical spectroscopy. The first example of single protein SERRS was performed on hemoglobin. However, the possibility of extracting the heme group by silver sols can not be excluded. Here we report on SM-SERRS spectra of enhanced green fluorescent protein (EGFP) in which the chromophore is kept in the protein. The time series of SM-SERRS spectra suggest the conversion of the EGFP chromophore between the deprotonated and the protonated form. Autocorrelation analysis of SM-SERRS trajectory reveals the presence of fast dynamics taking place in the protein. Our findings show the potential of the technique to study structural dynamics of protein molecules.

We report on the fluorescence dynamics of a red fluorescent protein DsRed from the coral Discosoma genus by means of ensemble and single molecule fluorescence spectroscopy. Single molecule experiments performed on 543-nm excitation point to the existence of DsRed as a tetramer and reveal the presence of a no/off blinking phenomenon in the millisecond time range. Collective effects involving the red chromophores within the individual tetramers were observed. Time-resolved fluorescence data reveal the presence of a population of 25 % of the immature green chromophores which relates to tetramers containing only this immature green form and which is responsible for the weak fluorescence emitted by DsRed at 500-nm when excited at 460-nm. The remaining 75 % of the immature green chromophores are involved in a FRET process to the red chromophores within the tetramers that contain them. Using time-resolved detection and spectroscopy at single molecule level we were able to demonstrate the presence of a photoconversion process of the red chromophore emitting at 583-nm into a super red species that emits weakly at 595-nm. The same phenomenon is further corroborated at the ensemble level with the observation of the creation of a super red form and a blue absorbing species upon irradiation with 532-nm pulsed light at high excitation power.

Regulated gene transcription is dependent on the steady-state concentration of DNA-binding and coregulatory proteins assembled in distinct regions of the cell nucleus. For example, several different transcriptional coactivator proteins, such as the Glucocorticoid Receptor Interacting Protein (GRIP), localize to distinct spherical intranuclear bodies that vary from approximately 0.2-1 micron in diameter. We are using multi-spectral wide-field microscopy of cells expressing coregulatory proteins labeled with the fluorescent proteins (FP) to study the mechanisms that control the assembly and distribution of these structures in living cells. However, variability between cells in the population makes an unbiased and consistent approach to this image analysis absolutely critical. To address this challenge, we developed a protocol for rigorous quantification of subnuclear organization in cell populations. Cells transiently co-expressing a green FP (GFP)-GRIP and the monomeric red FP (mRFP) are selected for imaging based only on the signal in the red channel, eliminating bias due to knowledge of coregulator organization. The impartially selected images of the GFP-coregulatory protein are then analyzed using an automated algorithm to objectively identify and measure the intranuclear bodies. By integrating all these features, this combination of unbiased image acquisition and automated analysis facilitates the precise and consistent measurement of thousands of protein bodies from hundreds of individual living cells that represent the population.

Harmful environmental factors - namely ionizing radiation - will continue to influence future manned space missions. The Cellular Biodiagnostic group at the German Aerospace Center (DLR) develops cellular monitoring systems, which include bacterial and mammalian cell systems capable of recognizing DNA damage as a consequence of the presence of genotoxic conditions. Such bioassay or biosensor systems will complement the physical detector systems used in space, insofar as they yield intrinsically biologically weighted measures of cellular responses. Furthermore, synergistic mutagenic and cancerogenic impacts of the radiation environment together with other potentially genotoxic constituents of the space habitat can be quantified using such systems, whose signals are especially relevant for the molecular damage to the DNA or the chromosomes. The experiment Cellular Responses to Radiation in Space (CERASP) has been selected by NASA to be performed on the International Space Station. It will supply basic information on the cellular response to radiation applied in microgravity. One of the biological end-points under investigation will be survival reflected by radiation-dependent reduction of constitutive expression of the enhanced variant of green fluorescent protein (EGFP), originally isolated from the bioluminescent jellyfish Aequorea victoria. A second end-point will be gene activation by space flight conditions in mammalian cells, based on fluorescent promoter reporter systems using the destabilized EGFP variant (d2EGFP). The promoter element to be investigated will reflect the activity of the NF-kappaB stress response pathway as an anti-apoptotic radiation response. DNA damage will be measured by fluorescent analysis of DNA unwinding (FADU). The systems have worked properly for terrestrial applications during the first experiments. Experiments using accelerated particles produced at the French heavy ion accelerator GANIL have given insights into cellular mechanisms relevant for the exceptional radiation field in space.

Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

Histamine is the primary etiological agent in the foodborne disease scombrotoxicosis, one of the most common food toxicities related to fish consumption. Procedures for detecting histamine in fish products are available, but are often too expensive or too complex for routine use. As an alternative, a bacterial bioluminescent bioreporter has been constructed to develop a biosensor system that autonomously responds to low levels of histamine. The bioreporter contains a promoterless Photorhabdus luminescens lux operon (luxCDABE) fused with the Vibrio anguillarum angR regulatory gene promoter of the anguibactin biosynthetic operon. The bioreporter emitted 1.46 times more bioluminescence than background, 30 minutes after the addition of 100mM histamine. However, specificity was not optimal, as this biosensor generated significant bioluminescence in the presence of L-proline and L-histidine. As a means towards improving histamine specificity, the promoter region of a histamine oxidase gene from Arthrobacter globiformis was cloned upstream of the promotorless lux operon from Photorhabdus luminescens. This recently constructed whole-cell, lux-based bioluminescent bioreporter is currently being tested for optimal performance in the presence of histamine in order to provide a rapid, simple, and inexpensive model sensor for the detection of foodborne toxins.

The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

Estrogen receptor (ER) is a ligand-activated transcriptional factor, able to dimerize after activation and to bind specific DNA sequences (estrogen response elements), thus activating gene target transcription. Since ER homo- and hetero-dimerization (giving a-a and a-b isoforms) is a fundamental step for receptor activation, we developed an assay for detecting compounds that induce human ERa homo-dimerization based on bioluminescence resonance energy transfer (BRET). BRET is a non-radiative energy transfer, occurring between a luminescent donor and a fluorescent acceptor, that strictly depends on the closeness between the two proteins and can therefore be used for studying protein-protein interactions. We cloned ERa coding sequence in frame with either a variant of the green fluorescent protein (enhanced yellow fluorescent protein, EYFP) or Renilla luciferase (RLuc). Upon ERa homo-dimerization, BRET process takes place in the presence of the RLuc substrate coelenterazine resulting in EYFP emission at its characteristic wavelength. The ER alpha-Rluc and ER alpha-EYFP fusion proteins were cloned, then the occurrence of BRET in the presence of ER alpha activators was assayed both in vivo, within cells, and in vitro, with purified fusion proteins.

A method for making temporal maps in bacteria, plasmids and bacteriophages is described. A cassette containing both the genes for bacterial luciferase and kanamycin resistance can be introduced at precise sites. The technique involves clonging followed by genetic recombination. The result is formation of structures that have the luciferase genes in place of the normal DNA and this allows the very precise measurement of transcription/translation of the substituted regions. Very low levels of transcription as well as the kinetics of induction can be easily ascertained. As a specific demonstration of this general method, the technique was used with bacteriophage λ, one of the best known organisms. By measuring light emission, the expression of luciferase was followed after induction for both early and late genes. The exact timing of initial expression of genes was also determined by sampling at very short intervals. The results show that the early genes express almost without delay implying that the function of the N antitermination system is not temporal regulation.

