An in-house constructed dual channel confocal fluorescence microscope for biomolecular imaging

Shakhi, P. K.; Bijeesh, M. M.; Varier, Geetha K.; Nandakumar, P.

doi:10.1364/osac.428601

Cited by 4 publications

(2 citation statements)

References 35 publications

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“…The time-lapse confocal imaging of the passive nuclear import and export is done using an Olympus confocal microscope (FV 3000, Olympus corporation) and/ or an in-house constructed confocal fluorescence microscope [ 38 ]. The import mixture for the transport is freshly prepared before the experiment.…”

Section: Methodsmentioning

confidence: 99%

Size-dependent steady state saturation limit in biomolecular transport through nuclear membranes

Shakhi,

Bijeesh,

Hareesh

et al. 2024

PLoS ONE

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The nucleus preserves the genomic DNA of eukaryotic organisms and maintains the integrity of the cell by regulating the transport of molecules across the nuclear membrane. It is hitherto assumed that small molecules having a size below the passive permeability limit are allowed to diffuse freely to the nucleus while the transport of larger molecules is regulated via an active mechanism involving energy. Here we report on the kinetics of nuclear import and export of dextran molecules having a size below the passive permeability limit. The studies carried out using time-lapse confocal fluorescence microscopy show a clear deviation from the passive diffusion model. In particular, it is observed that the steady-state concentration of dextran molecules inside the nucleus is consistently less than the concentration outside, in contradiction to the predictions of the passive diffusion model. Detailed analysis and modeling of the transport show that the nuclear export rates significantly differ from the import rates, and the difference in rates is dependent on the size of the molecules. The nuclear export rates are further confirmed by an independent experimental study where we observe the diffusion of dextran molecules from the nucleus directly. Our experiments and transport model would suggest that the nucleus actively rejects exogenous macromolecules even below the passive permeability limit. This result can have a significant impact on biomedical research, especially in areas related to targeted drug delivery and gene therapy.

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Section: Methodsmentioning

confidence: 99%

Size-dependent steady state saturation limit in biomolecular transport through nuclear membranes

Shakhi,

Bijeesh,

Hareesh

et al. 2024

PLoS ONE

Self Cite

View full text Add to dashboard Cite

show abstract

“…The time-lapse confocal imaging of the passive nuclear import and export is done using an Olympus confocal microscope (FV 3000, Olympus corporation) and/ or an in-house constructed confocal fluorescence microscope (Shakhi, Bijeesh, Varier, & Nandakumar, 2021). The import mixture for the transport is freshly prepared before the experiment.…”

Section: Methodsmentioning

confidence: 99%

Size dependent steady-state saturation limit in biomolecular transport through nuclear membranes

Shakhi,

Bijeesh,

Hareesh

et al. 2023

Preprint

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The nucleus preserves the genomic DNA of eukaryotic organisms and maintains the integrity of the cell by regulating the transport of molecules across the nuclear membrane. It is hitherto assumed that small molecules having a size below the passive permeability limit are allowed to diffuse freely to the nucleus while the transport of larger molecules are regulated via an active mechanism involving energy. Here we report on the kinetics of nuclear import and export of a model system of dextran molecules having a size below the passive permeability limit. The studies carried out using time-lapse confocal fluorescence microscopy show a clear deviation from the passive diffusion model. In particular, it is observed that the steady-state concentration of dextran molecules inside the nucleus is consistently less than the concentration outside, in contradiction to the predictions of the passive diffusion model. Detailed analysis and modeling of the transport show that the nuclear export rates significantly differ from the import rates and the difference in rates is dependent on the size of the molecules. The nuclear export rates are further confirmed by an independent experimental study where we observe the diffusion of dextran molecules from the nucleus directly. Our experiments and transport model would suggest that the nucleus actively rejects exogenous macromolecules even below the passive permeability limit. This result can have significant impact on biomedical research especially in areas related to targeted drug delivery and gene therapy.

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A low-cost confocal microscope for the undergraduate lab

Reguilon

Bethard

Brekke

2023

American Journal of Physics

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We demonstrate a simple and cost-efficient scanning confocal microscope setup for use in advanced instructional physics laboratories. The setup is constructed from readily available commercial products, and the implementation of a 3D-printed flexure stage allows for further cost reduction and pedagogical opportunity. Experiments exploring the thickness of a microscope slide and the surface of solid objects with height variation are presented as foundational components of undergraduate laboratory projects and demonstrate the capabilities of a confocal microscope. This system allows observation of key components of a confocal microscope, including depth perception and data acquisition via transverse scanning, making it an excellent pedagogical resource.

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An in-house constructed dual channel confocal fluorescence microscope for biomolecular imaging

Cited by 4 publications

References 35 publications

Size-dependent steady state saturation limit in biomolecular transport through nuclear membranes

Size-dependent steady state saturation limit in biomolecular transport through nuclear membranes

Size dependent steady-state saturation limit in biomolecular transport through nuclear membranes

A low-cost confocal microscope for the undergraduate lab

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