Quantum dots having four different surface coatings were tested for use in in vivo imaging. Localization was successfully monitored by fluorescence imaging of living animals, by necropsy, by frozen tissue sections for optical microscopy, and by electron microscopy, on scales ranging from centimeters to nanometers, using only quantum dots for detection. Circulating half-lives were found to be less than 12 min for amphiphilic poly(acrylic acid), short-chain (750 Da) methoxy-PEG or long-chain (3400 Da) carboxy-PEG quantum dots, but approximately 70 min for long-chain (5000 Da) methoxy-PEG quantum dots. Surface coatings also determined the in vivo localization of the quantum dots. Long-term experiments demonstrated that these quantum dots remain fluorescent after at least four months in vivo.

A series of new fluorescent labeling reagents based on sulfoindocyanine dyes has been developed. We describe the synthesis and properties of these reagents. They contain succinimidyl ester reactive groups and can be readily conjugated to antibodies, avidin, DNA, lipids, polymers, and other amino-group-containing materials. The labeling reagents are water soluble, pH insensitive, and show much reduced dye aggregation under labeling conditions. One of the reagents, Cy3, can be excited with the 488-, 514- and 532-nm laser lines and is optimally excited with the 546-nm mercury arc line. Another, Cy5, can be excited with the 633-nm HeNe and 647-nm Kr laser lines available with many flow cytometers and confocal laser-scanning microscopes. New laser diodes emitting near 650 nm should also be excellent excitation sources for Cy5.

This article describes an in vivo imaging method for visualizing and quantifying a specific cell population. Cells are labeled ex vivo with a perfluoropolyether nanoparticle tracer agent and then detected in vivo using 19

We demonstrate that quantum dots injected into two model tumors rapidly migrate to sentinel lymph nodes. PEG-coated quantum dots having terminal carboxyl, amino, or methoxyl groups all migrated from the tumor to surrounding lymph nodes similarly. Passage from the tumor through lymphatics to adjacent nodes could be visualized dynamically through the skin; at least two nodes could usually be defined. Imaging during necropsy confirmed confinement of the quantum dots to the lymphatic system and demonstrated easy tagging of sentinel lymph nodes for pathology. Examination of the sentinel nodes identified by quantum dot localization showed that at least some contained metastatic tumor foci.

We have previously used a gene-transfer scheme to isolate a human genomic DNA fragment that determines expression of a GDP-L-fucose:I3-D-galactoside 2-a-Lfucosyltransferase [a(1,2)FT; EC 2.4.1.69]. Although this fragment determined expression of an a(1,2)FT whose kinetic properties mirror those of the human H blood group a(1,2)FT, their precise nature remained undefined. We describe here the molecular cloning, sequence, and expression of a human cDNA corresponding to these human genomic sequences. When expressed in COS-1 cells, this cDNA directs expression of cell surface H structures and a cognate a(1,2)FT activity with properties analogous to the human H blood group a(1,2)FI. The cDNA sequence predicts a 365-amino acid polypeptide characteristic of a type II transmembrane glycoprotein with a domain structure analogous to that of other glycosyltransferases but without significant primary sequence similarity to these or other known proteins. To directly demonstrate that the cDNA encodes an a(1,2)FT, the COOH-terminal domain predicted to be Golgi-resident was expressed in COS-1 cells as a catalytically active, secreted, and soluble protein A fusion peptide. Southern blot analysis showed that this cDNA identifies DNA sequences syntenic to the human H locus on chromosome 19. These results strongly suggest that this cloned a(1,2)FI cDNA represents the product of the human H blood group locus.The antigens of the human ABO blood group system are carbohydrate molecules constructed by the sequential action ofa series of distinct glycosyltransferases (1,2 (Gal, moieties (1).Surface-expressed H determinants exhibit precise temporal and spatial changes in their expression patterns during human and murine development (4, 5). The functional significance of these changes is as yet unknown, although evidence suggests that other fucosylated molecules participate in adhesive events during development (6-8). Cloned gene segments that determine H antigen expression represent tools to address this question by genetic approaches that perturb H antigen expression during development. We, therefore, established a gene-transfer approach to isolate human DNA segments that determine expression of cell surface H molecules and their corresponding a(1,2)FTs (9, 10). These experiments yielded a cloned human DNA segment that determines expression ofan a(1,2)FT activity when transfected into a mammalian cell line deficient in this enzyme activity. This enzyme activity was kinetically similar to the human H blood group a(1,2)FT but distinct from the human secretor (SE) a(1,2)FT. Although these data were consistent with the hypothesis that this segment represented part or all of the structural gene encoding the H a(1,2)FT, they were consistent also with the possibility that the DNA sequences trans-determined enzyme expression by interaction with an endogenous gene, transcript, or protein. We report here our analysis of a cloned cDNA representing the product of this human genomic DNA segment. § These data indicate that this segment encode...

