scholarly journals Single-particle evanescent light scattering simulations for total internal reflection microscopy

2006 ◽  
Vol 45 (28) ◽  
pp. 7299 ◽  
Author(s):  
Laurent Helden ◽  
Elena Eremina ◽  
Norbert Riefler ◽  
Christopher Hertlein ◽  
Clemens Bechinger ◽  
...  
Keyword(s):  
Light Scattering ◽  
Single Particle ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Microscopy ◽  
Evanescent Light

scholarly journals A light scattering model for total internal reflection microscopy of geometrically anisotropic particles

2019 ◽  
Vol 66 (10) ◽  
pp. 1139-1151 ◽  
Author(s):  
Adrian Doicu ◽  
Alina A. Vasilyeva ◽  
Dmitry S. Efremenko ◽  
Christopher L. Wirth ◽  
Thomas Wriedt
Keyword(s):  
Light Scattering ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Anisotropic Particles ◽  
Scattering Model ◽  
Total Internal Reflection Microscopy

scholarly journals Functional Characterization of an Electromagnetic Perceptive Protein

2020 ◽  
Author(s):  
Sunayana Mitra ◽  
Carlo Barnaba ◽  
Jens Schmidt ◽  
Galit Pelled ◽  
Assaf A. Gilad
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Total Internal Reflection ◽  
Magnetic Stimulation ◽  
New Technologies ◽  
Cellular Function ◽  
Single Particle Tracking ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Diffusion Dynamics

AbstractMagnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in animal models. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique in determining that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic stimulation activation at a single particle level. Trajectory analysis show notably different EPG protein kinetics before and after magnetic stimulation in both 2 (free vs bound particle) and 3 state (free vs intermediate vs bound particle) tracking models. These data serve to provide additional information that support and understand the underlying biophysical mechanisms behind EPG activation by magnetic stimulation. In conclusion, our results provide evidence for the basis of magnetoreception in EPG protein that would aid in future studies that seek to understand this novel mechanism. This study is important for understanding the phenomenon of magnetoreception as well as developing new technologies for magnetogenetics – the utilization of electromagnetic fields to remotely control cellular function.Graphical TOCElucidation of magnetoreception in a fish derived Electromagnetic Perceptive Gene (EPG), using genetic tagging and single particle tracking with Total Internal Reflection Fluorescence (TIRF) suggests changes in kinetics of membranal motion upon stimulation by magnetic field.

Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy

2006 ◽  
Vol 291 (1) ◽  
pp. G146-G155 ◽  
Author(s):  
Jong Hak Won ◽  
David I. Yule
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Acinar Cells ◽  
Internal Reflection ◽  
Pancreatic Acinar Cells ◽  
Wide Field ◽  
Exocrine Cells ◽  
Parotid Acinar Cells ◽  
Signaling Dynamics ◽  
Total Internal Reflection Microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.

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