Author response: Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes Article Swipe

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (Vm) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FASTM). Different from present multiplexing approaches, FASTM uses phase-sensitive signal detection, which renders various combinations of common probes for Vm and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FASTM allows simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity and minimal crosstalk. FASTM is also suited for multiplexing using single-cell microscopy and genetically encoded FRET biosensors. Moreover, FASTM is compatible with optochemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FASTM also allows resolving rapid chemical reactions. Altogether, FASTM opens new opportunities for interrogating cellular signaling. Editor's evaluation The number and temporal resolution of fluorescent probes that can be used simultaneously to interrogate cell signaling pathways are currently limited by overlap of their excitation and emission spectra. This study introduces a new technique to overcome these limitations and enable simultaneous recording of multiple probes at millisecond time resolution. The technique will facilitate studies of the temporal and causal relationships among many signaling pathways that can be targeted with fluorescent probes. https://doi.org/10.7554/eLife.63129.sa0 Decision letter eLife's review process Introduction Cells respond to external stimuli by changes in membrane potential (Vm), ions, messenger molecules, or protein modification (e.g., phosphorylation or dephosphorylation). These signaling events can be monitored in real time using fluorescent probes (Tsien, 1989; Rothman et al., 2005; Mehta and Zhang, 2011; Depry et al., 2013; Ni et al., 2018). To delineate the network of cellular responses, it would be ideal to use different probes under identical conditions in the same sample (dubbed multiplexing) (Keyes et al., 2021). Such measurements can not only reveal the precise sequence of signaling events, for example, whether they are upstream or downstream of each other, but also whether events are mechanistically coupled like ion transport across membranes via exchangers or symporters (Welch et al., 2011; Depry et al., 2013). When recorded in separate experiments on different samples, inter-experimental and cell-to-cell variations may obscure temporal and mechanistic relationships of events. Moreover, by design, probes bind their target molecules, which might perturb the dynamics and sequence of cellular responses (Lew et al., 1985; Haugh, 2012; Delvendahl et al., 2015). Such probe-related perturbations can be inferred from multiplexing experiments. Signaling events, such as ligand-receptor binding and changes in Vm and ions, often occur on millisecond or even sub-millisecond timescales. Multiplexing of such rapid events requires kinetic techniques that allow both precisely timed stimulation of cells and simultaneous recording from different probes on a millisecond timescale. Discrimination of simultaneously excited probes relies on the spectral separation of their emissions using optical filtering (Figure 1A). However, the spectral space for simultaneous recording of probes is limited (Neher and Neher, 2004) because crosstalk arising from overlapping emission spectra compromises their discrimination. Therefore, although many spectrally distinct probes for Vm and various ions and biomolecules have been developed (Depry et al., 2013; Yuan et al., 2013; Yin et al., 2015; Kulkarni and Miller, 2017; Mehta et al., 2018), simultaneous recording with millisecond time resolution has been restricted to two probes, for example, for two ion species or one ion species and Vm (e.g., Vogt et al., 2011; Jaafari et al., 2015; Deal et al., 2016). For multiplexing of more than two probes, quasi-simultaneous recording has been used: probes are excited and detected sequentially by switching between different excitation wavelengths (Figure 1B; Canepari et al., 2007; Canepari et al., 2008; Lee et al., 2012; Sulis Sato et al., 2017; Miyazaki et al., 2018; Ait Ouares et al., 2019; Nguyen et al., 2019). Although quasi-simultaneous multiplexing overcomes fluorescence crosstalk, it limits the temporal resolution and, thereby, the application range for studying rapid signaling events occurring on a millisecond timescale (van Meer et al., 2019). Hitherto, a multiplexing strategy combining