Long wavelength fluorescence lifetime standards for front-face fluorometry

Abstract

With the increased development and use of fluorescence lifetime-based sensors, fiber optic sensors, fluorescence lifetime imaging microscopy (FLIM), and plate and array readers, , calibration standards are essential to ensure the proper function of these devices and accurate results. For many devices that utilize a "front face excitation" geometry where the excitation is nearly coaxial with the direction of emission, scattering-based lifetime standards are problematic and fluorescent lifetime standards are necessary. As more long wavelength (red and near-infrared) fluorophores are used to avoid background autofluorescence, the lack of lifetime standards in this wavelength range has only become more apparent . We describe an approach to developing lifetime standards in any wavelength range, based on Förster resonance energy transfer (FRET). These standards are bright, highly reproducible, have a broad decrease in observed lifetime, and an emission wavelength in the red to near infrared making them well suited for the laboratory and field applications as well. This basic approach can be extended to produce lifetime standards for other wavelength regimes.

Fig. 1

Schematic of fluorescence-based fiber optic sensor. Excitation from the laser (- - -) passes through the dichroic mirror and is launched into the fiber optic at the proximal end; fluorescence (⋯ ⋯) comes back from the sensor at the distal end though the fiber, is collected by the microscope objective, is reflected off the dichroic mirror, and passes through the emission filter to be collected by the detector. Reproduced from [29] with permission

Fig. 2

Spectral overlap of the emission spectrum of DTDCI in ethanol (dashed line) and absorption spectrum of Janus Green B in ethanol (solid line)

Fig. 3

Frequency-dependent phase and modulation data of the lifetime standards; DTDCI (■), together with 32.5µM (●), 97.5µM (▲), and 162.5µM (▼) Janus Green B. Open symbols represent the modulation ratio, closed symbols represent the phase delay, lines indicate the best single component fits to the data (except for 97.5µM and 162.5µM Janus Green B where two component fits were used). It should be noted that the modulation ratios for 97.5µM and 162.5µM were scaled by factors of 1.02 and 1.05 respectively