Sulforhodamine 101 Research Articles

Developmental expression of spontaneous activity in the spinal cord of postnatal opossums, Monodelphis domestica: An anatomical study

Using Sulforhodamine-101 (SR101) labeling and calcium imaging on in vitro preparations, we investigated the development of spontaneous activity in the spinal enlargements of a marsupial born more immature than eutherian mammals, the opossum Monodelphis domestica. Following the retrograde transport of Calcium Green dye from the limb nerves, we observed the occurrence of spontaneous calcium waves activating the motor columns of the cervical enlargement of opossums aged from P3 to P15 (day of birth: P0) and of the lumbar enlargement from at least P6 to P12. In other preparations, SR101 was added to the bath to identify the active cells. In P1 opossums, only a few SR101-labeled cells were observed in the cervical enlargement and none in the lumbar enlargement. At P5, their number increased cervically and they appeared in the lumbar enlargement. Motoneurons were the major cell type labeled by SR101 but dye leakage made their quantification inaccurate. SR101-labeled cells also occurred elsewhere in the ventral and dorsal grey. Their number increased until P12–14 in both enlargements and then decreased to disappear by P21, the last age examined. Thus in contrast to eutherian mammals, in which spontaneous activity is mostly prenatal, spontaneous activity occurs predominantly postnatally in opossums. It increases at the time when connections from the brain begin to impinge on spinal neurons and when the limbs, especially the hindlimbs, start moving and then decreases as the systems mature.

An integrated PLED – A light source for application specific lab-on-microchips (ALM)

We report a polymer organic light-emitting diode (PLED) excitation source for lab-on-microchip fluorescence analysis. A PDY-132 thin-film green PLED with an active area of 1 mm 2 was used as the excitation source. The excitation light of the PLED was used to illuminate a microfluidic device containing 150 μl of Sulforhodamine 101 (SRh101) dye dissolved in ethanol. Fluorescence signals of SRh101 (peak emission wavelength of 566 nm) dye is measured in a microfluidic device with the emission signal detected by a spectrometer. The measurements suggest that an integrated microfluidic device with a PLED excitation source can be fabricated to realize a compact and economical lab-on-microchip for point-of-care fluorescence assays.

Developmental profile and properties of sulforhodamine 101—Labeled glial cells in acute brain slices of rat hippocampus

The reliable identification of astrocytes for physiological measurements was always time-consuming and difficult. Recently, the fluorescent dye sulforhodamine 101 (SR101) was reported to label cortical glial cells in vivo [Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 2004;1:31–7]. We adapted this technique for use in acute rat hippocampal slices at early postnatal stages (P3, 7, 15) and in young adults (P24–27) and describe a procedure for double-labeling of SR101 and ion-selective dyes. Using whole-cell patch-clamp, imaging, and immunohistochemistry, we characterized the properties of SR101-positive versus SR101-negative cells in the stratum radiatum. Our data show that SR101, in contrast to Fura-2 or SBFI, only stains a subset of glial cells. Throughout development, SR101-positive and SR101-negative cells differ in their basic membrane properties. Furthermore, SR101-positive cells undergo a developmental switch from variably rectifying to passive between P3 and P15 and lack voltage-gated Na + currents. At P15, the majority of SR101-positive cells is positive for GFAP. Thus, our data demonstrate that SR101 selectively labels a subpopulation of glial cells in early juvenile hippocampi that shows the typical developmental changes and characteristics of classical astrocytes. Owing to its reliability and uncomplicated handling, we expect that this technique will be helpful in future investigations studying astrocytes in the developing brain.

Interfacial behavior of sulforhodamine 101 at the polarized water/1,2-dichloroethane interface studied by spectroelectrochemical techniques

The transfer mechanism of an amphoteric rhodamine, sulforhodamine 101 (SR101), across the polarized water/1,2-dichloroethane (DCE) interface was investigated using cyclic voltammetry, differential voltfluorometry and potential-modulated fluorescence (PMF) spectroscopy. The voltammetric response for the ion transfer of SR101 monoanion from water to DCE was observed as the diffusion-controlled transfer process. An unusual voltammetric response was found at 0.15 V more negative than the formal transfer potential of SR101(-) (deltaW(O)phi degrees') in the cyclic voltammogram and voltfluorogram. The frequency dependence of the PMF responses confirmed the presence of the adsorption processes at negative potentials. In addition, a further transient adsorption step was uncovered at deltaW(O)phi degrees'. The interfacial mechanism of SR101 is discussed by comparing the results obtained from each technique.

Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo

Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscopy. The red fluorescent dye sulforhodamine 101 (SR101) was specifically taken up by protoplasmic astrocytes after brief exposure to the brain surface. Specificity was confirmed by immunohistochemistry. In addition, SR101 labeled enhanced green fluorescent protein (EGFP)-expressing astrocytes but not microglial cells in transgenic mice. We used SR101 labeling to quantify morphological characteristics of astrocytes and to visualize their close association with the cortical microvasculature. Furthermore, by combining this method with calcium indicator loading of cell populations, we demonstrated distinct calcium dynamics in astroglial and neuronal networks. We expect SR101 staining to become a principal tool for investigating astroglia in vivo.

Nanochannels on a fused-silica microchip and liquid properties investigation by time-resolved fluorescence measurements.

We have fabricated nanometer-sized channels, demonstrated a technique for the introduction of liquid into the channels, and carried out time-resolved fluorescence measurements of aqueous solutions. In this study, 330-nm- and 850-nm-sized channels were fabricated on fused-silica substrates by fast atom beam etching and hydrofluoric acid bonding methods. A liquid introduction method utilizing capillary action was demonstrated. The liquid introduction was observed under an optical microscope, and the liquid velocity during the introduction was analyzed by surface energy and macroscale hydrodynamics. The liquid velocity due to capillary action in the nanometer-sized channel seemed more than four times slower than the estimation. Then, aqueous solutions of rhodamine 6G (R6G), sulforhodamine 101 (SR101), and rhodamine B (RB) in the channels were measured by time-resolved fluorescence spectroscopy; spectra of the same solution in a 250-microm-sized channel were also measured as a reference for the macrospace. Although the fluorescence spectra in the 330-nm-, 850-nm- and 250-microm-sized channels agreed with one another, the fluorescent decays in the nanometer-sized channels were faster for R6G and SR101 and slower for RB than the respective decays in the 250-microm-sized channels. The results suggested the solutions had lower dielectric constants and higher viscosities in the nanometer-sized channels.

Time-Resolved Total Internal Reflection Fluorometry Study on Chemical and Structural Characteristics at Water/Oil Interfaces

Abstract Structures and characteristics of water/oil interfaces at the molecular level are reviewed. Dynamic fluorescence anisotropy of Sulforhodamine 101 (SR101) and excitation energy transfer from SR101 to Acid Blue 1 (AB1) at water/oil interfaces were studied by using time-resolved total internal reflection (TIR) fluorometry. The results indicated that a water/carbon tetrachloride (CCl4) interface was sharp with respect to the molecular size of SR101 (∼10 Å), while a water/1,2-dichloroethane (DCE) interface was relatively rough compared to the water/CCl4 interface. The present results were also compared with those predicted from molecular dynamic simulations and the thermally capillary wave theory. Furthermore, on the basis of fluorescence dynamic measurements of Sulforhodamine B (SRB) adsorbed at a water/oil interface, a relationship between thickness/roughness and the polarity at the interface was discussed.

Anatomical evidence for brainstem circuits mediating feeding motor programs in the leopard frog, Rana pipiens.