millisecond temporal resolution with high flexibility regarding the number and combinations of probes has been lacking. Figure 1 Download asset Open asset Strategies for multiplexing of fluorescent probes, and outline of the chemosensory signal transduction in the flagellum of sea urchin sperm. (A) Spectrally separable emission spectra (dashed) of probes allow their simultaneous recording using optical filtering. (B) Spectrally separable excitation spectra (outlined) allow quasi-simultaneous recording of probes using excitation-switching. (C) Frequency-tagging and phase-sensitive detection of fluorescence combined with optical filtering using frequency- and spectrally-tuned multiplexing (FASTM) allow simultaneous recording of probes based on separable excitation and/or emission spectra. (D) Schematic of the chemosensory signaling pathway and (E) illustration of the time course of the signaling events in sea urchin sperm (reviewed in Strünker et al., 2015). Resact, the chemoattractant peptide released by the egg, triggers the synthesis of cGMP by activating a receptor guanylyl cyclase (GC). The rise in cGMP elicits a pulse-like Vm hyperpolarization mediated by a cyclic nucleotide-gated K+ channel (CNGK). The hyperpolarization activates a voltage-gated Na+/H+ exchanger (sNHE) and a hyperpolarization‐activated and cyclic nucleotide‐gated (HCN) channel. The Na+/H+ exchange increases [Na+]i and pHi. In turn, the increase in pHi primes pHi-controlled CatSper Ca2+ channels to open during the recovery from hyperpolarization driven by HCN channels. The resulting Ca2+ influx drives chemotactic steering towards the egg. (F) Schematic of the stopped-flow setup: one syringe is filled with a suspension of probe-loaded sperm, and a second syringe is filled with a solution of resact. The syringe pistons move synchronously to rapidly mix sperm with resact in a micromixer and subsequently push this mixture into an observation cuvette, where spectroscopic measurements are performed (see Hamzeh et al., 2019). Here, we introduce an approach that leverages phase-sensitive signal detection, which is commonly used to recover small signals buried in large noise (Meade, 1983), but also facilitates signal multiplexing (Aslund and Carlsson, 1993; Carlsson et al., 1994; Lewis et al., 2005; Hwang et al., 2015; Garbacik et al., 2018; Gómez-García et al., 2018; Tovar et al., 2019). We dubbed this method frequency- and spectrally-tuned multiplexing (FASTM). In brief, like conventional multiplexing, FASTM also involves the simultaneous excitation of different probes; however, the excitation light is modulated at distinct frequencies. The frequency-tagging of fluorescence combined with optical filtering allows discriminating probes based on their excitation and/or emission spectra (Figure 1C). We tested the time resolution and applicability of FASTM on signaling pathways of sperm and in single cultured cells. FASTM enabled multiplexing of at least three rapid signaling events at millisecond time resolution using various combinations of common non-ratiometric and ratiometric probes for ions and Vm as well as FRET-based biosensors. Moreover, FASTM can be combined with kinetic rapid-mixing techniques and flash-induced release of caged messengers, for example, cGMP, to instantaneously activate signaling pathways. Finally, FASTM is also suited to resolve rapid chemical reactions. These unique features of FASTM expand the scope of time-resolved multiplexing of cellular signaling. Results Multiplexing of rapid ionic and electrical signaling events using FASTM Chemosensory signaling in the flagellum of sea urchin sperm involves rapid changes in cellular messengers, ions, and Vm (Figure 1D and E) (reviewed in: Darszon et al., 2008; Strünker et al., 2015; Wachten et al., 2017; Darszon et al., 2020); therefore, sperm are an ideal model to develop and test novel strategies for multiplexing. In brief, a chemoattractant peptide, resact, activates a receptor guanylyl cyclase. The ensuing rise of cGMP elicits a brief transient hyperpolarization, followed by an increase of the intracellular pH (pHi) and Na+ concentration ([Na+]i) that, ultimately, trigger a Ca2+ influx and rise of the intracellular Ca2+ concentration ([Ca2+]i) (Figure 1E). The sequence of signaling events has been delineated by sequentially recording changes in either [Ca2+]i, pHi, [Na+]i, or Vm on different sperm samples using stopped-flow fluorimetry (Figure 1F; Hamzeh et