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Workflow and properties of the FlyClear procedure. a Main steps of the FlyClear protocol. b Upper panel shows wide-field image of optically cleared specimens placed on top of a USAF1951-chart in Solution-2 demonstrates the level of overall transparency. Middle panel shows higher magnification of red rectangular areas indicating the highest level of transparency reached in the corresponding sample. Lower panel shows GFP signal after RI matching. Bright field images were acquired with a 4x objective (Olympus, XLFluor4x/340, 0.28 NA, WD = 29.5 mm) using custom-made correction of optics for a refractive index of 1.45 (WD = 10 mm after correction). Fluorescent images were acquired with a stereomicroscope with a 1x objective (Leica, Plan APO 1.0×, WD 61.5 mm). Genotype: Peb-Gal4 UAS-mCD8::GFP;. Scale bars represent 500 µm in b
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The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analys...
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... This configuraGon enables simultaneous collecGon of emimed light across the enGre field of view (FOV), surpassing sequenGal point-scanning methods in photon efficiency and speed. Localized fluorescence excitaGon at the chosen plane significantly reduces photobleaching, which is criGcal for in vivo imaging, but LSFM is also an effecGve method for imaging large and cleared Gssues [6][7][8][9][10] . It enables scienGsts to obtain high-resoluGon images across the enGre intact sample, crucial for subsequent reconstrucGons and quanGficaGons. ...
Light sheet microscopy is the ideal technique for multiscale imaging of large and cleared tissues, and it is desirable to achieve the highest possible isotropic resolution across the entire sample. However, isotropic resolution for a centimeter-sized sample has only been achieved with slow and often aberrated, axially scanned light sheets, resulting in a low resolution of several micrometers. Here, we introduce a compact, high-speed light sheet fluorescence microscope with isotropic sub-micron resolution optimized for cleared tissue. We introduce three major opto-mechanical innovations using off-the-shelf optics to achieve an isotropic resolution of 850 nm across samples up to 1 cm3 and refractive indices ranging from 1.33 to 1.56. We show that combining an air objective and a meniscus lens achieves an axially swept light sheet with sub-micron diffraction-limited resolution, free of aberrations. The effective field of view is increased 2-fold by correcting the field curvature of the light sheet with a concave mirror in the remote focusing unit. Furthermore, the imaging speed is enhanced 10-fold by adapting the light sheet's motion with a closed-loop feedback, reaching 100 frames per second while maintaining isotropic resolution across the large field of view. Finally, we showcase the performance of our light sheet system for imaging from subcellular up to centimeter scale in cleared mouse cochlea and brain.
... Digestion and fixation steps of both L3 and white pre-pupae were adapted from Pende et al. [49]. Briefly, samples were treated at 37°C for 45 min with 0.03% proteinase K (Sigma, no. ...
Peroxisomes are essential organelles involved in critical metabolic processes in animals such as fatty acid oxidation, ether phospholipid production and reactive oxygen species detoxification. We have generated transgenic Drosophila melanogaster models expressing fluorescent reporters for the selective autophagy of peroxisomes, a process known as pexophagy. We show that these reporters are colocalized with a peroxisomal marker and that they can reflect pexophagy induction by iron chelation and inhibition by depletion of the core autophagy protein Atg5. Using light sheet microscopy, we have been able to obtain a global overview of pexophagy levels across the entire organism at different stages of development. Tissue-specific control of pexophagy is exemplified by areas of peroxisome abundance but minimal pexophagy, observed in clusters of oenocytes surrounded by epithelial cells where pexophagy is much more evident. Enhancement of pexophagy was achieved by feeding flies with the iron chelator deferiprone, in line with past results using mammalian cells. Specific drivers were used to visualize pexophagy in neurons, and to demonstrate that specific depletion in the larval central nervous system of Hsc70-5, the Drosophila homologue of the chaperone HSPA9/mortalin, led to a substantial elevation in pexophagy.
... However, both COLM and SPED are based on traditional LSM and dependent on the RI of the cleared tissue, achieving at cellular resolution. The ultramicroscopy technique 8,20 utilizes aspherical optics to generate a thin light sheet up to 3 mm long and when combed with multiview imaging techniques, is able to image millimeter-size samples at high speed and isotropic resolution. However, its resolution is limited to 3 μm and multiview image-fusion introduces additional complexity to the system. ...
Axially swept light-sheet microscope in conjunction with tissue clearing enables three-dimensional morphological investigation of millimeter-scaled tissues at isotropic sub-micron resolution. However, these microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci for axial sweeping. This enables full field of view imaging at 40 frames per second, a four-fold improvement over the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the existing techniques. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging platform on various cleared tissue samples and delineated its robustness over a wide range of clearing protocols.
