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

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.

For image comparison, green-fluorescence was captured in a 24-bit RGB format and converted to 8-bit grey-scale. Properties of the digital microscope images were compared with images taken from the beads with an optical fluorescent microscope (Amscope, 20x, and 40x optical magnification with a 6-megapixel camera) as reference. Fluorescent beads were excited with 475 nm light. Batches of ten images of the beads were recorded from both the digital and the optical imaging system for statistical analysis.
Temperature sensation in human embryonic kidney cell culture. To demonstrate the effect of temperature control of the imaging system, we chose human embryonic kidney (HEK) as they are well known to exhibit endogenous calcium channels 12 and to show high sensitivity to non-physiological temperatures [13][14][15] . HEK cells were cultured in mouse embryonic fibroblast (MEF, passage 18) media. When grown to 80% confluency, cells were trypsinated and reseeded into pre-coated 35 mm Petri dishes for the temperature sensation experiment and grown for two days. For calcium fluorometry in HEK cells, Fluo-4 AM with probenecid acid was loaded to the cells (1:1) and incubated for 60 min in a standard incubator (37 ºC, 5% CO2) following vendor protocol (ThermoFisher). Afterwards, HEK cells were gently washed with pre-warmed MEF media and then returned to the incubator for two hours. For the temperature sensation experiment, calcium fluorescent HEK cells were placed into the live-cell fluorescent imaging system, and somatic calcium dynamics were monitored at 0.1 frames per min, 1 s exposure time for 10 h. LED-light excitation at 480 nm was automatically controlled and switched on and off 10 s prior to image capture. The digital microscope was set to 160X magnification. HEK cells were either monitored without heat at room temperature (w/o heat), or at physiological temperature (37 °C, w/ heat) in the temperature-controlled livecell imaging system.

Temperature sensation in primary cortical neuron cultures.
Calcium fluorometry is an important imaging methodology to study neuronal cell and network signalling [16][17][18][19][20][21][22][23][24] . To test the robustness of our imaging system with neurons, we monitored calcium signalling in neural cultures grown from dissociated rat cortical neurons. Rat cortical hemispheres were dissected from whole embryonic rat brains (E18, BrainBits) and dissociated with 10% (v/v) papain (Carica papaya, Roche) in Hibernate®-E (BrainBits) at 35 °C for 15 min. The dissociated cortical neurons were centrifuged (6 min, 600 rpm, at room temperature) and seeded at a cell concentration of 1 million cells per ml into 35 mm Petri dishes. Petri dishes were coated with 0.05% (v/v) polyethyleneimine (PEI) for two hours and washed three times with phosphate-buffered solution (PBS) before cell seeding. Cortical neurons were seeded at a cell density of 180 cells/mm 2 and incubated (95% air, 5% CO2, 37 °C) in Neurobasal serum-free with 2% (v/v) serum-free B-27® and 1% (v/v) PenStrep antibiotics and grown until day 8 in vitro. For calcium fluorometry in the neuronal cultures, Fluo-4 AM with probenecid acid was loaded to the cells (1:1) and incubated for 60 min in a standard incubator (37 ºC, 5% CO2) following vendor protocol (ThermoFisher). For the temperature sensation experiment, calcium fluorescent neuron cultures were placed into the live-cell fluorescent imaging system, and somatic calcium dynamics were monitored at 0.1 frames per min, 1 s exposure time for 10 h with cyclic on/off LED-light 480 nm excitation, identical to the HEK cell experiment. Neuronal cells were either monitored without heat at room temperature (w/o heat), or at physiological temperature (37 °C, w/ heat) in the temperature-controlled live-cell imaging system over ten hours.

