Inorganic phosphors as new luminescent labels for immunocytochemistry and time-resolved microscopy

A new strongly luminescent marker consisting of inorganic crystals is described for time-resolved microscopy. These crystals, known as phosphors, show delayed luminescence, unlike prompt fluorescent labels such as FITC, TRITC and phycobiliproteins, and are therefore potentially suitable for time-resolved microscopy. The luminescence of these phosphors is strong and non-fading in comparison to FITC/TRITC, and not significantly influenced by pH or temperature. The phosphor yttriumoxisulfide activated with europium emits maximally at 620 nm with a typical half life-time of approximately 700 musec, upon excitation with near ultraviolet light (360 nm). Phosphors for immunocytochemical staining were made by ball milling and were stabilized in suspension with polycarboxylic acids. Proteins such as avidin, protein A or immunoglobulins were allowed to adsorb to the surface of the phosphors. The immunocytochemical properties of the conjugates were evaluated in a model system of latex beads with defined surface antigens and in a cellular system containing fixed human lymphocytes or erythrocytes. Specific cytochemical staining was observed in suspension as well as on glass slides. A specially constructed time-resolved microscope was used to suppress the fast decaying fluorescence, thereby permitting visualization of the specific, slowly decaying luminescence of the phosphor label without the necessity of integration. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.     

Conjugation of lectins with fluorochromes: an approach to histochemical double labeling of carbohydrate components.

Methodical investigations on the coupling of lectins (Con A, LcL, WGA, RcA) to tetramethylrhodamine isothiocyan ate (TRITC) are reported. 20-microgram of TRITC per mg of lectin were found to be the optimal amount of TRITC for the conjugation. With this fluorochrome: protein ratio conjugates were produced which resulted in a specific and brilliant fluorescence in tissue staining. The optimally conjugated lectins were separated on DEAE-Sephadex-A 50. Using two different lectins which were conjugated with TRITC or FITC, respectively, a double labeling of different lectin-binding sites in tissue sections was achieved.     

Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy.

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different double-haptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%. To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible.     

Conjugation of lectins with fluorochromes: An approach to histochemical double labeling of carbohydrate components

Summary Methodical investigations on the coupling of lectins (Con A, LcL, WGA, RcA) to tetramethylrhodamine isothiocyanate (TRITC) are reported. 20 g of TRITC per mg of lectin were found to be the optimal amount of TRITC for the conjugation. With this fluorochrome: protein ratio conjugates were produced which resulted in a specific and brilliant fluorescence in tissue staining. The optimally conjugated lectins were separated on DEAE-Sephadex-A 50. Using two different lectins which were conjugated with TRITC or FITC, respectively, a double labeling of different lectin-binding sites in tissue sections was achieved.     

Cleavage of pig embryos after labeling with fluorescent dyes

In studies on embryonic development, treated and control ova could be co-mixed before transfer to recipients if nontoxic labels for ova were available. These experiments were conducted to determine whether pig ova would continue to cleave after being stained with the fluorochromes tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC). In the first experiment, pig ova stained with TRITC and unstained control ova were transferred into opposite oviducts of recipient gilts. In the second experiment, ova stained with TRITC and ova stained with FITC were transferred into opposite oviducts of recipient gilts. Embryos were recovered 96 h after transfer (Day 6; Day 0 = onset of estrus), the presence of fluorescence was determined, and the number of nuclei per embryo was assessed. Stained ova retained sufficient fluorochrome to permit detection until the zonae pellucidae were shed. Development of embryos stained with TRITC was equal to that of unstained control embryos. However, development of embryos stained with FITC appeared slightly retarded in comparison to that of TRITC-stained embryos. These findings demonstrate the efficacy of the fluorescent staining technique for pig ova during the first six days of pregnancy.     

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with Tcell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.     

Improved indirect fluorescence immunocytochemical method using counterstains.

