Vishwas Joshi | Suffolk County Community College (original) (raw)
Papers by Vishwas Joshi
Microscopy and Microanalysis, 2020
Microscopy and Microanalysis, 2002
Six histidines added to expressed proteins have been a boon for rapidly purifying them from the e... more Six histidines added to expressed proteins have been a boon for rapidly purifying them from the expression organism lysate, since it was found that the 6x-His tag specifically binds (reversibly) to columns containing Ni +2 [1]. The nickel is chelated to the column with nitrilotriacetic acid (NTA), which is similar to EDTA. Since many proteins now have His-tags, and cells can be transfected to produce His-tagged proteins, it is of interest to have a gold label that also binds specifically to this tag. One application is identifying a specific protein against a background, for example in cell sections to see the localization of an expressed protein, much like green fluorescent protein; however, the gold label is visible in the EM for higher resolution studies; it can also be silver or gold enhanced to visualize it in the light microscope, or on blots. Another application is to use it as a molecular domain or subunit label for high resolution single particle analysis. Here, since the position of the His-tag is known, e.g., the amino terminus, that part of the molecule can be locally labeled and useful for cryoEM reconstructions. It would also serve as a high visibility fiducial mark for orienting single particles at low dose, perhaps permitting extension of the resolution obtainable by such techniques. For multi-subunit complexes, a particular subunit may be labeled to specifically identify it in the complex. In x-ray or electron protein crystallography, the label could be used to improve contrast or be used as a phasing aid. Another difference in this type of label is that the Ni-NTA group is quite small compared to an antibody, thus bringing the gold particle much closer to the 6x-His-tag giving higher resolution labeling with less ambiguity or floppiness. The smaller size will also improve its diffusion into cells or tissues.
Microscopy and Microanalysis, 2019
Microscopy and Microanalysis, 2003
The sexually dimorphic anterior transposition of the median unpaired fin, specifically the anal f... more The sexually dimorphic anterior transposition of the median unpaired fin, specifically the anal fin of the Western Mosquitofish, Gambusia affinis affinis, provides a versatile experimental model for gaining insight into a number of complex intercellular mechanisms, processes, and interactions during post-embryonic development. Of particular interest is the fate of the G. a. affinis nervous system as it undergoes remodeling to accommodate the radical changes in target tissues and organs and the animal changes from a non-internal fertilizing to an internal fertilizing species. However, a major restriction to realizing the full potential of this vertebrate model system, particularly with regard to understanding development and differentiation, is the lack of sufficiently refined and precise mapping techniques and probes. Thus, the cellular decisions underlying motor neuron remodeling during the anterior transposition of the anal fin have received no attention. In earlier work (Fig. 1A-1B), colloidal heavy metals, such as gold (18.0 nm) were directly conjugated to commercially available (Molecular Probes, Inc., Eugene, OR) recombinant cholera toxin B-subunit of green fluorescent Alexa Fluor® 488 [ex 495 nm/em 519 nm; Lot: 73B2] (AF 488 cgrCTB) and to recombinant cholera toxin B-subunit of red-fluorescent Alexa Fluor® 594 [ex 590 nm/em 617 nm; Lot: 73B1] (AF 594 cgrCTB) and shown to be transported retrogradely to the central nervous system, specifically spinal motor neurons, without quenching of the fluorescence (Fig 1). The lack of quenching seen in AF 488 cgrCTB and AF 594 cgrCTB is not well understood and is currently under investigation [1, 2].
Microscopy and Microanalysis, 2017
Microscopy and Microanalysis, 2017
Microscopy and Microanalysis, 2017
Electron microscopy (EM) affords a unique window into the structure and function of cells and tis... more Electron microscopy (EM) affords a unique window into the structure and function of cells and tissues at the macromolecular level. However, in order to realize its full potential, methods are required to identify and differentiate structures of interest at a resolution, labeling density and level of multiplexing appropriate to their size, proximity and complexity.
Microscopy and Microanalysis, 2012
Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, ... more Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.
A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold... more A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary s...
