Table 3.

General classes of fluorescence hyperspectral imaging assays and optimal operating parameters.

Type of Spectral Assay	Optimization Settings
Case I: High signal, high speed imaging	[Fluorophore A]∼ [Fluorophore B] High laser power Short acquisition time Low averaging Results in high SNR
Case II: High signal, variable concentration	[Fluorophore A] ≫ [Fluorophore B] Maximize dynamic range High laser power Short acquisition time Results in high SNR
Case III: Low signal (sensitive), high speed imaging	Low laser power Short acquisition time Low SNR Balance: - temporal resolution - photobleaching - SNR effects unmixing accuracy (RMS error)
Case IV: Low signal, variable concentration	Maximize dynamic range Low laser power Long acquisition time Results in low SNR Balance with photobleaching kinetics