The objective of this investigation is to develop a bioluminescent bioreporter system for the detection and monitoring of pathogenic microbial species. Current detection methodologies typically rely on time-consuming sample pre-enrichment steps to elevate pathogen concentrations to detectable levels or DNA based polymerase chain reaction (PCR) techniques that require extensive user training and expensive instrumentation. Detection utilizing bioluminescent bioreporter organisms, however, can provide a simple and rapid means of monitoring foodborne pathogens. Bioluminescent bioreporters are engineered to produce light in response to specific environmental inducers. The light signal is then measured with photodetector devices to generate a quantitative assessment of inducer concentration. The immediate goal of this research effort is to integrate key quorum sensing signal transduction elements into pathogen specific bacteriophages. Upon infection of a unique pathogenic species by the bacteriophages, quorum sensing signals will be generated that will subsequently stimulate bioluminescence in neighboring bioluminescent bioreporter cells. Utilizing both bacteriophages and bioluminescent bioreporters, we realize exceptional pathogen specificity while attaining enhanced bioluminescence production. This integrative approach will lead to rapid pathogen identification without requisite sample pre-enrichment. Additionally, since the bioluminescent response is completely intrinsic to the bioreporter organism, no user interventions are required for generating light signals; the protocol requires only addition of the food sample with the bacteriophage/bioluminescent bioreporter system. Measurement of light responses can be achieved using high-throughput microtiter plate readers, hand-held photomultiplier units, or microchip luminometers.

Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

pH-dependent aggregation and dissociation of yellow fluorescent protein zFP538 were studied by gel-filtration, dynamic light scattering, and fluorescence spectroscopy. According to the gel-filtration data for low concentration of zFP538 the molecular weight of aggregates decreases upon changing pH from alkaline to neutral. Dynamic light scattering showed that zFP538 aggregates strongly in concentrated solutions. Aggregation influences heavily the pH-profile of fluorescence of zFP538 and stabilizes zFP538 against fluorescence quenching on acidification. Reduction of the protein concentration results in the shifting of pH profile to the alkaline region. Conclusion: aggregation of the yellow fluorescent protein zFP538 depends on pH; dilution of the protein solution is accompanied by dissociation of zFP538 aggregates under neutral and alkaline pH.

Vascular endothelial growth factor (VEGF) is one of the most potent mediators of both physiologic and pathologic angiogenesis. Normal physiologic induction of VEGF occurs during periods of extreme growth, wound healing, as well as immune inflammatory response. Pathologically, however, VEGF is largely responsible for tumor induced angiogenesis and cell survival. Traditional methods of VEGF expression analysis involve either in vitro studies, or highly invasive in vivo methods. We have developed a unique transgenic mouse model (VGL) that possesses a truncated human VEGF promoter attached to a GFP-Luciferase fusion protein. Incorporating this model with both spontaneous and orthotopically injected tumors allow VEGF promoter activity to be visualized in vivo by luciferase luminescence in response to tumor growth non-invasively and over time. By also utilizing bioluminescent tumor cells, we were able to generate models that identify host, tumor, or combined VEGF promoter activity. Results indicate that tumor tissue is responsible for the majority of VEGF promoter activity during tumor growth. Additional studies into the mechanism by which tumor cells initiate VEGF production will yield much needed insight into tumor survival. In conclusion, we have shown that the VGL bioluminescent mouse model is indeed capable of yielding compelling information on host-tumor interactions.

Molecular excitation of photosensitizing agents provides reactive excited states, which can initiate chemical reactions, but it can also lead to molecular relaxation 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.

Fluorescent dyes and probes are key components in multiphoton based fluorescence microscopy imaging of biological samples. While many commercially available fluorescent dyes have sufficed, most exhibit relatively low two-photon absorption (2PA) cross-section values in the tunability range of Ti:sapphire lasers commonly used in multiphoton microscopy imaging. Furthermore, available fluorophores may be plagued with either low fluorescence quantum yields and/or the additional problem of rapid photobleaching upon exposure to the high peak powers provided by fs laser sources. In order to address the demand for better performing dyes for two-photon based imaging, we have prepared a new series of reactive fluorophores tailored for multiphoton imaging. These fluorophores are based upon the fluorene ring system, known to exhibit high fluorescence quantum yields, typically > 0.7, and possess high photostability. They have been functionalized with moieties to act, e.g., as efficient amine-reactive fluorescent probes for the covalent attachment onto, e.g., proteins and antibodies. The synthesis and the single-photon spectral characteristics, as well as measured two-photon absorption cross sections of the reactive fluorophores in solution will be presented. Spectral characterizations of bovine serum albumin (BSA) conjugated with the new reactive probe will also be presented.

