Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore

Abstract

We report a picosecond time-gated fluorescence lifetime imaging ͑FLIM͒ system extended to perform time-resolved fluorescence anisotropy imaging ͑TR-FAIM͒. Upon excitation with linearly polarized laser pulses, the parallel and perpendicular components of the fluorescence emission from a sample are imaged simultaneously using a polarization-resolved imager. The imaging technique presented here quantitatively reports the rotational mobility of a fluorophore as it varies according to the local environment. In a single acquisition run it yields maps of both rotational correlation time and fluorescence lifetime as they vary across a sample. TR-FAIM has been applied to imaging standard multiwell plate samples of rhodamine 6G dissolved in methanol, ethylene glycol, trimethylene glycol, and glycerol. The observed rotational correlation times and fluorescence lifetimes, which report the local viscosity and refractive index of the local rhodamine 6G environment, respectively, are in good agreement with previously published single point measurements. By considering the linear dependence of the rotational correlation time on viscosity up to 20 cP, we are able to obtain a two-dimensional viscosity map. Wide-field maps of rotational correlation time, and therefore viscosity, have been obtained. This illustrates the potential to image the local viscosity and fluorescence lifetime distributions of fluorophore tagged proteins in cells.

A lifetime in photochemistry; some ultrafast measurements on singlet states

“…at the immunological synapse. There was a need to develop bioimaging that not only located proteins in live cells, but could also probe their environment, for example viscosity via rotational mobility through anisotropy measurements [45]. Such a technique was demonstrated using fluorescence lifetime imaging of green fluorescent protein (GFP) [46].…”

The polarized AB plot for the frequency‐domain analysis and representation of fluorophore rotation and resonance energy homotransfer

“…The introduction of wide-field anisotropy imaging in frequency-domain (Clayton et al, 2002) and timedomain instruments (Siegel et al, 2003) has enabled the imaging of dynamic fluorescence depolarization arising from homotransfer FRET and/or molecular rotations.…”

Quantitative fluorescence nanospectroscopy of nucleotide excision repair : from single molecules to cells

“…where the G-factor accounts for different transmission and detection efficiencies of the imaging system at parallel and perpendicular polarization [157] and usually can be determined by calibration experiments using fluorescent samples with well-known FA values. In contrast to time-resolved FA (TFA), which requires a pulsed light source and high speed intensity measurements, SFA is performed using constant illumination and observation.…”

“…The outputs from both APDs are used to determine ACF in FCS by where I = I p + I s ; the terms I p and I s are the fluorescent intensity parallel and perpendicular to the excitation polarization as selected using the Glan-Thompson polarizer; G accounts for different transmission and detection efficiencies of the imaging system at parallel and perpendicular polarization [157], and I p,bk and I s,bk are average background intensity from the APDs, respectively, and separately measured in the binding buffer solution without fluorescent sample. The G-factor may be obtained by excitation of sample with first vertical and then horizontal polarization [157]. …”

“…In this work, linearly polarized laser line 488nm was used to excite GFPs in the XP4PA cells and a water immersion objective Zeiss C-Apochromat 63×, NA=1.20, working distance = 0.24 mm) was used for microscopic imaging. The SFA is formally evaluated by where I p and I s are the fluorescent intensity parallel and perpendicular to the excitation polarization as selected using the Glan-Thompson polarizer in the excitation path of the SFCM; G accounts for different transmission and detection efficiencies of the imaging system at parallel and perpendicular polarization [265] and it was the same for all pixels in a SFA image due to the scanning motion using a piezo nanopositioning stage (P-740.20 50×50μm 2 , Physik Instrumente, Waldbronn, Germany).…”