Scanning Fluorometer Research Articles

A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176€, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I2S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled high-accuracy temperature management in PCR tubes (±0.1 °C) allowing us to perform high-resolution thermal shift assays (TSA) from exemplary biological applications.

Firefly Luciferase-Based Sequential Bioluminescence Resonance Energy Transfer (BRET)-Fluorescence Resonance Energy Transfer (FRET) Protease Assays.

We describe here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyzes yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-Infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. This method successfully employs an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence to assay physiologically important protease activities and should be generally applicable to the measurement of any endoprotease lacking accessible cysteine residues.

Impaired Platelet Attack by fibronectin Binding Partner Msb2 of Candida Albicans

AbstractAbstract 3299 Introduction:Platelets can bridge innate and adaptive immunity. To attack microorganisms, platelets store microbicidal and immune stimulatory proteins in their α-granules. Platelet integrins and their plasma ligands can govern both binding of the pathogens and activation of platelets with subsequent secretion of their granular constituents. Fibronectin, a large dimeric glycoprotein, with specific binding sites for platelet integrins (α5β1, αvβ3, and αIIbβ3) and collagens, circulates in plasma and is a component of the extracellular matrix. C. albicans can also bind fibronectin. The ability to germinate and grow in hyphae is a relevant factor of virulence in vivo. The integrity of the C. albicans cell surface during filamentation is sensed by Msb2, a transmembrane protein. Recently, it has been shown that Msb2 can be shedded, thus providing a putative mechanism of C. albicans to escape from the host attack. We now hypothesized that Msb2 has a binding site for plasma fibronectin and that shedded (soluble) Msb2 can protect C. albicans from opsonization by fibronectin. Material and methods:Fibronectin purified from human fresh frozen plasma by gelatin-sepharose affinity column was labeled with alexa fluor 488 according the manufacturer’s instructions. To induce germination, C. albicans was cultured in complete medium (yeast extract, peptone, and dextrose) at 30°C, washed and starved for 1 h at room temperature before dilution in PBS supplemented with fetal calf serum (10 %) to a final optical density of 0.4 at 37°C. Msb2 heme agglutinin (HA)-tagged purified by affinity chromatography using anti-HA antibody-coupled agarose beads was kindly provided by J. Ernst (Szafranski-Schneider E, PLoS Pathog. 2012;8(2):e1002501). C. albicans yeast and hyphae forms or plates (24 wells) coated with 10 μg/ml purified HA-tagged Msb2 or 10μg/ml unlabeled fibronectin, and blocked with 1% BSA were incubated with various concentrations fibronectin-alexa fluor 488 for 60 min or 120 min. Flow cytometry (FACS, Beckman Coulter), confocal laser scanning microscope (LSM, Zeiss) and fluorometer (Fluoroskan Ascent, Thermo Scientific) were used to measure the fluorescent fibronectin. Results:Binding of 5, 10, 50, and 100 μg/ml fibronectin to 10 μg/ml immobilized Msb2 revealed saturation at 10 μg/ml fibronectin. Immobilized Msb2 bound 2.6 fold more fluorescent fibronectin than BSA (p=0.016). Immobilized fibronectin bound 30 % more fluorescent fibronectin than Msb2 and significantly more than BSA (p<0.01). C. albicans germination over time at 30 min, 60 min, and 120 min generated 5–10 %, 40 %, and 90 % hyphae, as determined by microscopy and confirmed by flow cytometry. Induction of C. albicans hyphae enhanced binding of fibronectin (40 μg/ml) from 1.7 % positive yeast cells (baseline), 6 % at 30 min, 11.7 % at 60 min, and 12.6 % at 120 min (p<0.05). As shown by LSM, C. albicans germ tubes are a main target for fluorescent fibronectin. Germination of C. albicans yeast form increased binding of platelets up to fivefold after 30 min and twentyfold after 60 min (p<0.05). Moreover, pre-treatment of C. albicans hyphae with fibronectin (40 μg/ml) enhanced the binding of platelets caused by integrin αIIbβ3 fibronectin interaction. Abciximab (2 μg/ml), a specific αIIbβ3 antagonist, blocked binding of platelets to C. albicans opsonized by fibronectin. Binding of fluorescent fibronectin was not homogeneously distributed on the cell surface of C. albicans and upon addition of soluble Msb2 binding of fluorescent fibronectin diminished as documented by LSM. Conclusion:Hyphae of C. albicans, a relevant factor of virulence, are attacked by platelets. Fibronectin, a ligand of the platelet integrins, opsonizes the hyphae of C. albicans and fosters platelet binding to C. albicans. Hence, platelets and fibronectin, with its intrinsic multivalent binding sites to collagen and platelet integrins (α5β1, αvβ3, and αIIbβ3) can support the platelet-mediated host defense against C. albicans. Msb2 is a novel fibronectin binding protein. Binding of fibronectin to C. albicans is impaired in the presence of soluble Msb2. Hence, shedding of Msb2 in vivo can protect C. albicans against the platelet-mediated host attack. Disclosures:No relevant conflicts of interest to declare.

