Imaging Of Fluorescent Proteins Research Articles

Ultraplex microscopy: versatile highly-multiplexed molecular labeling and imaging across scale and resolution.

The molecular organization of cells and tissue is challenging to study due to the inefficiency of multiplexed molecular labeling methods and the limited options for combining microscopy modalities in a single specimen, especially when high spatial resolution is needed. Here we describe ultraplex microscopy, which combines serial multiplexing, ultrathin sectioning, and reversible embedding to circumvent incompatibilities between labeling and imaging techniques, enhance resolution, and expand multiplexing capacity within and across modalities. Samples can be labeled with antibodies, RNA probes, and tissue stains for imaging by brightfield, epifluorescence, super-resolution, and electron microscopy without specialized reagents or materials. We demonstrate applications in brain tissue including molecular profiling of single cells and axonal boutons, high-resolution molecular colocalization, and correlative imaging of fluorescent proteins with confocal and ultraplex microscopy. The power and versatility of ultraplex microscopy will be valuable in addressing currently intractable experimental questions in many systems and contexts.

Scalable spatial single-cell transcriptomics and translatomics in 3D thick tissue blocks.

Characterizing the transcriptional and translational gene expression patterns at the single-cell level within their three-dimensional (3D) tissue context is essential for revealing how genes shape tissue structure and function in health and disease. However, most existing spatial profiling techniques are limited to 5-20 μm thin tissue sections. Here, we developed Deep-STARmap and Deep-RIBOmap, which enable 3D in situ quantification of thousands of gene transcripts and their corresponding translation activities, respectively, within 200-μm thick tissue blocks. This is achieved through scalable probe synthesis, hydrogel embedding with efficient probe anchoring, and robust cDNA crosslinking. We first utilized Deep-STARmap in combination with multicolor fluorescent protein imaging for simultaneous molecular cell typing and 3D neuron morphology tracing in the mouse brain. We also demonstrate that 3D spatial profiling facilitates comprehensive and quantitative analysis of tumor-immune interactions in human skin cancer.

Head-to-head Comparison of Green Fluorescent Protein (GFP) Imaging With Luciferase-luciferin Imaging In Vivo Using Single-nanometer Laser-excitation Tuning and an Ultra-low-light-detection Camera and Optics Demonstrates the Superiority of GFP.

Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.

GLT-1a glutamate transporter nanocluster localization is associated with astrocytic actin and neuronal Kv2 clusters at sites of neuron-astrocyte contact.

Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT-1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells. Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP). Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact. Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult.

Selective fluorescent labeling of cellular proteins and its biological applications.

Proteins, which are ubiquitous in cells and critical to almost all cellular functions, are indispensable for life. Fluorescence imaging of proteins is key to understanding their functions within their native milieu, as it provides insights into protein localization, dynamics, and trafficking in living systems. Consequently, the selective labeling of target proteins with fluorophores has emerged as a highly active research area, encompassing bioorganic chemistry, chemical biology, and cell biology. Various methods for selectively labeling proteins with fluorophores in cells and tissues have been established and are continually being developed to visualize and characterize proteins. This review highlights research findings reported since 2018, with a focus on the selective labeling of cellular proteins with small organic fluorophores and their biological applications in studying protein-associated biological events. We also discuss the strengths and weaknesses of each labeling approach for their utility in living systems.

Reducing Cellular Autolysis of Bacillus subtilis to Improve Keratinase Production.

Bacillus subtilis has been shown to be an excellent expression host for keratinases due to its powerful secretion system. However, cellular autolysis limits its production capacity. Here, we selected seven genes with significantly upregulated transcript levels from 15 genes associated with cellular autolysis as knockout targets by qRT-PCR and constructed a total of 127 strains to reduce cellular autolysis. Among them, the biomass of B. subtilis BSΔXLPC-ker deficient in xpf, lytC, pcfA, and cwlC increased by 57%. This was confirmed by cell staining, green fluorescent protein imaging, and extracellular nucleic acid leakage assay. Keratinase activity was increased by 1.46-fold in the 5 L fermenter. In addition, the activities of nattokinase and subtilisin E were also increased by 1.50-fold and 1.43-fold, respectively, in the modified chassis cells, which further confirms the generalizability of the strategy. Thus, reducing cellular autolysis to increase the ability of B. subtilis to produce subtilisins is promising.