Journal of the American Chemical Society, 1986
Journal of the American Chemical Society, 1986
Journal of Chemical Sciences, 1992
Abstract. Using optically active Ru(bpy)] § and Ru(phen)a 2+ as probe chelates, the pheno-menon o... more Abstract. Using optically active Ru(bpy)] § and Ru(phen)a 2+ as probe chelates, the pheno-menon of chiral recognition is demonstrated with days. Similarities and differences in the binding states of the above chelates are also discussed. Finally, the effect of such binding state ...
Journal of the American Chemical Society, 1986
The Journal of Physical Chemistry A, 2001
The previously observed facile photooxidation of Ru (bpy) 32+ to Ru (bpy) 33+ in oxygenated solut... more The previously observed facile photooxidation of Ru (bpy) 32+ to Ru (bpy) 33+ in oxygenated solutions of 9 M H2SO4 (Kotkar, D; Joshi, V.; Ghosh, PK Chem. Commun. 1987, 4; Indian Patent No. 164358 (1989)) is further studied. A similar phenomenon was ...
Journal of the Chemical Society, Chemical Communications, 1988
The synthesis of free standing and clay-supported chiral conducting polymer films is described. .... more The synthesis of free standing and clay-supported chiral conducting polymer films is described. ... We report here our preliminary results on the preparation and characterisation of free standing and clay-supported chiral conducting polymer films based on (S)-( +)-3-(2-...
Inorganic Chemistry, 1986
Contribution from the Alchemie Research Centre, Thane 400601, Maharashtra, India ... Thermal and ... more Contribution from the Alchemie Research Centre, Thane 400601, Maharashtra, India ... Thermal and Light-Induced Reduction of (+)-Tris(bipyridyl)ruthenium(III) in Aqueous Solution: Mechanistic Inferences from Optical Rotation Studies ... Dilip Kotkar, Vishwas Joshi, and Pushpito K. ...
Nature Methods, 2008
The difficulty in localizing specific cellular proteins by immunoelectron microscopy techniques l... more The difficulty in localizing specific cellular proteins by immunoelectron microscopy techniques limits applications of electron microscopy to cell biology. We found that in vivo immunogold labeling improves epitope accessibility, ultrastructural preservation and three-dimensional visualization, and allows correlated light and electron microscopy. We detected large-scale chromatin folding motifs within intact interphase nuclei of CHO cells and visualized the ultrastructure of DNA replication 'factories' labeled with GFP-proliferating cell nuclear antigen (PCNA).
Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab'... more Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab' immunoprobe by separate cross-linking reactions, to give a probe which labels antigenic sites in a single step for correlative fluorescence and electron microscope visualization [1,2]. These probes show high labeling density, labeling a pre-mRNA splicing factor in the HeLa cell nucleus[3]; Microtubules were also densely labeled using fluorescence, other optical modalities, and electron microscopy; in a parallel experiment, a 5 nm colloidal gold probe gave only occasional labeling [4]. We now describe Fab' and streptavidin probes containing both Nanogold® and the fluorescent cyanine dye, Cy3.
Synthetic Communications, 1998
ABSTRACT Tetramisole has been found to complex with chromium trioxide. Elemental analysis suggest... more ABSTRACT Tetramisole has been found to complex with chromium trioxide. Elemental analysis suggests a 1:1 complex formation and IR data suggest the ligand binding through N-7 nitrogen. The new complex is capable of selectively oxidizing alcohols, including benzyl and allylic alcohol, to carbonyl compounds under mild conditions.@ Abstract published in Advance ACS abstracts, Sept. 7, 1997.
Nature Methods, 2008
The difficulty in localizing specific cellular proteins by immunoelectron microscopy techniques l... more The difficulty in localizing specific cellular proteins by immunoelectron microscopy techniques limits applications of electron microscopy to cell biology. We found that in vivo immunogold labeling improves epitope accessibility, ultrastructural preservation and three-dimensional visualization, and allows correlated light and electron microscopy. We detected large-scale chromatin folding motifs within intact interphase nuclei of CHO cells and visualized the ultrastructure of DNA replication 'factories' labeled with GFP-proliferating cell nuclear antigen (PCNA).