Using injections of small molecular weight fluorescein dextran amines, combined with activity-dependent uptake of sulforhodamine 101 (SR101), brainstem circuits presumed to be involved in feeding motor output were investigated. As has been shown previously in other studies, projections to the cerebellar nuclei were identified from the cerebellar cortex, the trigeminal motor nucleus, and the vestibular nuclei. Results presented here suggest an additional pathway from the hypoglossal motor nuclei to the cerebellar nucleus as well as an afferent projection from the peripheral hypoglossal nerve to the Purkinje cell layer of the cerebellar cortex. Injections in the cerebellar cortex combined with retrograde labeling of the peripheral hypoglossal nerve demonstrate anatomical convergence at the level of the medial reticular formation. This suggests a possible integrative region for afferent feedback from the hypoglossal nerve and information through the Purkinje cell layer of the cerebellar cortex. The activity-dependent uptake of SR101 additionally suggests a reciprocal, polysynaptic pathway between this same area of the medial reticular formation and the trigeminal motor nuclei. The trigeminal motor neurons innervate the m adductor mandibulae, the primary mouth-closing muscle. The SR101 uptake clearly labeled the ventrolateral hypoglossal nuclei, the medial reticular formation, and the Purkinje cell layer of the cerebellar cortex. Unlike retrograde labeling of the peripheral hypoglossal nerve, stimulating the hypoglossal nerve while SR101 was bath-applied labeled trigeminal motor neurons. This, combined with the dextran labeling, suggests a reciprocal connection between the trigeminal motor nuclei and the cerebellar nuclei, as well as the medulla. Taken together, these data are important for understanding the neurophysiological pathways used to coordinate the proper timing of an extremely rapid, goal-directed movement and may prove useful for elucidating some of the first principles of sensorimotor integration.

Role of rare sites in single molecule spectroscopy measurements of spectral diffusion

Detailed investigations of the spectral diffusion of sulforhodamine 101 (SR101) in polymer films have been performed over a range of concentrations from typical concentrations for single molecule (SM) spectroscopic studies (10−10 M) to typical concentrations for bulk (10−6 M) experiments. A “single-molecule” ensemble spectrum, obtained by averaging 42 different SM spectra of SR101 in a poly(methyl methacrylate) film, exhibits extreme broadening and a large vibronic peak that is not observed in the more concentrated samples. The distribution of SM spectral means is extremely broad (FWHM=616 cm−1) with molecule-to-molecule variations that last at least several hours. Autocorrelation analysis of 170 SM spectral mean trajectories on the second times scale reveals that the most probable behavior for SR101 molecules is to exhibit negligibly small amounts of spectral diffusion. The observed effects, i.e., the large vibronic peak, the broad spectral distribution, and rare rapid jumps in the spectral mean, are assigned to differences in probe nanoenvironments. The effects of association with these “rare” sites are saturated at extremely low (10−9 M) concentrations. The unexpected concentration dependence observed here could have implications for data interpretation in many SM experiments.

Excitation Energy Transfer from Sulforhodamine 101 to Acid Blue 1 at a Liquid/Liquid Interface: Experimental Approach To Estimate Interfacial Roughness

Dynamic fluorescence anisotropy of Sulforhodamine 101 (SR101) and excitation energy transfer from SR101 to Acid Blue 1 (AB1) at water/carbon tetrachloride (CCl4) and water/1,2-dichloroethane (DCE) interfaces were studied by using time-resolved total-internal-reflection (TIR) fluorometry. A magic-angle dependence of the TIR fluorescence dynamic anisotropy revealed that rotational reorientation of SR101 at the water/CCl4 interface was restricted in the two-dimensional plane of the interface, while that at the water/DCE interface took place rather freely, similar to reorientation in an isotropic medium. Furthermore, the structural dimension derived from the excitation energy transfer dynamics measurements at the water/CCl4 or water/DCE interface was 1.93 or 2.48, respectively. These results indicated that the water/CCl4 interface was sharp with respect to the molecular size of SR101 (∼10 A), while the water/DCE interface was relatively rough compared to the water/CCl4 interface. Structural dimension analysis...

Endocytic Active Zones: Hot Spots for Endocytosis in Vertebrate Neuromuscular Terminals

We have used a sensitive activity-dependent probe, sulforhodamine 101 (SR101), to view endocytic events within snake motor nerve terminals. After very brief neural stimulation at reduced temperature, SR101 is visualized exclusively at punctate sites located just inside the presynaptic membrane of each terminal bouton. The number of sites (approximately 26 sites/bouton) and their location (in register with postsynaptic folds) are similar to the number and location of active zones in snake motor terminals, suggesting a spatial association between exocytosis and endocytosis under these stimulus conditions. With more prolonged stimulation, larger SR101-containing structures appear at the bouton margins. Thus endocytosis occurs initially at distinct sites, which we call "endocytic active zones," whereas further stimulation recruits a second endocytic paradigm.

