Determination of optimal rhodamine fluorophore for in vivo optical imaging

Optical imaging has the potential to improve the efficacy of surgical and endoscopic approaches to cancer treatment; however, the optimal type of fluorescent probe has not yet been established. It is well-known that rhodamine-core-derived fluorophores offer a combination of desirable properties such as good photostability, high extinction coefficient, and high fluorescence quantum yield. However, despite the ubiquitous use of rhodamine fluorophores for in vivo optical imaging, it remains to be determined if unique chemical properties among individual rhodamine core family members affect fluorophore parameters critical to in vivo optical imaging applications. These parameters include preserved fluorescence intensity in low pH environments, similar to that of the endolysosome; efficient fluorescence signal despite conformational changes to targeting proteins as may occur in harsh subcellular environments; persistence of fluorescence after cellular internalization; and sufficient signal-to-background ratios to permit the identification of fluorophore-targeted tumors. In the present study, we conjugated 4 common rhodamine-core based fluorescent dyes to a clinically feasible and quickly internalizing D-galactose receptor targeting reagent, galactosamine serum albumin (GmSA), and conducted a series of in vitro and in vivo experiments using a metastatic ovarian cancer mouse model to determine if differences in optical imaging properties exist among rhodamine fluorophores and if so, which rhodamine core possesses optimal characteristics for in vivo imaging applications. Herein, we demonstrate that the rhodamine-fluorophore, TAMRA, is the most robust of the 4 common rhodamine fluorophores for in vivo optical imaging of ovarian cancer metastases to the peritoneum.     

Galactosyl Human Serum Albumin-NMP1 Conjugate: A Near Infrared (NIR)-Activatable Fluorescence Imaging Agent to Detect Peritoneal Ovarian Cancer Metastases

Patient survival depends on the completeness of resection of peritoneal ovarian cancer metastases (POCM), and therefore, it is important to develop methods to enhance detection. Previous probe designs based on activatable galactosyl human serum albumin (hGSA)-fluorophore pairs, which target lectin receptors expressed on POCM, have used only visible range dyes conjugated to hGSA. However, imaging probes emitting fluorescence in the NIR range are advantageous because NIR photons have deeper in vivo tissue penetration and result in lower background autofluorescence than those emitting in the visible range. A NIR-activatable hGSA fluorophore was synthesized using a bacteriochlorin-based dye, NMP1. NMP1 has two unique absorption peaks, one in the green range and the other in the NIR range, but emits at a NIR peak of 780 nm. NMP1, thus, has two different Stokes shifts that have the potential to allow imaging of POCM both at the peritoneal surface and just below it. hGSA was conjugated with 2 NMP1 molecules to create a self-quenching complex (hGSA-NMP1). The activation ratio of hGSA-NMP1 was measured by the fluorescence intensity before and after exposure to 10% SDS. The activation ratio of hGSA-NMP1 was ～100-fold in vitro. Flow cytometry, fluorescence microscopy, and in vivo spectral fluorescence imaging were carried out to compare hGSA-NMP1 with hGSA-IR800 and hGSA-ICG (two always-on control agents with similar emission to NMP1) in terms of comparative fluorescence signal and the ability to detect POCM in mice models. The sensitivity and specificity of hGSA-NMP1 for POCM implant detection were determined by colocalizing NMP1 emission spectra with red fluorescent protein (RFP) expressed constitutively in SHIN3 tumor implants at different depths below the peritoneal surface. In vitro, SHIN3 cells were easily detectable after 3 h of incubation with hGSA-NMP1. In vivo submillimeter POCM foci were clearly detectable with spectral fluorescence imaging using hGSA-NMP1. Among 555 peritoneal lesions, hGSA-NMP, using NIR and green excitation light, respectively, detect 75% of all lesions and 91% of lesions ～0.8 mm or greater in diameter. Few false positives were encountered. Nodules located at a depth below the small bowel surface were only depicted with hGSA-NMP1. We conclude that hGSA-NMP1 is useful in imaging peritoneal ovarian cancer metastases, located both superficially and deep in the abdominal cavity.     

Biomedical applications of fluorescence imaging in vivo

Optical imaging can advance knowledge of cellular biology and disease at the molecular level in vitro and, more recently, in vivo. In vivo optical imaging has enabled real-time study to track cell movement, cell growth, and even some cell functions. Thus, it can be used in intact animals for disease detection, screening, diagnosis, drug development, and treatment evaluation. This review includes a brief introduction to fluorescence imaging, fluorescent probes, imaging devices, and in vivo applications in animal models. It also describes a quantitative fluorescence detection method with a reconstruction algorithm for determining the location of fluorophores in tissue and addresses future applications of in vivo fluorescence imaging.     

