Low-cost calcium fluorometry for long-term nanoparticle studies in living cells

Abstract

Calcium fluorometry is critical to determine cell homeostasis or to reveal communication patterns in neuronal networks. Recently, characterizing calcium signalling in neurons related to interactions with nanomaterials has become of interest due to its therapeutic potential. However, imaging of neuronal cell activity under stable physiological conditions can be either very expensive or limited in its long-term capability. Here, we present a low-cost, portable imaging system for long-term, fast-scale calcium fluorometry in neurons. Using the imaging system, we revealed temperature-dependent changes in long-term calcium signalling in kidney cells and primary cortical neurons. Furthermore, we introduce fast-scale monitoring of synchronous calcium activity in neuronal cultures in response to nanomaterials. Through graph network analysis, we found that calcium dynamics in neurons are temperature-dependent when exposed to chitosan-coated nanoparticles. These results give new insights into nanomaterial-interaction in living cultures and tissues based on calcium fluorometry and graph network analysis.

Multi-curvature micropatterns unveil distinct calcium and mitochondrial dynamics in neuronal networks

“…Rat embryonic brains (E18, BrainBits) were dissected following a previously established protocol. [31][32][33] Briefly, cortical tissues were dissociated in 10% (v/v) papain (Carica papaya, Roche) in Hibernate-E (BrainBits) at 37 °C for 15 min. Dissociated cortical neurons were centrifuged (6 min, 600 rpm, at room temperature) and then seeded at a concentration of 250 000 cells per ml per device.…”

“…S5a †). Mean fluorescent intensity for each ROI was extracted over the time series, and a numerical derivative was computed for each transient wave (ΔF/Δt) based on a similar algorithm reported in Beck et al 31 Calcium events based on increasing cytosolic calcium levels were denoted as a spike for visualization where the numerical derivative exceeded 2.5 times the standard deviation. The spike rate of calcium events was then computed as the average number of calcium increase events per second for each ROI (Fig.…”

“…Spike raster plots were compared using the Sørensen-Dice similarity coefficient. 31 Unique comparisons (i.e., a 3-length ROI index would follow as ROI 1 -ROI 2 , ROI 1 -ROI 3 , ROI 2 -ROI 3 ) was then linearized and run through the filter function (eqn (4)) where S i is the Sørensen-Dice similarity coefficient at the linear index i, and n max is the total length of the linearized list of spike events (Fig. S5c †).…”

Implementation of miniaturized modular-array fluorescence microscopy for long-term live-cell imaging

“…To circumvent the issue, diverse portable imaging systems have emerged to adapt existing cell-based assays and directly image live samples inside an incubator [6,7]. In particular, different miniaturized fluorescence microscopy have been proposed to image cells with high sensitivity and molecular specificity [8][9][10][11][12][13][14].…”