Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes
- Centre of Reproductive Medicine and Andrology, University of Münster, Germany
- CiM-IMPRS Graduate School, University of Münster, Germany
- Department of Chemistry, University of California, Berkeley, United States
- Department of Molecular & Cell Biology, University of California, Berkeley, United States
- Helen Wills Neuroscience Institute, University of California, Berkeley, United States
- Molecular Sensory Systems, Center of Advanced European Studies and Research, Germany
- Institute of Innate Immunity, Department of Biophysical Imaging, Medical Faculty, University of Bonn, Germany
- Institute of Biological Information Processing (IBI-1), Research Center Jülich, Germany
- Life & Medical Sciences Institute (LIMES), University of Bonn, Germany
- Cells in Motion Interfaculty Centre, University of Münster, Germany
Figures
Figure 1
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 …
Figure 2
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 …
Figure 3
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 …
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Figure 3—source data 1
Fluorescence signals in arbitrary fluorescence units.
- https://cdn.elifesciences.org/articles/63129/elife-63129-fig3-data1-v2.xlsx
Figure 4
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…
Figure 5
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 …
Figure 6 with 1 supplement
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 …
Figure 6—figure supplement 1
Spectral utility of voltage-sensitive probes.
Calibrated resact-evoked (500 pM) Vm response in sea urchin sperm reported by VF2.1.Cl (A), RhoVR (B), and BeRST (C) upon excitation at the principal peak (gray) versus excitation at the secondary …
Figure 7 with 1 supplement
Simultaneous ratiometric recording of [Ca2+]i and pHi signals in human sperm.
(A) Superimposed excitation (outlined) and emission (filled) spectra of Fura-FF and BCECF. Inset: individual spectra with respective filters (black bars). (B) Schematic of frequency- and …
Figure 7—figure supplement 1
Analysis of crosstalk between BCECF and Fura-FF channels recorded with frequency- and spectrally-tuned multiplexing (FASTM).
Human sperm loaded with BCECF or Fura-FF were mixed with either NH4Cl or progesterone, respectively. The first 5 s (BCECF-loaded sperm) and 20 s (Fura-FF-loaded sperm) of the fluorescence signals …
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Figure 7—figure supplement 1—source data 1
Fluorescence signals for the analysis of crosstalk between BCECF and Fura-FF channels.
- https://cdn.elifesciences.org/articles/63129/elife-63129-fig7-figsupp1-data1-v2.xlsx
Figure 8
Simultaneous recording of [Ca2+]i, pHi, and Vm signals in sea urchin sperm evoked by flash photolysis of caged cGMP.
(A) Superimposed absorbance spectrum of BECMCM-cGMP and excitation (outlined) and emission (filled) spectra of Fluo-4, pHrodo, and BeRST. Bandpass filters used for excitation (filled) and emission …
Figure 9 with 2 supplements
Simultaneous recording of the kinetics of Ca2+ dissociation from Fura-2, Fluo-4, and Calbryte 630.
(A) Superimposed excitation (outlined) and emission (filled) spectra of Fura-2, Fluo-4, and Calbryte 630. Inset: individual spectra depicted with respective filters (black bars). (B) Schematic of …
Figure 9—figure supplement 1
Analysis of crosstalk between Fura-2, Fluo-4, and Calbryte 630 channels simultaneously recorded with FASTM.
The first 18 ms of the fluorescence signals recorded in the different channels were plotted against each other, and crosstalk was evaluated by a linear fit to the data. The steepness of the linear …
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Figure 9—figure supplement 1—source data 1
Fluorescence signals for the analysis of crosstalk between Fura-2, Fluo-4, and Calbryte 630 channels.
- https://cdn.elifesciences.org/articles/63129/elife-63129-fig9-figsupp1-data1-v2.xlsx
Figure 9—figure supplement 2
Signal-to-noise (S/N) ratio of fluorescence signals recorded upon mixing of Ca2+-bound Fura-2 with BAPTA.
(A) Kinetics of Ca2+ dissociation from Fura-2 recorded upon excitation at 340 nm. The kinetics were recorded with frequency- and spectrally-tuned multiplexing (FASTM) using either different lock-in …
Figure 10
Single-cell frequency- and spectrally-tuned multiplexing (FASTM) fluorescence microscopy for simultaneous recording of cAMP and [Ca2+]i.