Avidin-dendrimer-(1B4M-Gd)(254): a tumor-targeting therapeutic agent for gadolinium neutron capture therapy of intraperitoneal disseminated tumor which can be monitored by MRI.

Peritoneal carcinomatosis is a late stage in cancer progress, for which no effective therapeutic modality exists. Targeting therapeutic agents to disseminated lesions may be a promising modality for treating peritoneal carcinomatosis. Gadolinium ((157,155)Gd) is known to generate Auger and internal conversion electrons efficiently by irradiation with a neutron beam. Auger electrons from neutron-activated Gd(III) are strongly cytotoxic, but only when Gd(III) atoms have been internalized into the cells. In the present investigation, we have developed a quickly internalizing tumor-targeting system to deliver large quantities of Gd(III) atoms into tumor cells to generate the Auger emission with an external neutron beam. Simultaneously, one would be able to image its biodistribution by MRI with a shortened T1 relaxation time. Avidin-G6-(1B4M-Gd)(254) (Av-G6Gd) was synthesized from generation-6 polyamidoamine dendrimer, biotin, avidin, and 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M). The Av-G6Gd was radiolabeled with Gd(III) doped with (153)Gd. All of the 1B4M's on the conjugate were fully saturated with Gd(III) atoms. An in vitro internalization study showed that Av-G6Gd accumulated and was internalized into SHIN3 cells (a human ovarian cancer) 50- and 3.5-fold greater than Gd-DTPA (Magnevist) and G6-(1B4M-Gd)(256) (G6Gd). In addition, accumulation of Gd(III) in the cells was detected by the increased signal on T1-weighted MRI. A biodistribution study was performed in nude mice bearing intraperitoneally disseminated SHIN3 tumors. Av-G6Gd showed specific accumulation in the SHIN3 tumor (103% ID/g) 366- and 3.4-fold greater than Gd-DTPA (0.28% ID/g, p < 0.001) and G6Gd (30% ID/g, p < 0.001) 1 day after i.p. injection. Seventy-eight percent of the tumor-related radioactivity of Av-G6Gd in the SHIN3 tumor was located inside the cells. The SHIN3 tumor-to-normal tissue ratio was greater than 17:1 in all organs and increased up to 638:1 at 1 day after i.p. injection. In conclusion, a sufficient amount (162 ppm) of Av-G6Gd was accumulated and internalized into the SHIN3 cells both in vitro and in vivo to kill the cell using (157/155)Gd with external irradiation with an appropriate neutron beam while monitoring with MRI. Thus, Av-G6Gd may be a promising agent for Gd neutron capture therapy of peritoneal carcinomatosis. This reagent also has the potential to permit monitoring of its pharmacokinetic progress with MRI.     

Fluorescence imaging in vivo: recent advances

In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.     

Lysosome imaging in cancer cells by pyrene-benzothiazolium dyes: An alternative imaging approach for LAMP-1 expression based visualization methods to avoid background interference

A series of pyrene-benzothiazolium dyes (1a–1d) were experimentally investigated to study their internalization mechanism into cellular lysosomes as well as their potential imaging applications for live cell imaging. The lysosome selectivity of the probes was further compared by using fluorescently tagged lysosome associated membrane protein-1 (LAMP-1) expression-dependent visualization in both normal (COS-7, HEK293) and cancer (A549, Huh 7.5) cell lines. These probes were successfully employed as reliable lysosome markers in tumor cell models, thus providing an attractive alternative to LAMP-1 expression-dependent visualization methods. One advantage of these probes is the elimination of significant background fluorescence arising from fluorescently tagged protein expression on the cell surface when cells were transfected with LAMP-1 expression plasmids. Probes exhibited remarkable ability to stain cellular lysosomes for long-term experiments (up to 24 h) and the highly lipophilic nature of the probe design allowed their accumulation in hydrophobic regions of the cellular lysosomes. Experimental evidences indicated that the probes are likely to be internalized into lysosomes via endocytosis and accumulated in the hydrophobic regions of the lysosomes rather than in the acidic lysosomal lumen. These probes also demonstrated significant stability and lysosome staining for fixed cell imaging applications as well. Lastly, the benzothiazolium moiety of the probes was identified as the key component for lysosome selectivity.     

Simultaneous Detection of Local Polarizability and Viscosity by a Single Fluorescent Probe in Cells

Many intracellular reactions are dependent on the dielectric (“polarity”) and viscosity properties of their milieu. Fluorescence imaging offers a convenient strategy to report on such environmental properties. Yet, concomitant and independent monitoring of polarity and viscosity in cells at submicron scale is currently hampered by the lack of fluorescence probes characterized by unmixed responses to both parameters. Here, the peculiar photophysics of a green fluorescent protein chromophore analog is exploited for quantifying and imaging polarity and viscosity independently in living cells. We show that the polarity and viscosity profile around a novel hybrid drug-delivery peptide changes dramatically upon cell internalization via endosomes, shedding light on the spatiotemporal features of the release mechanism. Accordingly, our fluorescent probe opens the way to monitor the environmental effects on several processes relevant to cell biochemistry and nanomedicine.     

