Analyzing optical imaging of Ca 2+ signals via TIRF microscopy: The limits on resolution due to chemical rates and depth of the channels

Abstract

High resolution total internal reflection (TIRF) microscopy (TIRFM) together with detailed computational modeling provides a powerful approach towards the understanding of a wide range of Ca2+ signals mediated by the ubiquitous inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel. Exploiting this fruitful collaboration further requires close agreement between the models and observations. However, elementary Ca2+ release events, puffs, imaged through TIRFM do not show the rapid single-channel openings and closings during and between puffs as are present in simulated puffs using data-driven single channel models. TIRFM also shows a rapid equilibration of 10ms after a channel opens or closes which is not achievable in simulation using standard Ca2+ diffusion coefficients and reaction rates between indicator dye and Ca2+. Furthermore, TIRFM imaging cannot decipher the depth of the channel with respect to the microscope, which will affect the change in fluorescence that the microscope detects, thereby affecting its sensitivity to fast single-channel activity. Using the widely used Ca2+ diffusion coefficients and reaction rates, our simulations show equilibration rates that are eight times slower than TIRFM imaging. We show that to get equilibrium rates consistent with observed values, the diffusion coefficients and reaction rates have to be significantly higher than the values reported in the literature, and predict the channel depth to be 200–250nm. Finally, we show that with the addition of noise, short events due to 1–2ms opening and closing of channels that are observed in computational models can be missed in TIRFM.

MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale

“…However, with recent innovations in camera technologies, notably the development of cameras using large, high resolution 250 Mpixel sensors such as the Canon 2U250MRXSAA CMOS sensor, tenfold higher imaging speeds (2.4 fps) can be achieved by avoiding the need for chip shifting. In combination with environmental control, this will offer opportunities to study faster dynamic processes, for example, the action of fast-acting antimicrobial peptides 23 or imaging of calcium transients in the plasma membrane 24 .…”

TraceSpecks: A Software for Automated Idealization of Noisy Patch-Clamp and Imaging Data

“…The propagation of Ca 2þ and buffers was solved implicitly using the Laplacian of Ca 2þ and buffers in spherical coordinates on a hemispherical volume of radius 5mm and a spatial grid size of 5nm. We estimated the fluorescence changes ðDF=F 0 Þ from TIRFM by following the procedure in (34,35), i.e.,…”

Total Internal Reflection Fluorescence (TIRF) Microscopy

“…TIRFM can be used to measure the kinetics of receptor endocytosis in response to ligand binding (Hellen & Axelrod, 1991; Rao, Nawara, & Mattheyses, 2022; Riven, Iwanir, & Reuveny, 2006; Tabor et al., 2016), receptor insertion into the plasma membrane (Yudowski, Puthenveedu, & von Zastrow, 2006), receptor channel opening and closing (Toglia, Ullah, & Pearson, 2017; Yao & Qin, 2009), receptor clustering (Drenan et al., 2008; Erdelyi, Simon, Barnard, & Kaminski, 2014; Salavessa et al., 2021; Sungkaworn, Rieken, Lohse, & Calebiro, 2014), the lateral movement of receptors (Fowler, Aryal, Suen, & Slesinger, 2007; Salavessa et al., 2021), and receptor stoichiometry (Salavessa & Sauvonnet, 2021). For these types of studies, TIRFM is sometimes combined with other advanced microscopy techniques (e.g., fluorescence recovery after photobleaching; see (Hellen & Axelrod, 1991; Riven et al., 2006)).…”