(A) Octopamine-signaling pathway in HEK cells coexpressing the DmOCTβ1 receptor, CNGA2-TM channel, and a FRET-based cAMP biosensor. (B) Superimposed excitation (outlined) and emission (filled) …
Figure 11 with 1 supplement
Simultaneous recording of cAMP and [Ca2+]i signals in single cells using frequency- and spectrally-tuned multiplexing (FASTM).
(A) Octopamine-induced (20 µM) [Ca2+]i signals and changes in the FRET ratio (donor/acceptor), that is, cAMP signals, in the absence or presence of a non-binding (R307Q), lower (M329C), or …
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Figure 11—source data 1
Onset of the optopamine-induced cAMP and [Ca2+]i signals.
- https://cdn.elifesciences.org/articles/63129/elife-63129-fig11-data1-v2.xlsx
Figure 11—figure supplement 1
Affinity of mlCNBD-M329C for 8-NBD-cAMP.
Increase of 8‐NBD‐cAMP fluorescence (emission at 550 nm) upon binding to mlCNBD-M329C (1 μM). 8‐NBD‐cAMP fluorescence in the absence of mlCNBD-M329C was subtracted. The solid line represents a …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (HEK293) | flp-In-293 | Invitrogen | #R750-07 | RRID:CVCL_U421 |
| Transfected construct (Drosophila melanogaster) | DmOCTβ1R | Balfanz et al., 2005 | ||
| Transfected construct (Bos taurus) | CNGA2-TM | Schröder-Lang et al., 2007 | ||
| Recombinant DNA reagent | pc3.1-ml CNBD-FRET | Mukherjee et al., 2016 | ||
| Recombinant DNA reagent | pc3.1-mlCNBD-FRET-R307Q | Mukherjee et al., 2016 | ||
| Recombinant DNA reagent | pc3.1-mlCNBD-FRET-M329C | This paper | Figure 11—figure supplement 1 and materials and methods part of this MS. | |
| Other | Pluronic F-127 | Sigma-Aldrich | P2443 | |
| Other | Fluo-4 AM | Thermo Fisher | F14202 | |
| Other | BCECF AM | Thermo Fisher | B1150 | |
| Other | Fura-2 AM | Thermo Fisher | F1201 | |
| Other | pHrodo Red AM | Thermo Fisher | P35372 | |
| Other | ANG-2 AM | MobiTec | 3502 | |
| Other | Calbryte 630 AM | AAT Bioquest | 20720 | |
| Other | Fura-FF, AM | AAT Bioquest | 21027 | |
| Other | VF2.1.Cl | Miller et al., 2012 | Sold by Thermo Fisher as FluoVolt | |
| Other | BeRST | Huang et al., 2015 | ||
| Other | RhoVR | Deal et al., 2016 | ||
| Other | Calbryte 630, potassium salt | AAT Bioquest | 20727 | |
| Other | Fluo-4, pentapotassium salt | Thermo Fisher | F14200 | |
| Other | Fura-2, pentapotassium salt | Thermo Fisher | F1200 |
Table 1
Loading protocols for fluorescent probes in A. punctulata sperm and FASTM modulation frequencies.
| Fura-2, BCECF, RhoVR | ANG-2, pHrodo, BeRST | |||||
| Loading order | First | Second | Third | First | Second | Third |
| Name | Fura-2 AM | RhoVR | BCECF AM | ANG-2 AM | pHrodo Red AM | BeRST |
| Probe type | Ca2+ | Vm | pH | Na+ | pH | Vm |
| Concentration (µM) | 10 | 5 | 5 | 10 | 10 | 5 |
| Incubation (min) | 90 | 10 | 5 | 90 | 25 | 10 |
| FASTM modulation frequency (kHz) | 30.4 | 37.3 | 50 | 37 | 50 | 23 |
| Fura-2, ANG-2, RhoVR | Fura-2, pHrodo, VF2.1.Cl | |||||
| Loading order | First | Second | Third | First | Second | Third |
| Name | Fura-2 AM | ANG-2 AM | RhoVR | Fura-2 AM | pHrodo Red AM | VF2.1.Cl |
| Probe type | Ca2+ | Na+ | Vm | Ca2+ | pH | Vm |
| Concentration (µM) | 10 | 10 | 5 | 10 | 10 | 5 |
| Incubation (min) | 90 (added together) | 5 | 50 | 35 | 5 | |
| FASTM modulation frequency (kHz) | 50 | 23 | 37 | 50.3 | 25.3 | 17.1 |
| Fluo-4, phrodo, BeRST, BECMCM-cGMP | ||||||
| Loading order | First | Second | Third | Fourth | ||
| Name | BECMCM-cGMP | Fluo-4 AM | pHrodo Red AM | BeRST | ||
| Probe type | Caged cGMP | Ca2 | pH | Vm | ||
| Concentration (µM) | 10 | 10 | 10 | 5 | ||
| Incubation (min) | 15 | 10 | 35 | 10 | ||
| FASTM modulation frequency (kHz) | None | 37.3 | 30.1 | 50.3 | ||
Table 2
Optical configurations for recording signals from A. punctulata sperm.
