Imaging lifetime and anisotropy spectra in the frequency domain

Summary We report the development of a system combining the capabilities of fluorescence imaging spectroscopy (x, λ, I), fluorescence lifetime (τ) and static and dynamic fluorescence anisotropy (r), enabling the wide‐field measurement of the spectroscopic parameters of fluorophores: (x, λ, I, τ, r). The system employs a frequency domain data collection strategy with a modulated light emitting diode as the light source. A polarization rotator placed in the excitation path after a polarizer allows alternating parallel and perpendicular images to be collected without moving parts. A second polarizer on the emission side serves as the analyzer, leading to estimations of the wavelength‐dependent dynamic anisotropies. The spectrograph has a nominal range of 365–920 nm; however, the light‐emitting diodes and filter sets used in this study restricted the usable range from about 510 to 700 nm. The system was tested on rhodamine 6G (R6G) solutions containing 0, 15, 37, 45, 59, 74 and 91 glycerol. These experiments gave rotational diffusion results comparing favourably with literature values while also demonstrating a trend towards shorter measured lifetimes at high refractive index. The ability of the system to resolve mixtures was tested on mixtures of anti‐human IgG‐FITC (γ‐chain‐specific) and R6G. These fluorophores have similar lifetimes but could be separated using anisotropy parameters. The imaging capabilities of the system were tested on mixtures of fluorescent beads with glycerol solutions of R6G.