Retroviral Integrases Promote Fraying of Viral DNA Ends

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.

A Preliminary Study of Silicone Hydrogel Lens Material and Care Solution Bioincompatibilities

The study purpose was to evaluate corneal barrier function and staining relative to potential bioincompatibilities. This was a randomized double-masked study (n = 25 subjects). Three lens material-care solution combinations were tested: (1) lotrafilcon B/polyhexamethylene biguanide (PHMB)-based multipurpose (MPS) solution (MPS-1); (2) lotrafilcon B/polyquaternium-1 and myristamidopropyl dimethylamine-based solution (MPS-2); and (3) lotrafilcon B and another PHMB-based solution (MPS-3). Saline served as the control. New lenses were soaked in the preserved solutions or saline and then worn for 2 hours before corneal measurements. Barrier function was characterized by the fluorescein penetration rate, corneal amount, both measured with an objective scanning fluorometer. The dye penetration rate ratio, test to control; amount ratio, test to control, and corneal staining were evaluated. The mean rate ratios (± SD) for the combinations were 2.98 (± 3.04), 1.23 (± 1.01), and 1.83 (± 1.77) for MPS-1, MPS-2, and MPS-3 solutions, respectively. Significant ratio differences were found across regimens (P = 0.007); for MPS-1 compared with baseline (P = 0.031) and for MPS-1 compared with MPS-2 (P = 0.007). The statistical results for staining were similar. Use of an objective quantitative physiological method suggests that significant differences in lens solution bioincompatibilities occur that mirror corneal staining data relative to corneal compromise.

Sequential bioluminescence resonance energy transfer–fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein

We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)–fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET–FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.

Sodium Effects on Calcium Dynamics in Mitochondrial ion Circuits

Mitochondrial Ca2+ is modulated by the mitochondrial Ca2+ uniporter(mCU) and the mitochondrial Na+/Ca2+ exchanger(mNCE) to influence NADH production through Ca2+-sensitive dehydrogenases. Alterations in cytosolic Na+ will significantly impact the rate of mitochondrial Ca2+ accumulation by changing the driving force for Ca2+ efflux through mNCE when the amplitude or frequency of the cytosolic Ca2+ transient changes. In the present study, a protocol to selectively measure Ca2+ influx and efflux rates from guinea pig heart mitochondria was developed by utilizing an extra-mitochondrial Ca2+ indicator(CaGreen) and the inhibitors Ru360 and CGP-37157, while varying extramitochondrial free Ca2+ from 2 to 20μM, and extramitochondrial Na+ from 0 to 60mM. The extra-mitochondrial Ca2+, mitochondrial membrane potential, NADH, and mitochondrial volume were monitored simultaneously using a wavelength scanning fluorometer. Maximal mCU velocity with efflux blocked by either CGP or 0mM Na+ varied as a function of extramitochondrial Ca2+ (0.5 nmol/mg/sec with a pulse of 15uM free Ca2+) while Ca2+ efflux rates initiated by addition of Ru360 after Ca2+ uptake varied as a function of Na+, increasing from 0.02 to 0.07 nmol/mg/sec from 5 to 15 mM Na+ and then paradoxically decreasing at extramitochondrial Na+ of 30mM. The biphasic Na+ effect was hypothesized to be the result of high Na+ exceeds the capacity of the proton pumps to support the Na+ extrusion coupled to Na+/H+ exchange. To test this hypothesis, the measured calcium rates were incorporated into our isolated mitochondria computational model and a single calcium pulse was simulated in a closed compartment under varied sodium concentrations as in the cuvette conditions. The simulations showed that Na+/H+ exchange rate and proton pump rate decrease at high sodium concentrations, which verify the hypothesis. The study supports Na+ playing a regulatory role in mitochondria Ca2+ through coupled mitochondrial ion circuits.