al., 2019). We set out to record the resact-induced [Ca2+]i, pHi, and Vm signals in the same sperm sample by multiplexing of the respective fluorescent probes Fura-2, BCECF, and RhoVR (Deal et al., 2016). The well-separated excitation spectra (Figure 2A) render these three probes accessible for quasi-simultaneous recording. The [Ca2+]i, pHi, and Vm signals occur, however, on a millisecond timescale, which requires their simultaneous recording; yet, due to the overlapping emission spectra (Figure 2A), simultaneous recording of these three probes using optical filtering alone seems intractable. Therefore, we chose to multiplex Fura-2, BCECF, and RhoVR based on simultaneous excitation by three LEDs each modulated at a distinct frequency in the kHz range (Figure 2B, Table 1); thereby, the emission of each probe is tagged with a unique frequency signature for discrimination. The fluorescence was collected on opposite sides of the cuvette by two photomultipliers (PMTs) equipped with appropriate optical filters: one PMT detected the emission of Fura-2 and BCECF and the other that of RhoVR (Figure 2B, Table 2). Lock-in amplifiers demodulated and amplified the PMT signals in a phase-sensitive fashion to discriminate, in real time, the probes based on their modulation frequencies. We refer to this approach as FASTM. Figure 2 Download asset Open asset Experimental configuration for frequency- and spectrally-tuned multiplexing (FASTM) of Fura-2, BCECF, and RhoVR in a stopped-flow device. (A) Superposition of excitation (outlined) and emission (filled) spectra of fluorescent probes for Ca2+ (Fura-2), pH (BCECF), and Vm (RhoVR). Bandpass filters used for excitation (filled) and emission (outlined) are shown above the spectra. Inset: excitation and emission spectra depicted individually with respective filters (black bars). (B) Schematic of FASTM: each probe is excited by an LED modulated at a different frequency. The modulated emission is optically filtered and collected by two photomultipliers (PMTs). The PMT signals are demodulated by lock-in amplifiers in a phase-sensitive fashion to recover in real time [Ca2+]i, pHi, and Vm signals. We tested whether FASTM permits simultaneous recording of the three probes. First, using sperm that had been loaded with one probe only, we compared crosstalk between all three recording 'channels,' with (Figure 3, colored traces, FASTM) and without (Figure 3, gray traces, optical filtering) modulating the LEDs at different frequencies. In BCECF-loaded sperm, relying on optical filtering alone, the basal fluorescence intensity (Fo) recorded in the BCECF channel and the Fura-2 channel (Figure 3A, gray) and the relative increase (∆F/Fo), reflecting the pHi response (Figure 3B, gray), were similar. Of note, in Figure 3A, the gray (optical filtering) and blue traces (FASTM) in the BCECF channel are superimposed. Unsurprisingly, the BCECF fluorescence detected in the BCECF and the Fura-2 channel was similar, considering that both were collected by the same detector and optical filter (Figure 2B, Table 2). Basal BCECF fluorescence and the resact-induced relative increase were also detected in the RhoVR channel (Figure 3A and B), demonstrating that optical filtering is not sufficient to isolate the RhoVR channel from BCECF's broad emission spectrum. To quantify the crosstalk between channels, we plotted the first two seconds of the fluorescence signal recorded in the BCECF channel against that recorded in the Fura-2 or the RhoVR channel (Figure 3C, optical filtering). The slope of a linear fit to these plots is a measure of the crosstalk: if the time course of the fluorescence perfectly correlates between channels, the slope and crosstalk is 1 and 100%, respectively. Vice versa, if the time course of the fluorescence is independent among channels, the slope/crosstalk is zero. For optical filtering alone, we determined a crosstalk between the BCECF and the Fura-2 and RhoVR channels of 100 and 31%, respectively (Figure 3J). Modulating the LEDs at different frequencies using FASTM did not affect the fluorescence signal in the BCECF channel (Figure 3A and B, blue trace). However, FASTM lowered the basal fluorescence and almost abolished its relative increase in both the Fura-2 (Figure 3A and B; cyan) and the RhoVR channel (Figure 3A and B, orange); with FASTM, the crosstalk between the BCECF and the Fura-2 or the RhoVR channel was only 9 and 1%, respectively (Figure 3C and J). Figure 3 