Macroscale fluorescence imaging against autofluorescence under ambient light

Ruikang Zhang (1) , Raja Chouket (1) , Marie-Aude Plamont (1) , Zsolt Kelemen (2) , Agathe Espagne (1) , Alison G Tebo (1) , Arnaud Gautier (1) , Lionel Gissot (2) , Jean-Denis Faure (2) , Ludovic Jullien (1) , Vincent Croquette (3, 4) , Thomas Le Saux (1)

1 PASTEUR - Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640)
2 IJPB - Institut Jean-Pierre Bourgin
3 LPS - Laboratoire de Physique Statistique de l'ENS
4 IBENS - Institut de biologie de l'ENS Paris

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.

Domaines: Chimie théorique et/ou physique; Instrumentations et Détecteurs [physics.ins-det]

Ruikang Zhang, Raja Chouket, Marie-Aude Plamont, Zsolt Kelemen, Agathe Espagne, et al.. Macroscale fluorescence imaging against autofluorescence under ambient light. Light: Science and Applications, 2018, 7 (1), ⟨10.1038/s41377-018-0098-6⟩. ⟨hal-02307924⟩