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.

Retention and Retention of Effect of Topical Formulations in Dry Eye Subjects

It was the purpose of this investigation to examine both retention time (RT) and retention of effect of two ophthalmic formulations in the same dry eye subjects. This was a randomized, subject-masked cross-over study. Dry eye subjects, characterized by sub-type, were recruited. For direct RT measurement, fluorescein isothiocyanate (FITC)-dextran, 70 kDa molecular weight, was admixed at 0.1% wt/vol concentration into buffered saline (active control) and a viscous marketed artificial tear formulations (test formulation). RT in minutes was estimated directly as the return to baseline intrinsic fluorescence with an objective scanning fluorometer. Retention of effect was measured with a xeroscope as non-invasive breakup time (NIBUT) and by use of a numerical rating scale for comfort. Eleven subjects, with most being classified as having non-inflammatory meibomian gland dysfunction, completed the study. The RTs averaged 17.7 (+/-10.0) and 26.8 (+/-16.5) minutes for the saline and test formulations, respectively. The averages for NIBUT were 8.73 (+/-6.1) and 19.5 (+/-4.9) minutes, for saline and the test formulations, respectively. Return to baseline for the numerical rating scale comfort averages were 9.91 (+/-4.1) and 20.21 (+/-6.4) minutes for the saline and test formulations, respectively. When residence time was subtracted from the retention of effect measures, no statistical significance was found for the polymeric solution (vs. comfort, p = 0.153; vs. NIBUT, p = 0.109). However, statistical differences were found for direct retention compared against comfort (p = 0.01) and NIBUT (p = 0.004) for buffered saline. It seems that these two retention of effect measures are much shorter than RT measured directly. Future work should aim to confirm these findings in additional dry eye sub-types and in those with more severe disease manifestations.

Precorneal Residence Time of Artificial Tears Measured in Dry Eye Subjects

The purpose of this investigation was to measure the precorneal residence time of saline and five marketed artificial tears in dry eye subjects using fluorometry. FITC-dextran, 70 kDa molecular weight, was admixed under sterile conditions (0.1% wt/vol) into buffered saline and the marketed artificial tear formulations of varying viscosity. Precorneal residence time (RT) was measured directly in 16 mild to moderate dry eye subjects, classified by sub-type, in a six-way cross-over, masked and randomized study. FITC-dextran tracer decay with a scanning fluorometer was used to estimate the gross RT (i.e., the time in minutes for the signal to return to baseline). All subjects were classified as having non-inflammatory meibomian gland dysfunction except one, who had a mixture of aqueous deficiency and meibomian gland dysfunction. In two separate determinations, the saline RTs were 19.1 +/- 7.4 and 17.6 +/- 8.2 min. The RTs for the formulations varied to some degree by viscosity, with two higher viscosity formulations demonstrating the longest RTs of 36 to 41 min, approximately twice that of saline (p < 0.001 for both 0.4% polyethylene glycol/0.3% propylene glycol, and 1.0% carboxymethylcellulose). An oil emulsion, low viscosity carboxymethylcellulose and moderate viscosity hydroxypropylmethylcellulose-containing formulation were not statistically different from saline (RTs of 18, 22 and 24 min, p values = 0.983, 0.818 and 0.099, respectively). More than two-fold RT differences were found for the higher viscosity, more muco-adhesive formulations compared to saline. However, other formulations provided RTs close to saline, suggesting that RT is influenced by factors other than simple viscosity. Future studies should examine the interplay of spreading characteristics, pseudoplasticity and muco-adhesion relative to RT to determine the individual and cumulative effects on formulation retention.