Immunocytochemistry in recent years has provided powerful tools for research in neurobiology. One of the more popular techniques is the indirect fluorescence technique in which fluorescein isothiocyanate (FITC) or tetrahodamine isothiocyanate (TRITC) is used. Although widely used, this technique has two disadvantages: (1) localization may be difficult in relation to background morphology, and (2) the fluorescence fades. The study reported here describes a modification of an indirect immunocytochemical technique using FITC, TRITC and 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) which enhances localization and significantly prolongs fluorescence. Evans blue was used as a counterstain. The results show that FITC and AMCA stained cells are seen against a background of clearly distinguishable cells after counterstaining with Evans blue. However, Evans blue is not compatible with TRITC. In addition, the fluorescence life of the FITC is extended from several days to several weeks with Evans blue. These results clearly indicate that Evans blue can be used to improve indirect fluorescence immunocytochemical techniques.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.     

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.     

Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.     

Fluorescent immunoanalysis. Synthesis, spectro-fluorescent and immunologic properties of conjugates of coproporphyrin I with antibodies

The synthesis of protein conjugates with the new high-efficient fluorescent labile coproporphyrin-I was optimized. A number of conjugates of monoclonal antibodies with different coproporphyrin-I content were synthesized, and their spectral properties were studied in water and micellar solutions, i.e. adsorption, excitation and emission spectra, fluorescence quantum yields, fluorescence pH-dependences. The binding constants of coproporphyrin-I and its protein conjugates with serum albumin were determined. The antibodies labelled with coproporphyrin-I retain the functional activity and photochemically stable in water solutions. The sensitivity of fluorometric detection of coproporphyrin-I and its conjugates with proteins is more than 10 times greater than in case of FITC.     

Evaluation of glucose sensitive affinity binding assay entrapped in fluorescent dissolved‐core alginate microspheres

The feasibility of dissolved‐core alginate‐templated fluorescent microspheres as “smart tattoo” glucose biosensors was investigated in simulated interstitial fluid (SIF). The sensor works on the principle of competitive binding and fluorescence resonance energy transfer. The sensor consists of multilayer thin film coated alginate microspheres incorporating dye‐labeled glucose receptor and competing ligand within the partially dissolved alginate core. In this study, different approaches for the sensing and detection chemistry were studied, and the response of encapsulated reagents was compared with the solution‐phase counterparts. The glucose sensitivity of the encapsulated TRITC‐Con A/FITC‐dextran (500 kDa) assay in DI water was estimated to be 0.26%/mM glucose while that in SIF was observed to be 0.3%/mM glucose. The glucose sensitivity of TRITC‐apo‐GOx/FITC‐dextran (500 kDa) assay was estimated to be 0.33%/mM glucose in DI water and 0.5%/mM glucose in SIF and both demonstrated a response in the range of 0–50 mM glucose. Therefore, it is hypothesized that the calcium ion concentration outside the microsphere (in the SIF) does not interfere with the response sensitivity. The sensor response was observed to exhibit a maximum response time of 120 s. The system further exhibited a sensitivity of 0.94%/mM glucose with a response in range of 0–50 mM glucose, using near‐infrared dyes (Alexa Fluor‐647‐labeled dextran as donor and QSY‐21‐conjugated apo‐GOx as acceptor), thereby making the sensor more amenable to in vivo use, when implanted in scattering tissue. Biotechnol. Bioeng. 2009; 104: 1075–1085. © 2009 Wiley Periodicals, Inc.     

Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.     

Acidification of endocytic vesicles by an ATP-dependent proton pump

One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2- macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p- trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma- imido)triphosphate (AMP-PNP) could support acidification. The ATP- dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP- dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump.     

Solvent accessibility of the adenosine 5'-triphosphate catalytic site of sarcoplasmic reticulum CaATPase

The CaATPase of rabbit skeletal sarcoplasmic reticulum was labeled at or near the ATP catalytic site with fluoresceinyl isothiocyanate (FITC), and the accessibility of the attached probe to the bulk solvent was determined by I- quenching of its fluorescence. The quenching of free FITC was also measured. In both cases, the quenching was of the Stern-Volmer type and collisional quenching rate constants were obtained over the pH range 5-8 in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and with added Ca2+, vanadate, or phosphate. The fluorescence intensity and susceptibility to quenching by I- of free FITC were insensitive to the added ligands. In all cases, the intensity decreased with pH, as predicted from the known properties of FITC mono- and dianions. The collisional quenching rate constants increased at lower pH, as expected for I- quenching of a molecule with decreasing negative charge due to protonation. When FITC was attached to the CaATPase, the FITC fluorescence intensity and I- collisional quenching rate constants were sensitive to ligand binding as well as pH. The changes in fluorescence intensity with acidity, when compared to the results for free FITC, indicated the pKa of the FITC was reduced 0.6 unit when it was attached to the CaATPase. Excited-state lifetime measurements indicated that ligand effects at constant pH were not due to protonation-induced changes in FITC quantum yield but to conformational changes of the CaATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