Low-level light-emitting imaging technique often detects the light emerged at the tissue surface that is generated internally from a specific target. However, in most cases, the high scattering nature of biological tissue limits the sensitivity and spatial resolution of this imaging modality. In this paper, we report that a significant improvement of chemiluminescence (CL) imaging performance in terms of both sensitivity and spatial resolution can be achieved by use of the topical application of glycerol solution onto tissue sample, i.e. optical clearing approach. Monte Carlo simulation of internally-launched point source shows that the decrease of scattering coefficient of turbid medium, which can be achieved by optical tissue clearing approach, causes stronger peak intensity with a narrower full width at half maximum. The improvement becomes more significant with the source depth increasing from 1 mm to 5 mm. The experimental results shows tissue clearing with 50% glycerol solution could largely improve the brightness and the spatial resolution of CL imaging when the target is covered with biological tissue having thickness of 1mm or 3mm. This method could have potential application in the in vivo low-level light imaging technique.

The goal of this work is to highlight those unique aspects of contrast-enhanced diagnostic optical imaging (OI) that favor a broad clinical utilization of this emerging diagnostic technique and to illustrate certain identified challenges opposing the enthusiastic clinical welcome for the OI method. We consider the single most appealing feature of OI to be its much-touted exquisite sensitivity for the detection of near-infrared fluorescing (NIRF) probes, a sensitivity supporting the development of disease- and molecule-specific NIR diagnostic probes, akin to nuclear imaging but without the ionizing radiation and with superior spatial resolution. But a qualitative OI diagnostic examination, merely defining the presence or absence of NIRF signal, may not be sufficient. The signal must be measurable. A quantitative OI examination, capable of accurately assaying the tissue concentration of the fluorescing probe and changes in that probe concentration related to disease progression or treatment would be extremely valuable. We discuss here at least three challenges to quantitative diagnostic OI, a non-linear relationship between probe concentration and signal intensity, background signal in the form of tissue auto-fluorescence, and the requirement to define precise location and depth of the signal origin from within the subject.

Expression of integrin αvβ3 is upregulated in a number of cancers including colon, pancreas, lung and breast. Additionally, αvβ3 integrin expression has been linked to tumor metastasis and targeting this cell surface protein could provide a viable approach to image and evaluate the metastatic potential of tumors. Accordingly, we evaluated the selective retention of some near infrared (NIR) fluorescent probes in nude mice bearing A549 lung cancer xenograft that express αvβ3 integrin. Our preliminary results indicate that a novel NIR probe designed to target this integrin selectively accumulated in A549 tumor while other non-integrin specific probes were not retained in the tumor. Blocking studies show that tumor uptake of the probe is mediated by αvβ3 integrin receptor.

We have prepared and characterized several lanthanide ion complexes of multidentate ligands or chelates in an effort to develop new luminescent reporters that will be immune to autofluorescence and photobleaching. Our study has involved the characterization of various chelates of Eu, Er, and Tm with respect to relative luminescent efficiency and excited state lifetimes. Included in the list of chelates studied are TTFA, EDTA, DPA, DOTA and DTPA as well as mixed and double chelates. In addition to determining the relative efficiencies and luminescence lifetimes of the lanthanide chelates, we have explored various excitation mechanisms and determined optimum excitation wavelengths. This paper will address the various hurdles encountered in the development of this new class of reporters.

Modern hyperspectral imaging is able to collect exceptional amounts of information at astonishing speed. Reducing these data from physical fields to high-level, useful information is difficult. Integrated computational imaging (ICI) is a process in which image information is encoded as it is sensed to produce information better suited for high-speed digital processors. Both spatial and spectral features of samples can be encoded in ICI. When spectral images are simultaneously obtained and encoded at many different wavelengths, the process is called hyperspectral integrated computational imaging (HICI). Lenslet arrays and masks are ideal for encoding spatial features of an image. This process is used here to analyze motion and metabolism in freely moving rats. Complex molecular absorption filters can be used as mathematical factors in spectral encoding to create a factor-analytic optical calibration in a high-throughput spectrometer. This process is used here for remote sensing of ethanol concentrations. In this system, the molecules in the filter effectively compute the calibration function by weighting the signals received at each wavelength over a broad wavelength range. One or two molecular filters are sufficient to produce a detector voltage that is proportional to an analyte concentration in the image field. Because a single detector voltage can reveal analyte concentration, HICI is able to calculate chemical images orders of magnitude more rapidly than conventional chemometric approaches.