... However, both COLM and SPED are based on traditional LSM and dependent on the RI of the cleared tissue, achieving at cellular resolution. The ultramicroscopy technique 8,20 utilizes aspherical optics to generate a thin light-sheet up to 3 mm long and when combed with multi-view imaging techniques, is able to image millimetre size samples at high speed and isotropic resolution. However, its resolution is limited to 3 m and multi-view image-fusion introduces additional complexity to the system. ...
Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging pipeline on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.
... Further advantages are the easy genetic manipulation using the UAS-GAL4 system, which allows a tissue/cell-type specific expression or depletion of target genes (Fig. 1B), as well as the huge number of transgenic flies available for purchase at organizations such as the Bloomington Drosophila Stock Center (BDSC, Indiana University), the Vienna Drosophila Resource Center (VDRC), the Kyoto Stock Center (DGGR) and the Drosophila Genomics Resource Center (DGRC). Also, the development of novel high-end techniques such as high-resolution imaging, single cell/nucleus RNA sequencing, spatial transcriptomics and proteomic approaches have been successfully used in the fruit fly [1][2][3][4]. ...
Biological and biomedical research using Drosophila melanogaster as a model organism has gained recognition through several Nobel prizes within the last 100 years. Drosophila exhibits several advantages when compared to other in vivo models such as mice and rats, as its life cycle is very short, animal maintenance is easy and inexpensive and a huge variety of transgenic strains and tools are publicly available. Moreover, more than 70% of human disease-causing genes are highly conserved in the fruit fly. Here, we explain the use of Drosophila in nephrology research and describe two kidney tissues, Malpighian tubules and the nephrocytes. The latter are the homologous cells to mammalian glomerular podocytes and helped to provide insights into a variety of signaling pathways due to the high morphological similarities and the conserved molecular make-up between nephrocytes and podocytes. In recent years, nephrocytes have also been used to study inter-organ communication as links between nephrocytes and the heart, the immune system and the muscles have been described. In addition, other tissues such as the eye and the reproductive system can be used to study the functional role of proteins being part of the kidney filtration barrier.
... To confirm that ingested acetaminophen accumulated in Drosophila tissues, we performed immunofluorescence on whole-mounted treated and untreated Drosophila adults using an anti-acetaminophen antibody, which recognizes native acetaminophen and acetaminophen-adducts in tissues 26,27 . Vehicle-fed Drosophila show very little background APAP immunoreactivity at 12 h (Fig. 1C), whereas APAP fed adults show increased APAP immunoreactivity, particularly in the abdomen (Fig. 1D). ...
... The tissue clearing CLARITY protocol was used to visualize epitope distribution in whole animals for acetaminophen immunofluorescence. Briefly, whole Drosophila were fixed in 4% PFA for 12 h at 4 °C, then rinsed 3× PBS for 3 h at RT with gentle shaking, and then immersed in FlyClear Solution-1 (8% THEED (2,2′,2″,2″′-(Ethylenedinitrilo)-tetraethanol) (Sigma-Aldrich), 5% Triton X-100 (Sigma-Aldrich) and 25% Urea (Fisher Scientific, in PBS) for 14 days at 37 °C, as described previously 26 . Cleared Drosophila tissues were rinsed 3 × in PBS with gentle shaking for 12 h at RT, then immersed in blocking solution and processed as outlined above. ...
Acetaminophen is the most common cause of acute drug-induced liver injury in the United States. However, research into the mechanisms of acetaminophen toxicity and the development of novel therapeutics is hampered by the lack of robust, reproducible, and cost-effective model systems. Herein, we characterize a novel Drosophila-based model of acetaminophen toxicity. We demonstrate that acetaminophen treatment of Drosophila results in similar pathophysiologic alterations as those observed in mammalian systems, including a robust production of reactive oxygen species, depletion of glutathione, and dose-dependent mortality. Moreover, these effects are concentrated in the Drosophila fat body, an organ analogous to the mammalian liver. Utilizing this system, we interrogated the influence of environmental factors on acetaminophen toxicity which has proven difficult in vertebrate models due to cost and inter-individual variability. We find that both increasing age and microbial depletion sensitize Drosophila to acetaminophen toxicity. These environmental influences both alter oxidative stress response pathways in metazoans. Indeed, genetic and pharmacologic manipulations of the antioxidant response modify acetaminophen toxicity in our model. Taken together, these data demonstrate the feasibility of Drosophila for the study of acetaminophen toxicity, bringing with it an ease of genetic and microbiome manipulation, high-throughput screening, and availability of transgenic animals.