| Probe combination | Fluorescent probe | LED (Thorlabs) | Excitation filter (Semrock) | Dichroics | Emission filter(Semrock) |
|---|---|---|---|---|---|
| Fura-2, BCECF, RhoVR | Fura-2 | M375L4 | 379/34 | 470 LPXR (Chroma)HC BS 409 (Semrock) | 524/24 |
| BCECF | M490L4 | 485/20 | |||
| RhoVR | M455L4 | 438/24 | 607/36 | ||
| ANG-2, pHrodo, BeRST | ANG-2 | M490L4 | 485/20 | 525 LPXR (Chroma)470 LPXR (Chroma) | 542/20 |
| pHrodo | M565L3 | 575/19 | 593LP | ||
| BeRST | M455L4 | 438/24 | |||
| Fura-2, ANG-2, RhoVR | Fura-2 | M340L4 | 340/22 | 470 LPXR (Chroma)HC BS 409 (Semrock) | 542/20 |
| ANG-2 | M505L3 | 513/17 | |||
| RhoVR | M455L4 | 438/24 | 607/36 | ||
| Fura-2, pHrodo, VF2.1.Cl | Fura-2 | M340L4 | 340/22 | 470 LPXR (Chroma)HC BS 409 (Semrock) | 542/20 |
| VF2.1.Cl | M455L4 | 438/24 | |||
| pHrodo | M565L3 | 575/19 | 593LP | ||
| BECMCM- cGMP, Fluo-4, pHrodo, BeRST | BECMCM-cGMP | M340L4 | 340/22 | 525 LPXR (Chroma)470 LPXR (Chroma)HC BS 409 (Semrock) | |
| Fluo-4 | M490L4 | 494/20 | 542/20 | ||
| pHrodo | M565L3 | 575/19 | 593LP | ||
| BeRST | M455L4 | 438/24 |
Table 3
Optical configuration for simultaneous ratiometric recording of [Ca2+]i and pHi signals in human sperm.
| Fluorescent probe | LED (Thorlabs) | FASTM modulation frequency (kHz) | Excitation filter (Semrock) | Dichroics | Emission filter(Semrock) |
|---|---|---|---|---|---|
| Fura-FF | M340L4 | 87.3 | 340/22 | HC BS 365 (Semrock)HC BS 409 (Semrock)470 LPXR (Chroma) | 524/24 |
| M375L4 | 73.51 | 370/10 | |||
| BCECF | M455L4 | 61.7 | 445/20 | ||
| M490L4 | 103.7 | 485/20 |
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FASTM, frequency- and spectrally-tuned multiplexing.
Table 4
Optical configuration for koff determination.
| Fluorescent probe | LED (Thorlabs) | FASTM modulation frequency (kHz) | Excitation filter (Semrock) | Dichroics | Emission filter(Semrock) |
|---|---|---|---|---|---|
| Fura-2 | M340L4 | 87.31 | 340/22 | HC BS 365 (Semrock)HC BS 409 (Semrock)525 LPXR (Chroma) | 524/24 |
| M375L4 | 103.7 | 370/10 | |||
| Fluo-4 | M490L4 | 59.51 | 485/20 | ||
| Calbryte 630 | M565L3 | 47.1 | 586/20 | 647/57 |
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FASTM, frequency- and spectrally-tuned multiplexing.