Download asset Open asset Resact-induced pHi, [Ca2+]i, and Vm signals recorded in sperm loaded with BCECF, Fura-2, or RhoVR using optical filtering alone or frequency- and spectrally-tuned multiplexing (FASTM). Time course of the fluorescence signals recorded from BCECF- (A–C), Fura-2- (D–F), or RhoVR-loaded sperm (G–I) after mixing with resact (50 pM). Fluorescence was recorded in the BCECF, Fura-2, and RhoVR channels using optical filtering alone (gray traces) or FASTM (colored traces). (A, D, G) Fluorescence signals in arbitrary fluorescence units (AFU); to ease the comparison, signals in (A), (D), and (G) were normalized (set to 1) to the baseline fluorescence (F0) in the BCECF, the Fura-2, and the RhoVR channel, respectively, recorded immediately after mixing with resact. (B, E, H) Resact-evoked change in fluorescence (ΔF) with respect to the baseline fluorescence (F0), that is, ΔF/F0 (%); #signals smoothed with a sliding average of 80 ms. (C, F, I) First 2 s of the fluorescence signal recorded in the BCECF channel plotted against that recorded in the Fura-2 or the RhoVR channel using either optical filtering (top panel) or FASTM (bottom panel). Gray line: linear fit of the plots to quantify the crosstalk between the channels (see explanation in the text). (J) Percent crosstalk between the channels according to the analysis shown in (C), (F), and (I). Figure 3—source data 1 Fluorescence signals in arbitrary fluorescence units. https://cdn.elifesciences.org/articles/63129/elife-63129-fig3-data1-v2.xlsx Download elife-63129-fig3-data1-v2.xlsx Next, we loaded sperm with Fura-2 alone and monitored the resact-induced [Ca2+]i response. With optical filtering alone, the basal fluorescence and its relative decrease, reflecting the [Ca2+]i response, were similar in all channels (Figure 3D and E; gray traces); the crosstalk between the Fura-2 and the BCECF or RhoVR channels was 98 and 79%, respectively (Figure 3F and J). Of note, Fura-2 fluorescence decreased with increasing [Ca2+]i because the probe was excited at 380 nm. FASTM did not affect the Fura-2 channel (Figure 3D and E; cyan), but lowered the basal fluorescence intensity and abolished its relative decrease in the BCECF channel (Figure 3D and E; blue) and the RhoVR channel (Figure 3D and E; orange); the crosstalk between the channels was ≤1% (Figure 3F and J). Finally, we monitored the resact-induced Vm response in RhoVR-loaded sperm. Due to the probe's red-shifted spectrum, crosstalk between channels was negligible; basal RhoVR fluorescence and its resact-induced decrease, reflecting the Vm response, were only detected in the RhoVR channel, both with and without FASTM (Figure 3G–J). We next loaded sperm with all three probes and simultaneously recorded resact-induced [Ca2+]i, pHi, and Vm signals (Figure 4A and B). Using optical filtering alone, the simultaneously recorded signals markedly differed from the respective signals recorded in sperm loaded with one probe only (compare Figure 4A and Figure 3B, E,H ); the pHi and [Ca2+]i signals represent a composite of the Fura-2-reported Ca2+ response (transient fluorescence decrease) and the BCECF-reported pHi response (sustained fluorescence increase), whereas the Vm signal featured a lower amplitude and slower kinetics (Figure 4A). Thus, the crosstalk among channels greatly misrepresented the true time course and size of signaling events. Figure 4 Download asset Open asset Simultaneous recording of resact-evoked pHi, [Ca2+]i, and Vm signals in sperm loaded with BCECF, Fura-2, and RhoVR. Relative changes in fluorescence ∆F/F0 evoked by 50 pM resact. The respective control signal evoked by mixing with artificial sea water (ASW) was subtracted, setting the control-signal level to ΔF/F0 (%) = 0 (dotted line). Signals were recorded using optical filtering alone (A) or frequency- and spectrally-tuned multiplexing (FASTM) (B). (C) Simultaneous FASTM recording of resact-evoked signals from pooled sperm loaded separately with either BCECF, Fura-2, or RhoVR. By contrast, using FASTM, we simultaneously recorded genuine resact-induced [Ca2+]i, pHi, and Vm signals in the respective channels (Figure 4B). The kinetics, waveforms, and amplitudes of the multiplexed signals were similar to those recorded with FASTM (compare Figure 4B with Figure 3B, E and H) or without FASTM (see previous studies, e.g., Hamzeh et al., 2019) in sperm loaded with one probe only. We further explored whether triple-loading per se