... Yet, brain-wide mapping of A14 output has yet to be performed to define if subtype-specific target segregation exists. To this end, we stereotaxically injected Credependent GFP-producing AAV2 viral particles into the PeVN of Dat1-IRES-Cre mice (Fig. 2a) and used tissue clearing and ultramicroscopy (light-sheet microscopy) to map individual axons at distant locations 45,46 . We found that A14 neurons give rise to long projections that leave the hypothalamus and densely innervate the LS (Fig. 2b, c, Supplementary Fig. 3a, Supplementary Video 1, n = 4 animals), with additional efferents coursing towards the central amygdaloid nucleus and the ME (Supplementary Video 2). ...
The lateral septum (LS) has been implicated in the regulation of locomotion. Nevertheless, the neurons synchronizing LS activity with the brain’s clock in the suprachiasmatic nucleus (SCN) remain unknown. By interrogating the molecular, anatomical and physiological heterogeneity of dopamine neurons of the periventricular nucleus (PeVN; A14 catecholaminergic group), we find that Th ⁺ / Dat1 ⁺ cells from its anterior subdivision innervate the LS in mice. These dopamine neurons receive dense neuropeptidergic innervation from the SCN. Reciprocal viral tracing in combination with optogenetic stimulation ex vivo identified somatostatin-containing neurons in the LS as preferred synaptic targets of extrahypothalamic A14 efferents. In vivo chemogenetic manipulation of anterior A14 neurons impacted locomotion. Moreover, chemogenetic inhibition of dopamine output from the anterior PeVN normalized amphetamine-induced hyperlocomotion, particularly during sedentary periods. Cumulatively, our findings identify a hypothalamic locus for the diurnal control of locomotion and pinpoint a midbrain-independent cellular target of psychostimulants.
... However, most viral infection models in flies are not focused on neurological outcomes. Nonetheless, novel approaches in genetic manipulation [159], behavioural assessment (automated monitoring systems [160]) and neuropathology (FlyClear tissue clearing protocols [161]) are expanding experimental design beyond the conventional readouts used to profile neuroinfection and immunity outcomes in flies ( Figure 1). Below, we will highlight examples of the benefits and drawbacks of using virus-infected Drosophila to study related neurological sequelae. ...
The study of human neurological infection faces many technical and ethical challenges. While not as common as mammalian models, the use of Drosophila (fruit fly) in the investigation of virus–host dynamics is a powerful research tool. In this review, we focus on the benefits and caveats of using Drosophila as a model for neurological infections and neuroimmunity. Through the examination of in vitro, in vivo and transgenic systems, we highlight select examples to illustrate the use of flies for the study of exogenous and endogenous viruses associated with neurological disease. In each case, phenotypes in Drosophila are compared to those in human conditions. In addition, we discuss antiviral drug screening in flies and how investigating virus–host interactions may lead to novel antiviral drug targets. Together, we highlight standardized and reproducible readouts of fly behaviour, motor function and neurodegeneration that permit an accurate assessment of neurological outcomes for the study of viral infection in fly models. Adoption of Drosophila as a valuable model system for neurological infections has and will continue to guide the discovery of many novel virus–host interactions.
... This possibility has been elegantly exploited by Pende and colleagues, who developed a combined tissues clearing procedure to decrease the AF with an ultramicroscopy approach (Figure 2). The resulting lowered signal of AF allowed to delineate the body of Drosophila melanogaster Meigen (Diptera: Drosophilidae) larvae and adults, favoring the multi-view topological localization of the green fluorescent protein (GFP)-expressing neuronal network with single-cell resolution in Drosophila larvae [30]. prevalently in green, attributed to the stiffer chitin. ...
... This possibility has been elegantly exploited by Pende and colleagues, who developed a combined tissues clearing procedure to decrease the AF with an ultramicroscopy approach (Figure 2). The resulting lowered signal of AF allowed to delineate the body of Drosophila melanogaster Meigen (Diptera: Drosophilidae) larvae and adults, favoring the multi-view topological localization of the green fluorescent protein (GFP)-expressing neuronal network with single-cell resolution in Drosophila larvae [30]. In insects, AF was also reported as a real disturbance through confounding and impeding the reliable detection of the fluorescent signal from the reporter gene [31], or limiting the detection of 16S rRNA-targeted fluorescent in situ hybridization (FISH) applied to tissues for the analysis of symbiotic bacteria [32,33]. ...
... On the other hand, Koga and colleagues have developed a protocol based on the use of hydrogen peroxide able to strongly reduce AF in insect tissues and allow a reliable FISH detection of endosymbionts of aphids, lice and bat flies [37]. Anyway, several studies performed in insects on the development of transgenic individuals through germline transformation and consequent expression of fluorescent protein markers (e.g., green and red fluorescent proteins) have reported that AF signals did not impair the detection of the experimentally-induced fluorescence [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43]. It is also worth to recall the particular case of the AF rising from the yolk granules and the vitelline membrane of dechorionated D. melanogaster embryos, which, together with the embryo thickness, size, and opacity, seemed to affect the application of optical fluorescence microscopy in the study of this developmental stage. ...