Multi
Live-cell nano fluorometry. At 9 days in vitro (DIV) cortical neurons were loaded with Fluo-4 AM for 60 min and gently washed as described above. Then chitosan-coated magnetic nanoparticles (5×10 11 NP per ml, Chemicell, core: 100 nm, hydrodynamic radius: 190 nm, Fig. S5) were added to the calcium fluorescent living neurons and placed into the live-cell fluorescent imaging system for further live-cell fluorescent monitoring. Extensive characterization of the chitosan-coated NPs can be found in Tay, Kunze et al. 25 . Somatic calcium dynamics were recorded with LED-light 480nm excitation at 1 fps, 1 s exposure time for 5 minutes in the incubator system without heat at room temperature (w/o heat), or at physiological temperature (37 °C, w/ heat). During a two-hour interval, neurons were left without excitation and imaged again with the same parameters. This process was repeated three times for a total imaging time of 8 h. For control, fluorescent neurons without magnetic nanoparticles were monitored under the same imaging parameters with and without physiological temperature settings.
Calcium spike event detection and synchronous network activity mapping. Fluorescent images acquired in our live-cell imaging system were converted from their native '.wmv' video format to '.tiff' using FFmpeg. All image analysis was performed in MATLAB 2019A, and final graphical data were plotted using Origin Lab 2018b. First, the tiff-based image stack was converted into an 8-bit image stack, and single-cell bodies were segmented and saved as individual regions of interests (ROIs) with their corresponding spatial x, y coordinates. Time-varying changes of somatic fluorescence (Fpixel) were recorded and saved as a time series data with averaged fluorescent intensity values across the pixels in each ROI. Equation 2 shows as the total number of pixels within the ROI and as the intensity value of each indexed pixel in the ROI and � as the averaged fluorescence intensity per ROI.
Second, � was normalized by the average background ( ) for each frame resulting in * as shown in Third, the rate of relative fluorescence change (∆ * /∆ ), where ∆ is the framerate -1 was used for subsequent calcium signal analysis (spike event detection, signal correlation, and synchronous network activity mapping). Calcium spike events were distinguished based on calcium influx and efflux. For both a double threshold analysis was applied based on a static ( ∆ * ∆ > ±0.05) and a varying threshold ( ∆ * ∆ > ±5x standard deviation). A calcium spike event was then set as a calcium influx event for a positive amplitude above the highest positive threshold, and as a calcium efflux event for a negative amplitude below the smallest negative threshold. From these calcium events, raster plots were generated, showing either influx, efflux or both event types.
Pathological calcium events were analyzed separately from transient calcium dynamics. If a cell body exhibited a high cytosolic influx in calcium followed by a substantial efflux, this event might indicate a relation of calcium signalling with apoptosis or necrosis. These calcium events were identified by a systematic scan of the time-varying fluorescent data for a single influx followed by single efflux events. Next, the time delay (∆T) between the influx and efflux event was extracted.
Calcium raster plots were compared for signal correlation and used to derive a connectivity map based on synchronous spiking activity between individual cell bodies. Only cell bodies exhibiting multiple positive or negative peaks were analyzed. Network nodes in our connectivity map were defined by the exact coordinates of segmented active cell bodies (at least one spike event occurred within the segmented ROI). Transient calcium spiking events were compared for a synchronic pair-wise occurrence and mapped onto a cross-correlation matrix in MATLAB 2019A. Calcium signal cross-correlation was determined based on computing the Sørensen-Dice similarity coefficient. For Dice-coefficients between 0.5 and 0.9, cells were assumed to be weakly connected, and a line with a transparency value of 40% and 5 pixels in width was drawn between their corresponding ROIs. For Dice-coefficients larger than 0.9, cells were assumed to be strongly connected, and a line with a transparency value of 40% and 7 pixels in width was drawn between their corresponding ROIs.

Cell network analysis.
To compare macro and micro environmental effects on the neural network activity and performance, we further quantified three parameters for each individual network and reported their change over time. The three parameters are the number of active cells, the number of synchronous network connections, and the number of calcium spike events. Cells were counted as active if at least one calcium spike event had been identified from this indexed ROI throughout the recording period. The number of synchronous network connections was summed for each synchronously active ROI pair. Calcium event activity was computed based on the count of network connections normalized by the count of active cell bodies.

Long-term validation of cell viability.
To determine toxicity levels upon long-term imaging, we cultured Normal Rattus norvegicus Kidney (NRK, ATCC® CRL-6509™) epithelial cells in the imaging incubator for up to 48 h. Passage 6 NRK cells were detached from tissue culture flasks with 0.25% trypsin, 0.53 EDTA solution (2 min) and dispensed in new culture flasks at a subcultivation ratio of 1:6 (90% Dulbecco's Modified Eagle's Medium, 10% fetal bovine serum (v/v)). Culture flasks were placed in both the imaging incubator and a standard reference incubator for 48 hours. We performed bright field imaging at 0 h, 24 h, and 48 h. Live-dead staining was implemented by adding 4 µL of 3,3′-dioctadecyloxacarbocyanine (DiOC18) in 1 mL of culture medium at 24 h and 2 µL propidium iodide in 1 mL culture medium at 48 h followed by 5 min incubation at 37 °C. Cell viability (CV) was imaged using fluorescent microscopy and computed based on equation 4.