affects the response waveforms. To this end, we pooled sperm suspensions that were separately loaded with either Fura-2, BCECF, or RhoVR. The overall time course of the [Ca2+]i, pHi, and Vm signals recorded simultaneously via FASTM from these pooled single-loaded sperm was similar to those recorded from triple-loaded sperm (Figure 4C). Competition of probes with downstream targets for signaling molecules might perturb response dynamics (Lew et al., 1985; Haugh, 2012; Delvendahl et al., 2015); in triple-loaded cells, this potential caveat might be enhanced. Therefore, using FASTM, we further examined in greater detail whether specific features of the signals were altered in single- vs. triple-loaded sperm. We compared resact-induced [Ca2+]i, pHi, and Vm signals in sperm loaded with one probe (single-loaded) to those in sperm loaded with three probes (triple-loaded); of note, for the ease of illustration, Fura-2 fluorescence was multiplied by –1 to depict an increase of [Ca2+]i as an increasing signal. Under both conditions, the respective signals were similar (Figure 5A and B). We took this comparison one step further and compared the resting membrane potential (Vrest) and threshold voltage (Vthr) at which [Ca2+]i and pHi commence to rise after stimulation with different resact concentrations; Vrest and Vthr are characteristic features of the signaling pathway (Figure 5C–E; Seifert et al., 2015). In single- and triple-loaded sperm, both Vrest (Figure 5C) and Vthr (Figure 5D and E) were similar. Thus, signaling is neither perturbed by Ca2+- and H+-binding to Fura-2 and BCECF, respectively, nor by partition of RhoVR into the membrane, at least under the experimental regimes used here. Figure 5 Download asset Open asset Interrogating putative probe-related perturbations of signaling. Resact-evoked Vm, pHi, and [Ca2+]i signals recorded individually from different sperm samples loaded with one probe only (A) or recorded simultaneously from triple-loaded sperm (B); to facilitate direct comparison, Fura-2 fluorescence was multiplied by –1 to depict an increase of [Ca2+]i as an increasing signal. (C) Comparison of Vrest of sperm loaded with RhoVR (single-loaded) or RhoVR, BCECF, and Fura-2 (triple-loaded). (D) Calibrated resact-induced (50 pM) Vm response and accompanying pHi and [Ca2+]i signals. The artificial sea water (ASW) control was subtracted, and the dotted black line indicates ΔF/F0 = 0 and Vrest. The Vm at the onset of the pHi and [Ca2+]i signals was deduced from the signal latencies. (E) Vm at the onset of pHi and [Ca2+]i signals in single- versus triple-loaded sperm. With increasing resact concentrations, the rise in pHi and [Ca2+]i commenced at increasingly negative Vm (Seifert et al., 2015). We conclude that Fura-2, BCECF, and RhoVR are not suitable for simultaneous recording based on optical filtering alone, whereas FASTM permits this probe combination for multiplexing of rapid [Ca2+]i, pHi, and Vm responses with millisecond time resolution. To illustrate the versatility of FASTM, we tested different triple combinations of Vm, Ca2+, pH, and Na+ probes, whose overlapping emission spectra prevent simultaneous recording using optical filtering alone (Figure 6A–C, Figure 6—figure supplement 1). By contrast, FASTM allowed for crosstalk-free multiplexing of resact-induced Vm-[Ca2+]i-pHi (Figure 6D), Vm-[Ca2+]i-[Na+]i (Figure 6E), or Vm-pHi-[Na+]i (Figure 6F) responses. Figure 6 with 1 supplement see all Download asset Open asset Simultaneous recording of resact-induced signaling events in sperm loaded with various triple combinations of probes for Ca2+, pH, Na+, and Vm using frequency- and spectrally-tuned multiplexing (FASTM). Superposition of excitation (outlined) and emission (filled) spectra of (A) Fura-2, VF2.1.Cl, and pHrodo; (B) Fura-2, RhoVR, and ANG-2; (C) BeRST, pHrodo, and ANG-2. Bandpass filters used for excitation (filled) and emission (outlined) are depicted above the spectra. Inset: individual excitation and emission spectra with respective filters (black bars). (D–F) Signals (∆F/F0) evoked by 500 pM resact corrected for the artificial sea water (ASW) control and normalized to their respective peak values (set to 1) for easier illustration. Finally, the shift of the excitation spectra of Fura probes and BCECF upon Ca2+ and H+ binding, respectively, can be harnessed to quantify [Ca2+]i or pHi in absolute terms using ratiometric recording (O'Connor and Silver, 2013). This relies on