Simultaneous Multicolor Imaging
Calculate spectral separation, crosstalk, timing budgets, and SNR for multi-channel fluorescence imaging.
Binned pixel
6.5 µm
Frame rate, simultaneous
45.5 fps
Frame rate, sequential
15.2 fps
SNR, own dye only
21.16
Channels
Channel 1
Channel 2
Channel 3
Spillover matrix (%)
Signal of each dye in each channel, as a percentage of its signal in its own channel, with all lasers on in one frame, so it depends only on the emission spectra.
| Channel ↓ / dye → | EGFP | mCherry | miRFP670 | SNR |
|---|---|---|---|---|
| 1: 525/50 | 100.00 | 0.00 | 0.00 | 21.16 |
| 2: 610/40 | 3.80 | 100.00 | 1.57 | 20.62 |
| 3: 700/75 | 0.81 | 43.43 | 100.00 | 17.63 |
Emission spectra and filters
Frame rate vs exposure
Model
R_ij ∝ ε_j Σ_k∈lasers(i) f_ex,j(λ_k) Φ_j ∫_filter i F_j dλ / ∫F_j dλ, spillover L_ij = R_ij / R_jj
Simultaneous: lasers(i) = all lasers, so the excitation cancels in L_ij. Sequential: lasers(i) = {laser i}.
S = flux × t × QE × B², SNR_i = S / √(S + Σ_j≠i L_ij S + I_dark t B² + σ_read²)
σ_read² = σ² (charge binning) or B²σ² (digital sum); f = 1/(t + t_read), sequential 1/(N(t + t_read))
Spectra are the sampled FPbase spectra (CC BY-SA 4.0) of the fluorescence-spectra page. Every laser delivers the same photon flux. Simultaneously, spillover depends only on the emission ratio between filters; excitation crosstalk (a laser exciting another dye) enters only in sequential mode. Filters are ideal top hats and each dye gives the same own-channel signal S; bleed-through photons count as background shot noise, before any linear unmixing. Laser switching time and the camera's overlap readout mode are not modelled.
Model and references
Textbook approximation. A standard approximation from the literature (for example paraxial, plane-wave or Gaussian-beam), valid within the range the page states. About the tiers
Spillover L_ij = R_ij/R_jj from sampled FPbase spectra, equal photon flux per laser, top-hat filters. Simultaneous: only the emission sets L (the lasers cancel); sequential adds excitation crosstalk. Bleed-through adds shot noise before unmixing; charge or digital binning sets the read noise.
- Roederer M. (2001). Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45, 194–205. Spillover as the signal in a secondary detector over that in the primary one. doi:10.1002/1097-0320(20011101)45:3<194::AID-CYTO1163>3.0.CO;2-C
- Lambert T. J. (2019). FPbase: a community-editable fluorescent protein database. Nat. Methods 16, 277–278. Peaks, extinction coefficients and quantum yields (read 2026-10-18); spectra from the FPbase API, CC BY-SA 4.0 (read 2026-11-05). doi:10.1038/s41592-019-0352-8
- Janesick J. R. (2007). Photon Transfer. SPIE Press. Pixel SNR from shot, dark and read noise. doi:10.1117/3.725073