2-Photon imaging of fluorescent proteins in living swine

A common point of failure in translation of preclinical neurological research to successful clinical trials comes in the giant leap from rodent models to humans. Non-human primates are phylogenetically close to humans, but cost and ethical considerations prohibit their widespread usage in preclinical trials. Swine have large, gyrencencephalic brains, which are biofidelic to human brains. Their classification as livestock makes them a readily accessible model organism. However, their size has precluded experiments involving intravital imaging with cellular resolution. Here, we present a suite of techniques and tools for in vivo imaging of porcine brains with subcellular resolution. Specifically, we describe surgical techniques for implanting a synthetic, flexible, transparent dural window for chronic optical access to the neocortex. We detail optimized parameters and methods for injecting adeno-associated virus vectors through the cranial imaging window to express fluorescent proteins. We introduce a large-animal 2-photon microscope that was constructed with off-the shelf components, has a gantry design capable of accommodating animals > 80 kg, and is equipped with a high-speed digitizer for digital fluorescence lifetime imaging. Finally, we delineate strategies developed to mitigate the substantial motion artifact that complicates high resolution imaging in large animals, including heartbeat-triggered high-speed image stack acquisition. The effectiveness of this approach is demonstrated in sample images acquired from pigs transduced with the chloride-sensitive fluorescent protein SuperClomeleon.

Instantaneous Clearing of Biofilm (iCBiofilm): an optical approach to revisit bacterial and fungal biofilm imaging

Whole-biofilm imaging at single-cell resolution is necessary for system-level analysis of cellular heterogeneity, identification of key matrix component functions and response to immune cells and antimicrobials. To this end, we developed a whole-biofilm clearing and imaging method, termed instantaneous clearing of biofilm (iCBiofilm). iCBiofilm is a simple, rapid, and efficient method involving the immersion of biofilm samples in a refractive index-matching medium, enabling instant whole-biofilm imaging with confocal laser scanning microscopy. We also developed non-fixing iCBiofilm, enabling live and dynamic imaging of biofilm development and actions of antimicrobials. iCBiofilm is applicable for multicolor imaging of fluorescent proteins, immunostained matrix components, and fluorescence labeled cells in biofilms with a thickness of several hundred micrometers. iCBiofilm is scalable from bacterial to fungal biofilms and can be used to observe biofilm-neutrophil interactions. iCBiofilm therefore represents an important advance for examining the dynamics and functions of biofilms and revisiting bacterial and fungal biofilm formation.

Green fluorescent protein and phase contrast image fusion via Spectral TV filter-based decomposition

The spectral filter is one of the crucial tools for signal scale and texture analysis. The main function is to decompose a raw image into a series of multi-scale signatures in the spectral domain. For cross-modal fusion, most of the existing approaches naively employ the decomposition of spectral features to achieve image reconstruction. Unfortunately, it does not sufficiently explore the potential cues of different level components in the spatial domain. In this work, we describe an effective decomposition approach using the spectral filter. Specifically, we aim to build a multi-scale image decomposition framework from the spectral domain to the spatial domain, which is called the multi-scale spatial decomposition approach (MSD). Moreover, building upon spectral total variation (TV), we characterize the spectral signatures into various spatial levels with different filters. A fusion scheme of green fluorescent protein and phase contrast image is introduced, leveraging the multi-scale spatial decomposition approach. The source image is first separated by applying MSD into three spatial bands, namely high-frequency bands, low-frequency bands and structure bands. Our method is simple and effective. The divided bands are obtained by a set of filters and could be processed with the specific fusion strategies to extract the features of the source image. In addition, experiments show that our method produces promising results than other current popular methods.

Entamoeba histolytica EHD1 Is Involved in Mitosome-Endosome Contact.