obtaining the ratio of the probe's emission recorded at two different excitation wavelengths, which, in previous studies, required switching between excitation wavelengths. We whether FASTM allows for simultaneous ratiometric recording of and Moreover, we used of sea urchin sperm, FASTM in different cells. In sperm, the CatSper Ca2+ channel is at pHi and also by the et al., 2011; Strünker et al., We and BCECF-loaded sperm in the stopped-flow with or and BCECF were simultaneously excited each at two different wavelengths and with the emission was collected at by one detector (Figure and B, Table evoked an rapid and more increase in the emission of BCECF and Fura-2, respectively, reflecting the pHi increase and Ca2+ influx via respectively (Figure By contrast, evoked an increase of the reflecting Ca2+ whereas the BCECF ratio was (Figure These that FASTM minimal crosstalk between the and the BCECF channels (Figure Figure supplement 1). FASTM allows for simultaneous recording of rapid signaling events with millisecond temporal resolution using various combinations of non-ratiometric and ratiometric probes. Figure with 1 supplement see all Download asset Open asset Simultaneous ratiometric recording of [Ca2+]i and pHi signals in sperm. (A) excitation (outlined) and emission (filled) spectra of and Inset: individual spectra with respective filters (black bars). (B) Schematic of frequency- and spectrally-tuned multiplexing (FASTM) configuration for simultaneous ratiometric recording of and BCECF in sperm. (C) among channels based on the analysis shown in Figure supplement these conditions, the approach to quantify crosstalk an see Figure supplement 1). (D) ratiometric [Ca2+]i and pHi signals in sperm evoked by or corrected for the fluorescence signals in the individual and BCECF channels the of FASTM with of caged and tools (e.g., ion channels, caged to cellular signaling pathways 2007; et al., et al., 2018). In combining such tools with fluorescent probes requires the from the trigger such as the used for filtering alone is not sufficient to prevent recording by the (e.g., see Strünker et al., et al., et al., We used sea urchin sperm to whether FASTM can were loaded with pHrodo, BeRST, and cGMP to simultaneously record [Ca2+]i, pHi, and Vm responses evoked by the intracellular of cGMP that receptor et al., 2019; Figure and B). due to the phase-sensitive signal detection, the was by the lock-in amplifiers et al., 2019) and FASTM allowed for simultaneous recording of [Ca2+]i, pHi, and Vm signals (Figure Figure Download asset Open asset Simultaneous recording of [Ca2+]i, pHi, and Vm signals in sea urchin sperm evoked by of caged (A) of and excitation (outlined) and emission (filled) spectra of pHrodo, and Bandpass filters used for excitation (filled) and emission (outlined) are depicted above the spectra. Inset: individual spectra and respective filters (black bars). (B) Schematic of frequency- and spectrally-tuned multiplexing (FASTM) configuration for experiments. (C) Vm, pHi, and [Ca2+]i signals evoked by intracellular cGMP with a 50 (gray Multiplexing of chemical using FASTM We next explored whether FASTM also allows multiplexing of chemical in Using the stopped-flow we simultaneously monitored the kinetics of Ca2+ from Fura-2 and (Figure Figure supplement 1). solution Fura-2, and was with an of the Ca2+ that with the probes for binding of of Ca2+ was by a decrease of and Fura-2 fluorescence decreased and at and respectively (Figure of the traces the (Figure The of Fura-2 2 was similar to that et al., and whereas that of 3 and 2 = had not been determined to the of These experiments the of FASTM for multiplexing of rapid chemical reactions. Figure 9 with 2 see all Download asset Open asset Simultaneous recording of the kinetics of Ca2+ from Fura-2, and (A) excitation (outlined) and emission (filled) spectra of Fura-2, and Inset: individual spectra depicted with respective filters (black bars). (B) Schematic of the frequency- and spectrally-tuned multiplexing (FASTM) configuration for simultaneous recording of Ca2+ from Fura-2, and (C) between channels according to Figure supplement (D) in Fura-2, and fluorescence and emission ratio of Fura-2 upon mixing of the probes with the Ca2+ (E) values determined by of the individual fluorescence traces and the ratio of Fura-2 Fura-2, 2 Fura-2, 3 Fura-2 2 3 2 = FASTM fluorescence microscopy Finally, we tested FASTM for single-cell fluorescence microscopy (Figure