Imaging Membrane Curvature inside a Model Immunological Synapse in RBL-2H3 Cells using TIRF Microscopy with Polarized Excitation

Machado, Raphael C. S.; Bendesky, Justin; Brown, Madison; Spendier, Kathrin; Hagen, Guy M.

doi:10.1101/608463

2019

DOI: 10.1101/608463

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Imaging Membrane Curvature inside a Model Immunological Synapse in RBL-2H3 Cells using TIRF Microscopy with Polarized Excitation

Raphael C. S. Machado

Justin Bendesky

Madison Brown

et al.

Abstract: Total internal reflection fluorescence microscopy with polarized excitation (P-TIRF) can be used to image nanoscale curvature phenomena in live cells. We used P-TIRF to visualize rat basophilic leukemia cells (RBL-2H3 cells) coming into contact with a supported lipid bilayer, modeling an immunological synapse. These studies help correlate the dynamics of cell surface molecules with the mechanical properties of the plasma membrane during synapse formation.

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TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells

Drawbond

Spendier

2019

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Total internal reflection fluorescence (TIRF) microscope image sequences are commonly used to study receptors in live cells. The dataset presented herein facilitates the study of the IgE-FcεRI receptor signaling complex (IgE-RC) in rat basophilic leukemia (RBL-2H3) cells coming into contact with a supported lipid bilayer with 25 mol% N-dinitrophenyl-aminocaproyl phosphatidylethanolamine, modeling an immunological synapse. TIRF microscopy was used to image IgE-RCs within this FcεRI-centric synapse by loading RBL-2H3 cells with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) in suspension for 24 h. Fluorescent anti-DNP IgE (IgE 488 ) concentrations of this suspension increased from 10% to 100% and corresponding non-fluorescent anti-DNP IgE concentrations decreased from 90% to 0%. After the removal of unbound anti-DNP IgE, multiple image sequences were taken for each of these ten conditions. Prior to imaging, anti-DNP IgE-primed RBL-2H3 cells were either kept for a few minutes, for about 30 min, or for about one hour in Hanks buffer. The dataset contains 482 RBL-2H3 model synapse image stacks, dark images to correct for background intensity, and TIRF illumination profile images to correct for non-uniform TIRF illumination. After background subtraction, non-uniform illumination correction, and conversion of pixel units from analog-to-digital units to photo electrons, the average pixel intensity was calculated. The average pixel intensity within FcεRI-centric synapses for all three Hanks buffer conditions increased linearly at a rate of 0.42 ± 0.02 photo electrons per pixel per % IgE 488 in suspension. RBL-2H3 cell degranulation was tested by detecting β-hexosaminidase activity. Prolonged RBL-2H3 cell exposure to Hanks buffer inhibited exocytosis in RBL-2H3 cells.Dataset: http://dx.

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TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells

Drawbond

Spendier

2019

Data

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An Improved Bio-Orientation Method Based on Direct Sunlight Compensation for Imaging Polarization Sensor

Li,

Zhang,

Fan

et al. 2024

J. Imaging

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Direct sunlight in complex environmental conditions severely interferes with the light intensity response for imaging Polarization Sensor (PS), leading to a reduction in polarization orientation accuracy. Addressing this issue, this article analyzes the impact mechanism of direct sunlight on polarization sensor detection in a complex environment. The direct sunlight interference factor is introduced into the intensity response model of imaging polarization detection, enhancing the accuracy of the polarization detection model. Furthermore, a polarization state information analytical solution model based on direct sunlight compensation is constructed to improve the accuracy and real-time performance of the polarization state information solution. On this basis, an improved bio-orientation method based on direct sunlight compensation for imaging polarization sensor is proposed. The outdoor dynamic reorientation experiment platform is established to validate the effectiveness of the proposed method. Compared with the traditional methods, the experimental results demonstrate a 23% to 47% improvement in the polarization orientation accuracy under various solar zenith angles.

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Imaging Membrane Curvature inside a Model Immunological Synapse in RBL-2H3 Cells using TIRF Microscopy with Polarized Excitation

Cited by 2 publications

References 33 publications

TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells

TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells

An Improved Bio-Orientation Method Based on Direct Sunlight Compensation for Imaging Polarization Sensor

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