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.     

Live cell imaging: approaches for studying protein dynamics in living cells

In the last decade, the long-standing biologist's dream of seeing the molecular events within the living cell came true. This technological achievement is largely due to the development of fluorescence microscopy technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed us to determine the subcellular localization, mobility and transport pathways of specific proteins and even visualize protein-protein interactions of single molecules in living cells. Direct observation of such molecular dynamics can provide important information about cellular events that cannot be obtained by other methods. Thus, imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and cellular functions. In this special issue of review articles, we review various imaging technologies of microscope hardware and fluorescent probes useful for cell biologists, with a focus on recent development of live cell imaging.     

Preparation and preliminary evaluation of a biotin-targeted, lectin-targeted dendrimer-based probe for dual-modality magnetic resonance and fluorescence imaging

A novel approach for the preparation of a biotinylated dendrimer-based MRI agent 5 is described, in which a unique disulfide bond in the core of the Gd(III)-1B4M-DTPA chelated G2 PAMAM dendrimer was reduced and then attached to a maleimide-functionalized biotin. The new MRI agent 5 features a well-defined dendron structure and a unique biotin functionality. Immobilization of up to four copies of biotinylated dendrimer 5 to fluorescently labeled avidin yields a supramolecular avidin-biotin-dendrimer-Gd(III) complex. Validation of the complex in mice bearing ovarian cancer tumors demonstrates that the avidin-biotin-dendrimer targeting system efficiently targets and delivers sufficient amounts of chelated Gd(III) and fluorophores (e.g., Rhodamine green) to ovarian tumors to produce visible changes in the tumors by both MRI and optical imaging, respectively. Thus, the avidin-biotin-dendrimer complex may be used as a tumor-targeted probe for dual-modality magnetic resonance and fluorescence imaging.     

Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation

Two novel taspine diphenyl derivatives (Ta‐dD) were designed and synthesized by introducing different coumarin fluorescent groups into the basic structure of Ta‐dD. The main advantage of these two compounds is that they can be used as fluorescence probes and inhibitors simultaneously. In the present study, the fluorescent properties of the probes were measured and their inhibition of four breast cancer cell lines was tested. Different concentrations of the fluorescence probe were added to MCF‐7 breast cancer cells for fluorescence imaging analysis under normal conditions. The results suggested that both of the new compounds have not only fluorescence but also the ability to inhibit effects on different breast cancer cell lines, which indicates their possible further use as dual functional fluorescence probes in tracer analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Endocytosis and intracellular fate of liposomes using pyranine as a probe

Lipid vesicles (liposomes) containing pH-sensitive fluorophores were used as probes for the study of liposome entry and intracellular fate. Pyranine [8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)] was entrapped in the liposome aqueous core during preparation to provide a means of detecting internalization into living cells. HPTS is highly water soluble and shows a strong pH-dependent shift in its fluorescence excitation spectrum. Fluorescence emission (FEM) is slightly pH dependent with excitation (lambda EX) at 350-415 nm but highly pH dependent with lambda EX at 450 nm. Liposomes bearing a net negative charge bound rapidly to CV-1 cells and underwent endocytosis. One hour after liposome addition, high FEM with lambda EX at 413 nm and low FEM with lambda EX at 450 nm suggest that most cell-associated liposomes had been internalized and resided at a mean pH of approximately 6.6. Collapse of cellular H+ gradients with NH4Cl or monensin treatment rapidly and reversibly increased FEM with lambda EX at 450 nm. Direct examination by fluorescence microscopy corroborates the fluorometric data on internalization; over time, FEM remained high with lambda EX at 350-405 nm but decreased with lambda EX at 450-490 nm, showing that all lipid vesicles were internalized within 40 min at 37 degrees C. Acidification of intracellular liposomes increased over 3 h, reaching a minimum value of approximately pH 5.5. HPTS persisted within acidic cellular vesicles for 2-3 days, and cytoplasmic dye was observed infrequently, suggesting that liposome fusion with cellular membranes seldom occurs. Material delivered to the endocytic pathway via lipid vesicles labeled an assortment of intracellular organelles of varying motility and morphology, including dynamic tubular structures whose lumen is acidic.     

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.     

Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells

A crucial step in the immune response is the binding of antigenic peptides to major histocompatibility complex (MHC) proteins. Class II MHC proteins present their bound peptides to CD4(+) T cells, thereby helping to activate both the humoral and the cellular arms of the adaptive immune response. Peptide loading onto class II MHC proteins is regulated temporally, spatially and developmentally in antigen-presenting cells. To help visualize these processes, we have developed a series of novel fluorogenic probes that incorporate the environment-sensitive amino acid analogs 6-N,N-dimethylamino-2-3-naphthalimidoalanine and 4-N,N-dimethylaminophthalimidoalanine. Upon binding to class II MHC proteins these fluorophores show large changes in emission spectra, quantum yield and fluorescence lifetime. Peptides incorporating these fluorophores bind specifically to class II MHC proteins on antigen-presenting cells and can be used to follow peptide binding in vivo. Using these probes we have tracked a developmentally regulated cell-surface peptide-binding activity in primary human monocyte-derived dendritic cells.     

Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10 min at 4 °C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37 °C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90 min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.     

Three-dimensional in vivo imaging of green fluorescent protein-expressing T cells in mice with noncontact fluorescence molecular tomography

Given that optical tomography is capable of quantitatively imaging the distribution of several important chromophores and fluorophores in vivo, there has been a great deal of interest in developing optical imaging systems with increased numbers of measurements under optimal experimental conditions. In this article, we present a novel system that enables three-dimensional imaging of fluorescent probes in whole animals using a noncontact setup, in parallel with a three-dimensional surface reconstruction algorithm. This approach is directed toward the in vivo imaging of fluorophore or fluorescent protein concentration in small animals. The system consists of a rotating sample holder and a lens-coupled charge-coupled device camera in combination with a fiber-coupled laser scanning device. By measuring multiple projections, large data sets can be obtained, thus improving the accuracy of the inversion models used for quantitative three-dimensional reconstruction of fluorochrome distribution, as well as facilitating a higher spatial resolution. In this study, the system was applied to determining the distribution of green fluorescent protein (GFP)-expressing T lymphocytes in a transgenic mouse model, thus demonstrating the potential of the system for studying immune system function. The technique was used to image and reconstruct fluorescence originating from 32 x 10(6) T cells in the thymus and 3 x 10(5) T cells in the spleen.     

Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation

Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11):2439–2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.     

Far-Red Fluorescent Protein Excitable with Red Lasers for Flow Cytometry and Superresolution STED Nanoscopy

Far-red fluorescent proteins are required for deep-tissue and whole-animal imaging and multicolor labeling in the red wavelength range, as well as probes excitable with standard red lasers in flow cytometry and fluorescence microscopy. Rapidly evolving superresolution microscopy based on the stimulated emission depletion approach also demands genetically encoded monomeric probes to tag intracellular proteins at the molecular level. Based on the monomeric mKate variant, we have developed a far-red TagRFP657 protein with excitation/emission maxima at 611/657 nm. TagRFP657 has several advantages over existing monomeric far-red proteins including higher photostability, better pH stability, lower residual green fluorescence, and greater efficiency of excitation with red lasers. The red-shifted excitation and emission spectra, as compared to other far-red proteins, allows utilizing TagRFP657 in flow cytometry and fluorescence microscopy simultaneously with orange or near-red fluorescence proteins. TagRFP657 is shown to be an efficient protein tag for the superresolution fluorescence imaging using a commercially available stimulated emission depletion microscope.     

Highly sensitive detection of hybridization of oligonucleotides to specific sequences of nucleic acids by application of fluorescence resonance energy transfer

We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light.     

Fluorescent membrane probes incorporating dipyrrometheneboron difluoride fluorophores.

The spectroscopic properties of a new series of fatty acid analogs in which a dipyrrometheneboron difluoride fluorophore forms a segment of the acyl methylene chain are presented and their characteristics as fluorescent membrane probes are examined. When incorporated as a low mole fraction component in model phospholipid membranes, the probes retain the principal characteristics of the parent fluorophore: green fluorescence emission with high quantum yield, extensive spectral overlap, and low environmental sensitivity. The fluorescence quantum yield is typically two to three times that of comparable membrane probes based on the nitrobenzoxadiazole fluorophore. The spectral overlap results in a calculated F?rster energy transfer radius (Ro) of about 57 A. Consequently, increasing fluorescence depolarization and quenching are observed as the mole fraction of the probe species incorporated in the membrane is increased. Low environmental sensitivity is manifested by retention of high quantum yield emission in aqueous dispersions of fatty acids. Partition coefficient data derived from fluorescence anisotropy measurements and iodide quenching experiments indicate that in the presence of fluid phase phospholipid bilayers the aqueous fraction of fatty acid is very small. Fluorescence intensity and anisotropy responses to phospholipid phase transitions are examined and found to be indicative of nonrandom fluorophore distribution in the gel phase. It is concluded that the spectroscopic properties of the fatty acid probes and their phospholipid derivatives are particularly suited to applications in fluorescence imaging of cellular lipid distribution and membrane level studies of lateral lipid segregation.