Macroscale fluorescence imaging against autofluorescence under ambient light

Ruikang Zhang,Agathe Espagne,Raja Chouket,Jean-Denis Faure,Alison G Tebo,Vincent Croquette,Lionel Gissot,Zsolt Kelemen,Arnaud Gautier,Thomas Le Saux,Ludovic Jullien,Marie-Aude Plamont

doi:10.1038/s41377-018-0098-6

Abstract

Macroscale fluorescence imaging is increasingly used to observe biological samples. However, it may suffer from spectral interferences that originate from ambient light or autofluorescence of the sample or its support. In this manuscript, we built a simple and inexpensive fluorescence macroscope, which has been used to evaluate the performance of Speed OPIOM (Out of Phase Imaging after Optical Modulation), which is a reference-free dynamic contrast protocol, to selectively image reversibly photoswitchable fluorophores as labels against detrimental autofluorescence and ambient light. By tuning the intensity and radial frequency of the modulated illumination to the Speed OPIOM resonance and adopting a phase-sensitive detection scheme that ensures noise rejection, we enhanced the sensitivity and the signal-to-noise ratio for fluorescence detection in blot assays by factors of 50 and 10, respectively, over direct fluorescence observation under constant illumination. Then, we overcame the strong autofluorescence of growth media that are currently used in microbiology and realized multiplexed fluorescence observation of colonies of spectrally similar fluorescent bacteria with a unique configuration of excitation and emission wavelengths. Finally, we easily discriminated fluorescent labels from the autofluorescent and reflective background in labeled leaves, even under the interference of incident light at intensities that are comparable to sunlight. The proposed approach is expected to find multiple applications, from biological assays to outdoor observations, in fluorescence macroimaging.

Highlights

Macroimaging has become an essential tool for studying various types of samples of biological origin
The average intensities of the light sources, which are denoted as I10 and I20 and modulated at angular frequency ω must be kept constant over the whole imaged field since the Speed OPIOM resonance conditions fix both I10=I20 and I10=ω20
The two light-emitting diodes (LEDs) that emit at 405 and 480 nm are directly involved in the implementation of Speed OPIOM with reversibly photoswitchable fluorescent proteins (RSFPs); the LED that emits at 550 nm has been added to excite red fluorescent proteins in view of agronomic applications to be published elsewhere

Summary

Introduction

Macroimaging has become an essential tool for studying various types of samples of biological origin (from plate readers, microorganism and cell cultures, tissues, and organs, up to plants and animals). The dynamic response of a fluorescent label to light modulation has been exploited since kinetics brings as many discriminative dimensions as the number of rate constants involved in the label reactions[1] This discriminative strategy was explored early in fluorescence spectroscopy, in which the signal originates from the relaxation of an excited label after light absorption by its ground state. Several microscopy protocols (e.g., optical lock-in detection (OLID)[21], synchronously amplified fluorescence image recovery (SAFIRe)[22], and out-of-phase imaging after optical modulation, which is denoted as OPIOM20,23,24) have been recently introduced for kinetically discriminating reversibly photoswitchable fluorescent labels against several types of spectrally interfering backgrounds without a deconvolution or subtraction scheme. Since a reversible photoswitchable fluorescent label has unique photochemical and kinetic properties (and, correspondingly, its own resonance conditions), tuning the illumination control parameters to the resonant values enables one to selectively image a targeted label, filtering out the contributions of nonresonant fluorophores and ambient light (Fig. 1b)

Methods

Results

Conclusion