scholarly journals Effect of Photon Counting Shot Noise on Total Internal Reflection Microscopy

Soft Matter ◽  
2021 ◽  
Author(s):  
Fan Cui ◽  
David J Pine
Keyword(s):  
Shot Noise ◽  
Total Internal Reflection ◽  
Colloidal Particle ◽  
Photon Counting ◽  
Internal Reflection ◽  
Scattering Intensity ◽  
Transparent Substrate ◽  
Total Internal Reflection Microscopy

Total internal reflection microscopy (TIRM) measures changes in the distance between a colloidal particle and a transparent substrate by measuring the scattering intensity of the particle illuminated by an evanescent...

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.

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
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