The phytochrome family of red/far-red photoreceptors has been optimized to support photochemical isomerization of a bound bilin chromophore, a process that triggers a conformational change and modulates biochemical output from the surrounding protein scaffold. Recent studies have established that the efficiency of this photochemical process is profoundly altered by mutation of a conserved tyrosine residue (Tyr176) within the bilin-binding GAF domain of the cyanobacterial phytochrome Cph1 [Fischer, A. J., and Lagarias, J. C. (2004) Harnessing phytochrome's glowing potential, Proc. Natl. Acad. Sci. U.S.A. 101, 17334-17339]. Here, we show that the equivalent mutation in plant phytochromes behaves similarly, indicating that the function of this tyrosine in the primary photochemical mechanism is conserved. Saturation mutagenesis of Tyr176 in Cph1 establishes that no other residue can support comparably efficient photoisomerization. The spectroscopic consequences of Tyr176 mutations also reveal that Tyr176 regulates the conversion of the porphyrin-like conformation of the bilin precursor to a more extended conformation. The porphyrin-binding ability of the Tyr176Arg mutant protein indicates that Tyr176 also regulates the ligand-binding specificity of apophytochrome. On the basis of the hydrogen-bonding ability of Tyr176 substitutions that support the nonphotochemical C15-Z,syn to C15-Z,anti interconversion, we propose that Tyr176 orients the carboxyl side chain of a conserved acidic residue to stabilize protonation of the bilin chromophore. A homology model of the GAF domain of Cph1 predicts a C5-Z,syn, C10-Z,syn, C15-Z,anti configuration for the chromophore and implicates Glu189 as the proposed acidic residue stabilizing the extended conformation, an interpretation consistent with site-directed mutagenesis of this conserved acidic residue.

We review recent progress in tumor imaging in vivo using fluorescent tags, highlight the problems of fluorescence imaging in small animals, discuss recent advances in near-infrared fluorochromes and quantum dots, and point to some future possibilities. GFP-based fluorescence imaging is briefly discussed. The authors believe that improvements in near-infrared fluorochromes are required to enable practical imaging in tissues at centimeter depths.

Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytornetry 1011-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.Key terms: Fluorescent biolabeling probes, fluorescent cyanine dyes, succinimidyl ester biolabeling probes, twocolor immunofluorescent imaging, longwavelength fluorescent probes, flow cytometry, chromophore equivalent weight Earlier papers from this laboratory (4,111 have described the preparation of two series of cyanine and merocyanine dyes which are useful as fluorescent labeling reagents for biological investigations. Functional groups utilized in these reagents for covalent linking to biomolecules have been the iodoacetamido group, for sulfhydryl tagging, and the isothiocyanato moiety, for attachment to primary or secondary amino groups. The incorporation of carboxylic acid groups into the basic cyanine structure was expected to permit fluorescent labeling through the use of derived active esters. The synthesis of (2,3,3-trimethyl-3-H-indol-5-yl)-acetic acid and derived intermediates suitable for the construction of highly fluorescent carboxyl-containing indocyanine dyes has been described recently (12).Here we describe the preparation of the dyes themselves, the procedures used for converting the carboxylic acid groups into active ester functions (esters of N-hydroxysuccinimide) which are reactive toward proteins, and procedures for attaching these dyes to various proteins. Advantages which characterize reagents in this group include ease of preparation, higher activity toward protein substrates in aqueous media under mild conditions, and the high extinction coefficients and quantum yields required for intense fluorescence. As with the other dyes treated in this series of papers, the fluorescence emission maxima are to be found from the long-wavelength portion of the visible range into the near infrared, well separated from the shorterwavelength autofluorescence which is characteristic of many biological specimens and which presents a difficulty in working with such fluorophores as fluorescein.