A current limitation of NIR imaging is the lack of sufficient tumor-to-tissue contrast due to the nonspecific nature of delivering the dye to the tumor. Utilizing one of the most important cancer signatures, the overexpression of GLUTs, we have developed a series of 2-deoxyglucose conjugated NIR dyes (NIR- 2DG) to enhance tumor selectivity. This uptake mechanism is first confirmed in vitro by confocal microscopy and flow cytometry studies with various cancer cells. Following intravenously administration to animals, NIR-2DGs are selectively accumulated in the tumor compared to the surrounding normal tissue as observed by ex vivo and in vivo fluorescence imaging techniques.

A new carbocyanine optical molecular probe with enhanced water solubility and constrained structural conformations was designed and synthesized. The near infrared (NIR) fluorescent probe contains a nonionic D-galactopyranose, which could improve water solubility of the probe and enhance uptake in tumors mediated by glucose transporter. The possibility of multiple attachment points provides the potential to conjugate diverse bioactive molecules to the probe. We developed an efficient synthetic method that is optimized for large-scale synthesis. Preliminary in vivo biodistribution studies show that the probe is rapidly cleared from blood and localize in the liver as early as 5 minutes post-injection of the probe in nude mice. Additional studies to evaluate the tumor uptake of the probe and its bioactive peptide conjugates are in progress.

Degradation of Indocyanine green (ICG) in aqueous media, limits its application in early tumor diagnosis and therapy. Thus, the objective of this study is to develop biodegradable nanoparticles entrapping ICG and to establish its effectiveness in providing overall stability to ICG. Nanoparticles entrapping ICG were engineered and characterized. The degradation kinetics of ICG in the nanoparticles was investigated in aqueous media. The degradation of ICG in aqueous nanoparticle suspension followed first-order kinetics. Nanoparticles enhanced aqueous, photo and thermal-stability of ICG.

We have recently observed both one and multicolor luminescent blinking from a variety of noble-metal nanostructures upon laser-light illumination. Our results suggest a new class of metallic probes, based on intrinsic metal luminescence, with several advantages over conventional organic fluorophores, such as enhanced photostability, higher luminescence intensity, tunable emission wavelengths and the possibilities for a variety of functional surface chemistries. Our findings for silver, gold and copper nanostructures have revealed some notable and attractive differences in their individual luminescence properties.

In recent years, a variety of Green Fluorescent Protein (GFP)-like pigments have been discovered from corals and other marine organisms. They are widely used to expand the range of available GFP-type proteins in imaging applications, such as in vivo markers for gene expression and protein localization studies, FRET-based (Förster resonance energy transfer) multicolor imaging and biosensors. They have known diverse optical and biochemical properties but their in vivo spectral properties and biological function in marine organisms is only beginning to be understood. We have investigated their spectral diversity, optical properties and cellular microstructure in corals of the Great Barrier Reef with the aim of elucidating their photo-biological function/s as well as to identify novel proteins suitable for GFP-based technologies. We found numerous spectral variants, with emissions covering almost the full range of the visible spectrum. Many of these GFP-like proteins, especially in corals from the more extreme habitats, such as sun-exposed shallows or in deep water, showed a range of light-related spectral characteristics: high photostability, spectral tuning for energy transfer and dynamic photo-induced transformation properties. Intra-cellularly they were organized into spectral donor-acceptor pairs or even arrays, tuned for FRET. Coral color proteins thus offer an exciting potential to expand the use of the available GFPs in bio-imaging applications and as a basis for improved protein engineering.