Impact of single-cell sample selection in analyzing digital live-cell fluorescent images during longterm image acquisition
Heterogeneity in calcium signalling within cell cultures may impact average calcium intensity plots. Within our data set, the MATLAB algorithm randomly selects single cell bodies. Figure S1 A1 -A2 show the distribution of normalized calcium intensity over time for three different samples with a fixed number of randomly selected single-cell bodies (ncell = 10). The plots only show minor differences in average intensity distribution. When accumulated over time, the data sets are not statistically different (Fig. S1 B). Decreasing the fixed number of randomly selected cell bodies from 10 to 5, as well as increase the number from 10 to 20, resulting in a similar average intensity over time. However, for a fixed number of 40 cells, we observed a significant shift in average signal intensity (Fig. S2). This artefact may be due to differences in sub neuronal cell types, differences in metabolism, or calcium signalling heterogeneity in neuronal networks. To exclude this artefact in our temperature experiments, we compared smaller sample sizes. However, we also used this artefact as a motivation to evaluate calcium signalling synchronicity over time in response to nanoparticle uptake.  Figure S3 and Figure S4 show comprehensive calcium data sets which include the rate of fluorescent change over time, extracted raster plots highlighting calcium influx and efflux events, and resulting cell-to-cell cross-correlation matrices from Fluo-4 loaded neurons which were recorded without temperature control (at room temperature, Fig. S3) and with temperature control at the physiological level (Fig. S4). Figure S3: Calcium data sets were obtained in our study under room temperature. Each panel consists of a rate of change in calcium intensity recording cumulated over the entire recording time (first row), which was used to extract calcium signal spiking (influx and efflux events). Time-correlation between each calcium event resulted in a cross-correlation matrix, where blue colours indicate a low probability, and red colours indicate a high probability of correlated calcium events. Each column shows calcium signal recording at 0 h, 2 h, 4 h, 6 h, and 8 h. Calcium signals were recorded at 1 fps for 150 s. Figure S4: Calcium data sets were acquired in the digital live-cell fluorescent imaging system under heated (37 °C) incubation. Each panel consists of a rate of change in calcium intensity recording cumulated over the entire recording time (first row), which was used to extract calcium signal spiking events (influx and efflux). Timecorrelation between each calcium event resulted in a cross-correlation matrix, where blue colours indicate a low probability, and red colours indicate a high probability of correlated calcium events. Each column shows calcium signal recording at 0 h, 2 h, 4 h, 6 h, and 8 h. Calcium signals were captured at 1 fps for 150 s.

Nanoparticle sizing with dynamic light scattering (DLS)
Incubating primary neuron cultures with nanomaterials has been shown to impact calcium signalling in previous studies [25][26][27] . To validate the capturing of similar calcium events in the digital live-cell imaging system, we incubated primary cortical neurons with chitosan-coated superparamagnetic nanoparticles. Figure S5 shows the size distribution of the hydrodynamic radius of the utilized nanoparticles measure in Neurobasal.

Supplementary characterization of key imaging parameters.
Histogram plots were used to assess image contrast for monitoring grey-scale, green, and red fluorescent images captured with the digital fluorescent microscope. Figure S6 shows the histogram distribution associated with the representative false-colour and grey-scale images. Although the image resolution of the digital fluorescent microscope is lower in comparison to images taken with a traditional optical fluorescent microscope, image contrast, as shown in the histogram plots based on green fluorescence in Figure S7, remains similar. Figure S7: Histogram plots were extracted from 8-bit green-colour fluorescent images which were taken with (A) the portable digital fluorescent imaging system, and with (B) the non-portable traditional optical fluorescent microscope (20x objective). Images show single primary cortical neurons that were loaded with Fluo-4 AM and grown for two weeks under standard culture conditions in a Petri dish. Figure S8 shows representative fluorescent images extracted from the video recordings done with the digital live-cell fluorescent imaging system to validate long-term image acquisition and portability. HEK cells were monitored in a lab-extern cell culture facility. Primary neuron cultures were monitored in our lab. Figure S9 shows representative fluorescent images extracted from the video recordings done with the digital live-cell fluorescent imaging system to validate short-term image acquisition using primary cortical neurons. Figure S8: Representative calcium images of Fluo-4 loaded human embryonic kidney (HEK) cells and primary cortical neurons. Green-fluorescent signals were monitored over time in the digital imaging system without (w/o heat) and with heat (w/ heat) setting to physiological temperature (37 °C). Figure S9: Representative calcium images of Fluo-4 primary cortical neurons. Green-fluorescent signals were monitored over time in the digital imaging system without (w/o heat) and with heat (w/ heat) setting to physiological temperature (37 °C). Figure S10A (right panel) shows NRK cells growing in culture flasks in the portable imaging incubator system at the start (0 h) and after 24 h and 48 h. Comparing NRK cells grown in the portable incubator with a standard incubator system yields no significant differences in their cell density, proliferation rate, morphology and cell viability (Fig. S10A-B). After 48 h, fluorescent imaging displayed cell viability of 98.7% for the established incubation system and 98.6% for the portable incubation system based on Live/ Dead staining (Fig. S10C, Live: green = DiOC18, Dead: red = Propidium Iodide). Hence, the portable imaging incubator can support live-cell imaging in a remote setting over up two days, if needed.  Table S1 compares quantitative measures of the assembled and used low-cost digital live-cell imaging systems against other reported low-cost digital and traditional optical imaging systems. All digital imaging systems have a clear cost advantage with varying long-term imaging capabilities; however, they lack behind in spatial resolution in comparison to traditional optical systems.