Light-based phenomena in insects have long attracted researchers’ attention. Surface color distribution patterns are commonly used for taxonomical purposes, while optically-active structures from Coleoptera cuticle or Lepidoptera wings have inspired technological applications, such as biosensors and energy accumulation devices. In Diptera, besides optically-based phenomena, biomolecules able to fluoresce can act as markers of bio-metabolic, structural and behavioral features. Resilin or chitinous compounds, with their respective blue or green-to-red autofluorescence (AF), are commonly related to biomechanical and structural properties, helpful to clarify the mechanisms underlying substrate adhesion of ectoparasites’ leg appendages, or the antennal abilities in tuning sound detection. Metarhodopsin, a red fluorescing photoproduct of rhodopsin, allows to investigate visual mechanisms, whereas NAD(P)H and flavins, commonly relatable to energy metabolism, favor the investigation of sperm vitality. Lipofuscins are AF biomarkers of aging, as well as pteridines, which, similarly to kynurenines, are also exploited in metabolic investigations. Beside the knowledge available in Drosophila melanogaster, a widely used model to study also human disorder and disease mechanisms, here we review optically-based studies in other dipteran species, including mosquitoes and fruit flies, discussing future perspectives for targeted studies with various practical applications, including pest and vector control.
... This may generate new hypotheses regarding roles for these receptors in behavioral and/or physiological processes, including lifespan. I generated preliminary data on the expression patterns of each 5-HT receptor in fly heads and bodies using a recently developed optically clearing protocol (Pende, Becker et al. 2018), which allows for fluorescent imaging of intact flies by depigmentation of the cuticle. This revealed that certain receptors are expressed in regions of the head which might typically be missed with a standard brain dissection. ...
Sensory perception of environmental cues such as nutrients, mates, and threats, modulates aging across taxa. These effects on lifespan require the action of conserved neuromodulators, such as serotonin. Despite clear links between serotonin and lifespan in specific environmental contexts, little was known about the influence of serotoninergic signaling pathways on lifespan in normal environments. I therefore, aimed to understand how manipulation of serotonin signaling through each of its receptors influenced lifespan in Drosophila. I first discovered that loss of specific serotonin receptors induces profound differences in lifespan. Of the five receptors for serotonin, loss of three led to a shortened lifespan in both males and females; however, loss of two receptors (5-HT2A and 5-HT7) caused a lifespan extension in female flies, with no effect on male lifespan. Behavioral characterization of these receptor mutants revealed most mutants do not display broad changes in sleep, activity, and feeding behavior. This 1) implies loss of individual receptors does lead to drastic negative health consequences, and 2) decouples the lifespan extension from any behavioral changes. This supports the notion that loss of receptor signaling pathways influence lifespan, independent of behavioral changes. Of the changes in lifespan across the 5-HT receptor mutants, I was most interested in the manipulations that extended lifespan, loss of 5-HT2A or 5-HT7. I focused on the mechanisms underlying the lifespan extension in the 5-HT2A-/- mutant in greater detail, as additional data in the lab revealed 5-HT2A was an important modulator of lifespan in two separate contexts. I was involved with one of these projects studying the mechanisms by which flies’ perception of presumed threat cues in the environment influenced lifespan. Specifically, visual detection of dead conspecifics shortens lifespan, and this effect requires 5-HT2A signaling. Further, a previous project identified that lifespan is shorted when flies must choose between sugar and protein consumption and this lifespan phenotype requires 5-HT2A. Based on these data, I suspected 5-HT2A plays an important role in evaluation of the nutritional environment, and disruption of this by loss of 5-HT2A may influence lifespan. and I found that 5-HT2A plays an important role in determining a protein consumption target in the fruit fly. My findings indicate that 5-HT2A signaling is recruited to promote protein consumption, perhaps by establishing a heightened protein consumption target, and to enact a physiological state of higher protein utilization that subsequently accelerates aging. In the absence of this receptor, protein demand remains chronically unsatisfied, which leads to an adaptive lowering of protein utilization and metabolism, extending lifespan. Together, the results from these projects support the notion that manipulating distinct aspects of serotonin signaling induces variable effects on lifespan. Closer examination of one receptor, 5-HT2A, revealed new insights into the ways in which organisms cope with nutrient stress and highlight how perception of nutrient demand influence lifespan. This work also opens the door for further investigations into the direct effects on lifespan that result from manipulations of serotonin signaling pathways.