ABSTRACTInterorganellar cross talk is often mediated by membrane contact sites (MCSs), which are zones where participating membranes come within 30 nm of one another. MCSs have been found in organelles, including the endoplasmic reticulum, Golgi bodies, endosomes, and mitochondria. Despite its seeming ubiquity, reports of MCS involving mitochondrion-related organelles (MROs) present in a few anaerobic parasitic protozoa remain lacking. Entamoeba histolytica, the etiological agent of amoebiasis, possesses an MRO called the mitosome. We previously discovered several Entamoeba-specific transmembrane mitosomal proteins (ETMPs) from in silico and cell-biological analyses. One of them, ETMP1 (EHI_175060), was predicted to have one transmembrane domain and two coiled-coil regions and was demonstrated to be mitosome membrane integrated based on carbonate fractionation and immunoelectron microscopy (IEM) data. Immunoprecipitation analysis detected a candidate interacting partner, EH domain-containing protein (EHD1; EHI_105270). We expressed hemagglutinin (HA)-tagged EHD1 in E. histolytica, and subsequent immunofluorescence and IEM data indicated an unprecedented MCS between the mitosome and the endosome. Live imaging of a green fluorescent protein (GFP)-EHD1-expressing strain demonstrated that EHD1 is involved in early endosome formation and is observed in MCS between endosomes of various sizes. In vitro assays using recombinant His-EHD1 demonstrated ATPase activity. MCSs are involved in lipid transfer, ion homeostasis, and organelle dynamics. The serendipitous discovery of the ETMP1-interacting partner EHD1 led to the observation of the mitosome-endosome contact site in E. histolytica. It opened a new view of how the relic mitochondria of Entamoeba may likewise be involved in organelle cross talk, a conserved feature of mitochondria and other organelles in general.

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

Visualizing polymeric components that define distinct root barriers across plant lineages.

ABSTRACTHydrophobic cell wall depositions in roots play a key role in plant development and interaction with the soil environment, as they generate barriers that regulate bidirectional nutrient flux. Techniques to label the respective polymers are emerging, but are efficient only in thin roots or sections. Moreover, simultaneous imaging of the barrier constituents lignin and suberin remains problematic owing to their similar chemical compositions. Here, we describe a staining method compatible with single- and multiphoton confocal microscopy that allows for concurrent visualization of primary cell walls and distinct secondary depositions in one workflow. This protocol permits efficient separation of suberin- and lignin-specific signals with high resolution, enabling precise dissection of barrier constituents. Our approach is compatible with imaging of fluorescent proteins, and can thus complement genetic markers or aid the dissection of barriers in biotic root interactions. We further demonstrate applicability in deep root tissues of plant models and crops across phylogenetic lineages. Our optimized toolset will significantly advance our understanding of root barrier dynamics and function, and of their role in plant interactions with the rhizospheric environment.

Quantifying endothelial cell proliferation in the zebrafish embryo.

Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research. In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of the in vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of an in vivo model in terms of complexity but are also genetically manipulable and optically transparent. Methods:Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches. Results:Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos. Discussion:We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existing in vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research.

IRFP (near-infrared fluorescent protein) imaging of subcutaneous and deep tissue tumours in mice highlights differences between imaging platforms

BackgroundIn vivo imaging using fluorescence is used in cancer biology for the detection, measurement and monitoring of tumours. This can be achieved with the expression of fluorescent proteins such as iRFP, which emits light at a wavelength less attenuated in biological tissues compared to light emitted by other fluorescent proteins such as GFP or RFP. Imaging platforms capable of detecting fluorescent tumours in small animals have been developed but studies comparing the performance of these platforms are scarce.ResultsThrough access to three platforms from Xenogen, Bruker and Li-Cor, we compared their ability to detect iRFP-expressing subcutaneous tumours as well as tumours localised deeper within the body of female NSG mice. Each platform was paired with proprietary software for image analyse, but the output depends on subjective decisions from the user. To more objectively compare platforms, we developed an ‘in house’ software-based approach which results in lower measured variability between mice.ConclusionsOur comparisons showed that all three platforms allowed for reliable detection and monitoring of subcutaneous iRFP tumour growth. The biggest differences between platforms became apparent when imaging deeper tumours with the Li-Cor platform detecting most tumours and showing the highest dynamic range.

Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy.

We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans.

ABSTRACTFluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

Inhibition of efflux pumps aids small-molecule probe-based fluorescence labeling and imaging in the Gram-negative bacterium Escherichia coli.