Impeded Rotation of a Protein in a Sol−Gel Matrix

The sol−gel encapsulation process has been exploited in recent years for the immobilization of proteins to be used as biosensors. Sol−gels derived from tetramethyl orthosilicate provide a stable environment for the macromolecule combined with the free flow of small substrates to a protein's binding site. The functionality of a number of enzymes within the solid matrix has been demonstrated. However, very little biophysical characterization of the encapsulated proteins has been done. In this study, time-resolved fluorescence anisotropy was used to compare the rotational mobility of two probes in sol−gel matrices derived from three different preparative methods. A small fluorescent probe, sulforhodamine 101 (SR101), was used to gauge the relative solvent viscosity within the sol−gels. Magnesium protoporphyrin IX-substituted myoglobin (MgMb) provides a convenient fluorescent probe for measuring rotational dynamics of a typical globular protein. The anisotropy decay of the Mg−heme is sensitive only to the glo...

Total Internal Reflection Fluorescence Dynamic Anisotropy of Sulforhodamine 101 at a Liquid/Liquid Interface: Rotational Reorientation Times and Interfacial Structures

The dynamic anisotropy of Sulforhodamine 101 (SR101) at water/phthalate ester (PE, bis(2-ethylhexyl) phthalate, di-n-heptyl phthalate, di-n-butyl phthalate, or di-n-ethyl phthalate) interfaces was studied by using time-resolved total internal reflection (TIR) fluorometry. A magic-angle dependence of the TIR fluorescence dynamics revealed that rotational reorientation of SR101 at the water/PE interface was restricted in the X−Y plane (in-plane) of the interface. The results indicated that the interface was sharp with respect to the molecular size of SR101 (∼10 A). In-plane rotational reorientation of SR101 at the interface showed two time constants (τrot). The fast component (τ1rot) was similar to that in water irrespective of the nature of PE, while the slow one (τ2rot) was affected by the viscosity of PE but not directly by the macroscopic viscosity. The two rotational reorientation times of SR101 characteristic to the water/PE interface were explained in terms of different adsorption modes of the dye on...

Phase Fluorometric Optical Carbon Dioxide Gas Sensor for Fermentation Off-Gas Monitoring.

We demonstrated an optical carbon dioxide gas sensor suitable for replacement of gas chromatographs and mass spectrometers for the measurement of carbon dioxide in the off-gas of a bioreactor for fermentation and cell culture applications. The sensor is based upon the change in lifetime of a donor fluorophore, sulforhodamine 101 (SR101), induced by fluorescence resonance energy transfer to a pH-sensitive, nonfluorescent acceptor, m-cresol purple (MCP). Carbon dioxide diffusing into the sensor produces carbonic acid, changing the absorbance spectrum of the MCP, and thus its spectral overlap with the SR101, changing its lifetime. This lifetime change was measured in the frequency, rather than the time domain, as a change in the phase angle of the fluorescence relative to the modulated excitation light. The sensor was calibrated by correlating the phase response to carbon dioxide concentrations. The calibration remained valid over the life of the sensor, which has been shown to be greater than 2 weeks. The sensor was most sensitive at low CO2 concentrations and responded to concentration changes in seconds. The sensor film is very inexpensive to produce and the light source is an inexpensive light-emitting diode. Furthermore, lower cost detection electronics can be developed since only one modulation frequency is required. In addition, this sensor can potentially be used in vivo, with a fiber optic both delivering the excitation light and collecting the emission.

Intracellular levels of calmodulin are increased in transformed cells

By using Hoechst 33342, rabbit anti calmodulin antibody, FITC-labeled goat anti rabbit IgG and SR101 (sulfo rhodamine 101) simultaneously to stain individual normal and transformed cells, the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus, calmodulin and total protein respectively, could be recognized in individualj cells without interference. The phase of the cell cycle was determined by DNA content (Hoechst 33342). We found that in transformed cells (NIH3T3) tsRSV-LA90, cultured at 33°C and transformed C3H10T1/2 cells), the ratio of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV−LA90 cells, cultured at 39°C and C3H10T1/2 cells)in every cell cycle phase. This ratio increased obviously only from G1 to S phase in either normal or transformed cells. The results showed that calmodulin really increased during the transformation, and its increase was specific. In the meantime when cells proceeded from G1 to S, the intracellular calmodulin content also increased specifically.