Ocular surface retention time and extensions of TFBUT of a lubricating eye drop

Abstract Purpose: The tear film in subjects with ocular dryness is often unstable and incapable of maintaining the protective qualities that are intrinsic to its structure and function. Until recently, lubricating eye drops have provided only transient relief from ocular dryness. The purpose of these studies is to determine if a lubricating eye drop, containing PG/PEG/HP‐Guar, will extend ocular protection. Methods: To compare the ocular residence time of lubricating eye drops, subjects with dry feeling eyes were administered a solution of Fluoroscein Isothiocyanate‐dextran mixed with a the eye drops. The residence time was measured by a scanning fluorometer. To compare TFBUT, subjects with moderately dry feeling eyes were administered a single drop of a lubricating eye drop. After instillation, TFBUT was measured at 0, 5, 10, 15, 20, 30, 45, 60, and 90 minutes. Results: Tear film retention of the PG/PEG/HP‐Guar product was significantly greater than a HPMC and a CMC lubricating eye drop. The product containing PG/PEG/HP‐Guar was measured in the tear film as much as 37 minutes post instillation, while the HPMC and CMC drops were only 25 and 23 minutes, respectively. The PG/PEG/HP‐Guar tear yielded a significantly greater increase in TFBUT from baseline at 45, 60, and 90 minutes post‐installation compared to eyes dosed with a CMC/Glycerin drop. Conclusions: Based on these results, it can be postulated that the extended duration of retention observed with the PG/PEG/HP‐Guar tear allows the necessary time for restructuring of the unstable tear film into a more stable tear film. This leads to a long‐term tear film stabilization and enhancement of TFBUT and ultimately, extended ocular surface protection

Differential coupling of β3A- and β3B-adrenergic receptors to endogenous and chimeric Gαs and Gαi

Chimeric G proteins made by replacing the COOH-terminal heptapeptide of G(alpha)q with the COOH-terminal heptapeptide of G(alpha)s or G(alpha)i were used to assess the relative coupling of beta(3)-adrenergic receptor (beta(3)-AR) splice variants (beta(3A) and beta(3B)) to G(alpha)s and G(alpha)i. The G(alpha)q/s and G(alpha)q/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium (Ca(2+)(i)). Complementary high-throughput and single-cell approaches were used to evaluate agonist-induced coupling of the receptor to the G protein chimeras. In cells stably transformed with rat beta(3)-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, beta(3)-AR-induced coupling to G(alpha)q/s produced a rapid eightfold increase in Ca(2+)(i) followed by a slow decay to levels 25% above baseline. G(alpha)q/i also linked rat beta(3)-AR to mobilization of Ca(2+)(i) in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in Ca(2+)(i) was only 30% of the response obtained with G(alpha)q/s. Activation of the rat beta(3)-AR also increased GTP binding to endogenous G(alpha)i threefold in membranes from CHO cells stably transformed with the receptor. A complementary single-cell imaging approach was used to assess the relative coupling of mouse beta(3A)- and beta(3B)-AR to G(alpha)i under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to G(alpha)q/s and to increases in intracellular cAMP through endogenous G(alpha)s. The beta(3A)- and beta(3B)-AR coupled equivalently to G(alpha)q/i, but the temporal patterns of Ca(2+)(i) mobilization indicated that coupling was significantly less efficient than coupling to G(alpha)q/s. Collectively, these findings indicate less efficient but equivalent coupling of beta(3A)- and beta(3B)-AR to G(alpha)i vs. G(alpha)s and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to beta(3)-AR activation.

P-364 Clinicopathological and immunogenetical study of tiopronin induced hepatic injury

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNAhi, Thy-1hi, CD3−/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23, gp45 complex expressed on murine Stromal cells.

Microfluorometric Evaluation of Calcein Acetoxymethyl Ester as a Probe for P-Glycoprotein-Mediated Resistance: Effects of Cyclosporin A and Its Nonimmunosuppressive Analogue SDZ PSC 833

A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and two of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high expression) and dox6 (low expression), were used as models. Nonfluorescent calcein acetoxymethyl ester (calcein/AM) was added to the cells and subsequent accumulation of calcein was measured in a 96-well scanning fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-dependently in the presence of cyclosporin A (CsA) and the nonimmunosuppressive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrations. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inhibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells could reproducibly be detected. The results indicate that microtiter-plate determination of calcein accumulation is a simple and sensitive method for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples.

Thymic stromal cells in culture: 2. Binding of normal thymocytes to a cloned thymic stromal cell line

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNA hi, Thy-1 hi, CD3 −/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23, gp45 complex expressed on murine Stromal cells.