Intralysosomal accumulation of polyanions. I. Fusion of pinocytic and phagocytic vacuoles with secondary lysosomes

The long-term exposure of macrophages to low concentrations of a number of polyanions leads to their accumulation in high concentration within secondary lysosomes. This was associated with enlargement of the lysosomes, the presence of membranous whorls, and intense toluidine blue staining of the organelles at pH 1.0. After the ingestion of a particulate load by these cells, newly formed phagocytic vacuoles failed to fuse with polyanion-laden lysosomes. The lack of fusion was evident in both fluorescence and electron micrographic studies which followed the transfer of acridine orange or Thorotrast from 2 degrees lysosomes to phagosomes. Agents that inhibited phagosome-lysosome (P-L) fusion included molecules containing high densities of sulfate, sulfonate, or carboxylate residues. Dextran sulfate (DS) in microgram/ml quantities was an excellent inhibitor, whereas nonsulfated dextran (D) was without effect at 1,000-fold higher concentrations. In contrast to their effects on P-L fusion, polyanions failed to influence the fusion of pinocytic vesicles with 2 degrees lysosomes. The uptake, intravacuolar distribution, and intralysosomal digestion of fluid-phase pinocytic markers were unaltered in lysosomes containing either D or DS. Furthermore, subcellular fractionation studies showed that the fluid-phase pinocytic marker HRP was efficiently transferred from pinosomes to large, dense 2 degrees lysosomes containing DS.     

Evaluation of Fluorescein Isothiocyanate-labeled Whole Antiserum in the Immunofluorescent Identification of Microorganisms

Portions of a whole antiserum to Histoplasma capsulatum were reacted with amounts of fluorescein isothiocyanate (FITC) that ranged from 50 to 400 mug/mg of protein. Portions of the globulin from the same antiserum were reacted with amounts of FITC that ranged from 12.5 to 50 mug of FITC per mg of protein. The globulin conjugates (postlabeled globulins), the whole serum conjugates, and the globulins from the whole serum conjugates (prelabeled globulins) were compared with respect to their fluorescein-protein (F:P) ratios and fluorescent-antibody (FA) activities. The whole serum sample treated with 50 mug of FITC per mg of protein was least reactive in FA tests, and its globulin had the lowest F:P. All other conjugates had globulins with F:P ratios that were considered to be adequate for high FA activity. It was found, however, that the prelabeled globulins were considerably less reactive than the postlabeled globulins or the whole serum conjugates. A larger amount of brightly staining reagent per milliliter of original serum could be obtained from labeled whole serum than from postlabeled globulin. Lissamine-rhodamine conjugated to bovine serum albumin (LRBSA) was evaluated as a counterstain to be used in conjunction with FITC-labeled whole antisera. The counterstain was effective in masking nonspecific FITC fluorescence in Formalin-fixed tissues and in culture smears of fungi. Masking was incomplete in culture smears of a bacterium and in blood smears containing a protozoan.     

A continuous fluorescent assay for measuring protease activity using natural protein substrate.

A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. Beta-casein labeled with both N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and fluorescein isothiocyanate (FITC) is used as the substrate in this assay. The effect of proteolysis of the substrate is a reduction of the intermolecular energy transfer from DACM to FITC. The caseinolytic activity is then monitored by the fluorescence increase. The activities of both proteases obey Michaelis-Menten kinetics with Km = 1.6 +/- 0.2 microM for trypsin and Km = 13.2 +/- 1.9 microM for transin. Protease concentrations as low as 10 ng/mL can be utilized. The pH dependence of the caseinolytic activity has been determined for both enzymes.