Microscopy and Microanalysis, 2020
Microscopy and Microanalysis, 2002
Six histidines added to expressed proteins have been a boon for rapidly purifying them from the e... more Six histidines added to expressed proteins have been a boon for rapidly purifying them from the expression organism lysate, since it was found that the 6x-His tag specifically binds (reversibly) to columns containing Ni +2 [1]. The nickel is chelated to the column with nitrilotriacetic acid (NTA), which is similar to EDTA. Since many proteins now have His-tags, and cells can be transfected to produce His-tagged proteins, it is of interest to have a gold label that also binds specifically to this tag. One application is identifying a specific protein against a background, for example in cell sections to see the localization of an expressed protein, much like green fluorescent protein; however, the gold label is visible in the EM for higher resolution studies; it can also be silver or gold enhanced to visualize it in the light microscope, or on blots. Another application is to use it as a molecular domain or subunit label for high resolution single particle analysis. Here, since the position of the His-tag is known, e.g., the amino terminus, that part of the molecule can be locally labeled and useful for cryoEM reconstructions. It would also serve as a high visibility fiducial mark for orienting single particles at low dose, perhaps permitting extension of the resolution obtainable by such techniques. For multi-subunit complexes, a particular subunit may be labeled to specifically identify it in the complex. In x-ray or electron protein crystallography, the label could be used to improve contrast or be used as a phasing aid. Another difference in this type of label is that the Ni-NTA group is quite small compared to an antibody, thus bringing the gold particle much closer to the 6x-His-tag giving higher resolution labeling with less ambiguity or floppiness. The smaller size will also improve its diffusion into cells or tissues.
Microscopy and Microanalysis, 2019
Microscopy and Microanalysis, 2003
The sexually dimorphic anterior transposition of the median unpaired fin, specifically the anal f... more The sexually dimorphic anterior transposition of the median unpaired fin, specifically the anal fin of the Western Mosquitofish, Gambusia affinis affinis, provides a versatile experimental model for gaining insight into a number of complex intercellular mechanisms, processes, and interactions during post-embryonic development. Of particular interest is the fate of the G. a. affinis nervous system as it undergoes remodeling to accommodate the radical changes in target tissues and organs and the animal changes from a non-internal fertilizing to an internal fertilizing species. However, a major restriction to realizing the full potential of this vertebrate model system, particularly with regard to understanding development and differentiation, is the lack of sufficiently refined and precise mapping techniques and probes. Thus, the cellular decisions underlying motor neuron remodeling during the anterior transposition of the anal fin have received no attention. In earlier work (Fig. 1A-1B), colloidal heavy metals, such as gold (18.0 nm) were directly conjugated to commercially available (Molecular Probes, Inc., Eugene, OR) recombinant cholera toxin B-subunit of green fluorescent Alexa Fluor® 488 [ex 495 nm/em 519 nm; Lot: 73B2] (AF 488 cgrCTB) and to recombinant cholera toxin B-subunit of red-fluorescent Alexa Fluor® 594 [ex 590 nm/em 617 nm; Lot: 73B1] (AF 594 cgrCTB) and shown to be transported retrogradely to the central nervous system, specifically spinal motor neurons, without quenching of the fluorescence (Fig 1). The lack of quenching seen in AF 488 cgrCTB and AF 594 cgrCTB is not well understood and is currently under investigation [1, 2].
Microscopy and Microanalysis, 2017
Microscopy and Microanalysis, 2017
Microscopy and Microanalysis, 2017
Electron microscopy (EM) affords a unique window into the structure and function of cells and tis... more Electron microscopy (EM) affords a unique window into the structure and function of cells and tissues at the macromolecular level. However, in order to realize its full potential, methods are required to identify and differentiate structures of interest at a resolution, labeling density and level of multiplexing appropriate to their size, proximity and complexity.
Microscopy and Microanalysis, 2012
Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, ... more Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.
A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold... more A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary s...
