An Analog Filter Approach to Frequency Domain Fluorescence Spectroscopy

Abstract

The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modelled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as SPICE can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modelling of the entire system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. The techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response. The simplification of the analysis mathematics, and the ability to model the entire detection chain, make it possible to develop more compact instruments for remote sensing applications.

Fluorescence-based whole plant imaging and phenomics

“…Previously, researchers have developed an inexpensive imaging system using blue LED arrays to excite GFP engineered into Arabidopsis 14 , in which dichroic filter cubes were coupled with an inexpensive camera, which could image cm-scale seedling ‘canopies.’ At the other end of the cost spectrum a portable laser-induced fluorescence imaging (LIFI) system containing a tripled Nd:YAG laser (355 nm) has been used to excite UV-excitable GFP in plants at a standoff (3 m), but this instrument was very expensive and was limited to UV-excitation because FPs excitable at 532nm were not available 10 . In order to move to higher efficiency light sources and multiple wavelengths, non-imaging techniques were explored to frequency modulate 405nm laser diodes and a fluorescence spectrometer was used to detect materials at distances greater than 2 km in field experiments with a 1 m spot size 15,16 . All current remote FP-imaging systems currently available lack flexibility with regards to imaging a variety of FPs and cannot simultaneously image multiple FPs in multiple plants at the canopy level.…”

Imaging of multiple fluorescent proteins in canopies enables synthetic biology in plants

“…Previously, researchers have developed an inexpensive imaging system using blue LED arrays to excite GFP engineered into Arabidopsis (Baker et al ., 2012), in which dichroic filter cubes were coupled with an inexpensive camera, which could image cm‐scale seedling ‘canopies.’ At the other end of the cost spectrum, a portable laser‐induced fluorescence imaging (LIFI) system containing a tripled Nd:YAG laser (355 nm) has been used to excite UV‐excitable GFP in plants at a stand‐off (3 m), but this instrument was very expensive (Stewart et al ., 2005). In order to move to higher efficiency light sources and multiple wavelengths, non‐imaging techniques were explored to frequency modulate 405 nm laser diodes and a fluorescence spectrometer was used to detect signals at distances greater than 2 km in field experiments (DiBenedetto et al ., 2012; Trainham et al ., 2015). All current remote FP‐imaging systems lack flexibility with regard to imaging a variety of FPs and cannot simultaneously image multiple FPs in multiple plants at the canopy level.…”