A major challenge in fluorescence imaging experiments, which are essential to determine protein activity, expression, and localization, is the penetration of small-molecule probes through the outer membrane permeability barrier of bacteria. Here, we describe a novel strategy for small-molecule probe-based fluorescence protein labeling and imaging in the Gram-negative bacterium Escherichia coli. We targeted a siderophore enterobactin biosynthetic enzyme EntE in E. coli. When coupled with an efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone, small-molecule probes were able to efficiently enter the cells, leading to the fluorescence labeling and imaging of overproduced EntE in E. coli. This study demonstrates that the combination of small-molecule probes with appropriate efflux pump inhibitors may substantially enhance their interaction with the target proteins in live bacteria.

Green Fluorescent Protein and Phase Contrast Image Fusion Via Detail Preserving Cross Network

In cell and molecular biology, the fusion of green fluorescent protein (GFP) and phase contrast (PC) images aims to generate a composite image, which can simultaneously display the functional information in the GFP image related to the molecular distribution of biological living cells and the structural information in the PC image such as nucleus and mitochondria. In this paper, we propose a detail preserving cross network (DPCN), which consists of a structural-guided functional feature extraction branch (SFFEB), a functional-guided structural feature extraction branch (FSFEB) and a detail preserving module (DPM), to address the GFP and PC image fusion issue. Unlike traditional parallel multi-branch architectures used for multiple inputs, the SFFEB and the FSFEB are interacted via a cross manner to fuse the functional information from the GFP image and the structural information from the PC image more adequately. Moreover, the DPM is composed of eight multi-scale convolutional blocks (MSCBs) associated with short, medium, and long skip connections to further extract the detail information from the source images. Experimental results on the popular Arabidopsis thaliana cell database demonstrate that the proposed method outperforms the state-of-the-art methods in terms of both qualitative and quantitative evaluations. The proposed method is also extended to deal with the functional and structural image fusion issue in medical imaging, and the promising results obtained exhibit its good generalizability. The code of our method is available at https://github.com/yuliu316316/DPCN-Fusion.

Organelle extensions in plant cells.

The life strategy of plants includes their ability to respond quickly at the cellular level to changes in their environment. The use of targeted fluorescent protein probes and imaging of living cells has revealed several rapidly induced organelle responses that create the efficient sub-cellular machinery for maintaining homeostasis in the plant cell. Several organelles, including plastids, mitochondria, and peroxisomes, extend and retract thin tubules that have been named stromules, matrixules, and peroxules, respectively. Here, I combine all these thin tubular forms under the common head of organelle extensions. All extensions change shape continuously and in their elongated form considerably increase organelle outreach into the surrounding cytoplasm. Their pleomorphy reflects their interactions with the dynamic endoplasmic reticulum and cytoskeletal elements. Here, using foundational images and time-lapse movies, and providing salient information on some molecular and biochemically characterized mutants with increased organelle extensions, I draw attention to their common role in maintaining homeostasis in plant cells.

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

SummaryReverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields. Genetically encoded fluorescent proteins (FPs) have revolutionized plant biology paradigms in gene expression, protein trafficking and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 25 years ago, modern phenomics has largely ignored fluorescence as a transgene expression device despite the burgeoning FP colour palette available to plant biologists. Here, we show a new platform for stand‐off imaging of plant canopies expressing a wide variety of FP genes. The platform—the fluorescence‐inducing laser projector (FILP)—uses an ultra‐low‐noise camera to image a scene illuminated by compact diode lasers of various colours, coupled with emission filters to resolve individual FPs, to phenotype transgenic plants expressing FP genes. Each of the 20 FPs screened in plants were imaged at >3 m using FILP in a laboratory‐based laser range. We also show that pairs of co‐expressed fluorescence proteins can be imaged in canopies. The FILP system enabled a rapid synthetic promoter screen: starting from 2000 synthetic promoters transfected into protoplasts to FILP‐imaged agroinfiltrated Nicotiana benthamiana plants in a matter of weeks, which was useful to characterize a water stress‐inducible synthetic promoter. FILP canopy imaging was also accomplished for stably transformed GFP potato and in a split‐GFP assay, which illustrates the flexibility of the instrument for analysing fluorescence signals in plant canopies.