Evaluation of Quality of Cataract Surgery. I. Corneal Endothelial Permeability

Fluorophotometry was carried out in 19 eyes with no pathology except for the presence of advanced cataracts. The age range was 60–75 and there was no evidence of systemic disorders such as diabetes mellitus or hypertension. The scanning fluorometer used (Fluorotron Master) was modified in order to improve depth resolution (0.585 mm). Corneal endothelial permeability (Pac) was calculated before surgery (Pac: 3.14 ± 0.60 × 10 -4 cm/min). In 11 eyes, corneal endothelial permeability was again measured one week after planned extracapsular cataract extraction and intraocular lens (IOL) implantation. The corneal endothelial permeability increased after surgery and this increase correlated well with duration of surgery. Longer surgical procedures (>20 min) were associated with greater increases in corneal endothelial permeability.

A scanning fluorometer for imaging ischaemic areas in traumatized muscle.

The criteria used to evaluate the state of muscle surrounding a bullet wound are: a lack of contractility, a lack of capillary bleeding, and changes in colour and consistency. Muscle with all these properties may be assumed to be irreversibly damaged. However, the boundary between such tissue and tissue with potentially reversible levels of damage may not be clear cut. In general, oxygen lack due to blood vessel damage may be sufficient to cause irreversible damage or may result in a site of anaerobic infection. Such areas may occur at some distance from the missile track. Tissue responses to oxygen lack can be monitored by observing changes in intrinsic cellular fluorescence. The blue autofluorescence of intracellular pyridine nucleotides increases with anoxia, whereas the green autofluorescence of intracellular flavoprotein has been found to decrease with anoxia. We have developed a scanning fluorometer which rapidly provides an image of the distribution of anoxic areas in soft tissues. In experiments with anaesthetized rabbits, occlusion of the blood vessels to the gracilis muscle caused an increase in its pyridine nucleotide fluorescence and a decrease in its flavoprotein fluorescence. The changes were hastened by stimulation of the muscle to fatigue. Arterial infusion of colloidal carbon confirmed that adjacent muscles still had a blood flow. The apparatus has potential for the identification of hypoxic zones peripheral to the permanent wound cavity. Conversely, it may also help to indicate whether vascular repair after trauma of large blood vessels has led to improvement of the metabolic status of tissue being reperfused.

Scanning fluorometer for the rapid assessment of pyridine nucleotide and flavoprotein fluorescence changes in tissues in vivo

This paper describes a scanning fluorometer which produces images in real time of the distribution of pyridine nucleotide or flavoprotein fluorescence at the surface of tissues in vivo. The basic difference between this device and others reported in the literature is that fluorescence changes at any selected point within the image can be quantified as they occur. We suggest that the apparatus has potential application in those areas of surgery where vascular replacement or repair is required and where it would be advantageous to have an immediate measure of the cellular response to a return of blood flow.

Rapid scanning fluorometer based on constant energy synchronous scanning

A rapid scanning fluorometer based on constant energy synchronous fluorescence scanning at 200 nm/s has been developed. Constant energy synchronous fluorescence spectrometry involves measurement of fluorescence as both excitation and emission monochromators are scanned with a constant energy difference being maintained. Spectral information is collected in the forward to reverse directions and is processed in real time. A discussion of the instrumentation and software is given. A theoretical comparison between the photomultiplier tube (PMT) detection system used with this system and a silicon intensified target vidicon (SIT) detection system has been given. Calculations indicate that the signal-to-noise ratio S/N for the PMT system should be about 5–40× greater than for the SIT system. A comparison of the experimental figures of merit (detection limits, scan speed, analysis time) of the rapid scan constant energy synchronous fluorescence spectrometer with video fluorometers has also been made, again showing the superiority of the rapid scan synchronous system.

A batch assay using Calcofluor fluorescence to characterize cell wall regeneration in plant protoplasts

A batch assay to study and measure the regeneration of cell walls during the early days of culture of primary protoplasts is presented. The assay involves the measurement of Calcofluor White fluorescence on a scanning fluorometer when the Calcofluor is adsorbed to the cellulosic component of the newly synthesized cell walls. The Calcofluor fluorescence, when standardized with microcrystalline cellulose, provided a measure of cell wall cellulose. The assay was used to study cell wall regeneration in Hyoscyamus muticus L. protoplasts during 8 days of culture.