Macroscale fluorescence imaging against autofluorescence under ambient light

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.

Simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy

“…The preceding analysis prompted us to implement dynamic contrast of fluorophores in fluorescence endomicroscopy. Unlike regular fluorescence imaging, which exploits the spectral dimension to generate contrast in images, dynamic contrast discriminates reversibly photoswitchable fluorescent labels against spectrally interfering backgrounds by exploiting the dynamics of their photoswitching properties without any deconvolution or subtraction schemes [28][29][30][31][32][33]. More specifically, we adopted the fast modality of out-of-phase imaging after optical modulation (Speed OPIOM) [32,33], which uses two modulated light sources synchronized in antiphase at two wavelengths in order to drive photoswitching [ Fig.…”

Dynamic Contrast for Plant Phenotyping

“…The fluorescence lifetime has been exploited early on to discriminate fluorophores and it was applied to eliminate an autofluorescence background in plants . To overcome the limitations of a narrow dispersion of the fluorescence lifetimes and facilitate discrimination, dynamic contrast has been more recently proposed for selective imaging of reversibly photoswitchable fluorescent proteins (RSFPs). , The latter benefit from a wider range of relaxation times and they have been successfully used for selective imaging in plants. − In this account, we exploit the Speed OPIOM (out-of-phase imaging after optical modulation) imaging protocol of dynamic contrast, which we recently implemented on a fluorescence macroscope. , Designed to image macroscale samples with minimal preparation, enhanced sensitivity, and excellent signal-to-noise ratio originating from lock-in amplification, , we here demonstrate that it can be used to image RSFPs in transient as well as in stable plant systems in a context relevant to plant phenotyping. We show that Speed OPIOM can efficiently report on transcriptional activities, both in a qualitative and quantitative manner, and reliably monitor early plant response to biotic stresses, even in the presence of endogenous fluorescence and under ambient light.…”

Dynamic contrast with reversibly photoswitchable fluorescent labels for imaging living cells

“…Thus several protocols (e.g. optical lock-in detection -OLID, 41 synchronously amplied uorescence image recovery -SAFIRe, 42,43 , transient state imaging microscopy -TRAST, 44 and out-of-phase imaging aer optical modulation -OPIOM [45][46][47][48] ) have exploited the time response of uorescence to light variations for discriminating up to three spectrally similar RSFs, 46 by relying on neither deconvolution nor subtraction schemes.…”

“…46 Speed OPIOM proved relevant for macroscale uorescence, which is increasingly used to observe biological samples. 47 In particular, it enhanced the sensitivity and the signal-to-noise ratio for uorescence detection in blot assays by factors of 50 and 10, respectively, over direct uorescence observation under constant illumination. Moreover it easily discriminated uorescent labels from the autouorescence and reective background in labeled leaves of plant seedlings, even under the interference of incident light at intensities that are comparable to that of sunlight.…”