Journal of the American Chemical Society, 1986
Journal of the American Chemical Society, 1986
Journal of Chemical Sciences, 1992
Abstract. Using optically active Ru(bpy)] § and Ru(phen)a 2+ as probe chelates, the pheno-menon o... more Abstract. Using optically active Ru(bpy)] § and Ru(phen)a 2+ as probe chelates, the pheno-menon of chiral recognition is demonstrated with days. Similarities and differences in the binding states of the above chelates are also discussed. Finally, the effect of such binding state ...
Journal of the American Chemical Society, 1986
The Journal of Physical Chemistry A, 2001
The previously observed facile photooxidation of Ru (bpy) 32+ to Ru (bpy) 33+ in oxygenated solut... more The previously observed facile photooxidation of Ru (bpy) 32+ to Ru (bpy) 33+ in oxygenated solutions of 9 M H2SO4 (Kotkar, D; Joshi, V.; Ghosh, PK Chem. Commun. 1987, 4; Indian Patent No. 164358 (1989)) is further studied. A similar phenomenon was ...
Journal of the Chemical Society, Chemical Communications, 1988
The synthesis of free standing and clay-supported chiral conducting polymer films is described. .... more The synthesis of free standing and clay-supported chiral conducting polymer films is described. ... We report here our preliminary results on the preparation and characterisation of free standing and clay-supported chiral conducting polymer films based on (S)-( +)-3-(2-...
Inorganic Chemistry, 1986
Contribution from the Alchemie Research Centre, Thane 400601, Maharashtra, India ... Thermal and ... more Contribution from the Alchemie Research Centre, Thane 400601, Maharashtra, India ... Thermal and Light-Induced Reduction of (+)-Tris(bipyridyl)ruthenium(III) in Aqueous Solution: Mechanistic Inferences from Optical Rotation Studies ... Dilip Kotkar, Vishwas Joshi, and Pushpito K. ...
Nature Methods, 2008
The difficulty in localizing specific cellular proteins by immunoelectron microscopy techniques l... more The difficulty in localizing specific cellular proteins by immunoelectron microscopy techniques limits applications of electron microscopy to cell biology. We found that in vivo immunogold labeling improves epitope accessibility, ultrastructural preservation and three-dimensional visualization, and allows correlated light and electron microscopy. We detected large-scale chromatin folding motifs within intact interphase nuclei of CHO cells and visualized the ultrastructure of DNA replication 'factories' labeled with GFP-proliferating cell nuclear antigen (PCNA).
Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab'... more Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab' immunoprobe by separate cross-linking reactions, to give a probe which labels antigenic sites in a single step for correlative fluorescence and electron microscope visualization [1,2]. These probes show high labeling density, labeling a pre-mRNA splicing factor in the HeLa cell nucleus[3]; Microtubules were also densely labeled using fluorescence, other optical modalities, and electron microscopy; in a parallel experiment, a 5 nm colloidal gold probe gave only occasional labeling [4]. We now describe Fab' and streptavidin probes containing both Nanogold® and the fluorescent cyanine dye, Cy3.
Synthetic Communications, 1998
ABSTRACT Tetramisole has been found to complex with chromium trioxide. Elemental analysis suggest... more ABSTRACT Tetramisole has been found to complex with chromium trioxide. Elemental analysis suggests a 1:1 complex formation and IR data suggest the ligand binding through N-7 nitrogen. The new complex is capable of selectively oxidizing alcohols, including benzyl and allylic alcohol, to carbonyl compounds under mild conditions.@ Abstract published in Advance ACS abstracts, Sept. 7, 1997.
Nature Methods, 2008
The difficulty in localizing specific cellular proteins by immunoelectron microscopy techniques l... more The difficulty in localizing specific cellular proteins by immunoelectron microscopy techniques limits applications of electron microscopy to cell biology. We found that in vivo immunogold labeling improves epitope accessibility, ultrastructural preservation and three-dimensional visualization, and allows correlated light and electron microscopy. We detected large-scale chromatin folding motifs within intact interphase nuclei of CHO cells and visualized the ultrastructure of DNA replication 'factories' labeled with GFP-proliferating cell nuclear antigen (PCNA).