This work was supported by National Science Foundation (NSF) grant no. IOS 1354956. We thank Dr. Andrew D. Warren (McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida) for permission to use the butterfly images.

1. Insect Species, Preparation of Solutions and Feeding Station Setup
NOTE: Cabbage butterflies (Pieris rapae L., Pieridae) are selected as the species of representative Lepidoptera because they have been used in previous studies of fluid-uptake abilities and mouthpart morphology22,23. House flies (Musca domestica L., Muscidae) and blue bottle flies (Calliphora vomitoria L., Calliphoridae) are used because they are often observed feeding on porous surfaces13.
<ol>
	<li>Order P. rapae as larvae from an insect supplier and rear them on artificial diet (see Table of Materials) until they pupate and emerge as adults in an environmental chamber set to 23 &#176;C and a 18L:6D photoperiod. Order M. domestica and C. vomitoria as pupae and rear them at the same environmental conditions as P. rapae. Do not feed adult butterflies and flies after they emerge from the pupae prior to the feeding experiments.</li>
	<li>Prepare a 20% sucrose solution (control) and a 20% sucrose nanoparticle solution to test for fluid uptake. Prepare the nanoparticle solution by adding fluorescent magnetic nanoparticles (iron oxide with a polyacrylic acid coating, approximately 20 nm in diameter)24 to a 20% sucrose solution (1 mg/mL dH2O nanoparticles with 20% sucrose solution, 1:1). Prepare a 1x solution of phosphate buffered saline (PBS) (10x diluted to 1x in dH2O, pH 7.4), which is used for dissections.</li>
	<li>Set up a feeding station that consists of a manual manipulator with a clamp and a separate feeding stage (a flat platform) (Figure 1). Place a concave slide on the feeding stage and have nylon net filters with pore size diameters of 11, 20, 30, 41, or 60 &#181;m and membrane filters with pores size diameters of 1, 5, 8, or 10 &#181;m nearby for the feeding experiments.</li>
</ol>
2. Feeding Protocol
<ol>
	<li>Wrap the insect&#39;s bodies, legs, and wings into a folded tissue paper. Position the insect so that only the head and mouthparts are exposed. Place the wings of the insect between two microscope slides, which are held together by the clamp on the manipulator so that the insect is suspended above the feeding stage (Figure 1).</li>
	<li>Administer a 30 &#181;L droplet of either the 20% sucrose solution or the 20% sucrose nanoparticle solution with a micropipette to the center of the concave slide. Place a single filter paper of a specific pore size onto the concave slide so that the center of the filter paper is aligned with the droplet of the nanoparticle solution. The contact between the droplet and the filter paper results in the solution spreading along the filter paper, filling the pores (Figure 1). 
	NOTE: The filter paper is placed on top of the droplet, rather than the other way around, to minimize the potential presence of nanoparticles on top of the filter paper where the insects feed.</li>
	<li>Position the insect with the manipulator so that only the distal regions of the mouthparts can contact the wetted filter paper on the feeding stage (Figure 1). Use an insect pin to extend the mouthparts onto the filter paper and allow the insect to feed for 45 s.</li>
	<li>To minimize the chance of insects feeding on liquid films that might be present on the surface of the filter paper, position the mouthparts so that they are in contact with a part of the filter paper that is touching the flat part of the slide (i.e., not directly above the concave part of the slide). If the insect does not express an interest in feeding, the mouthparts can be held to the filter paper with the insect pin for the duration of the feeding time.</li>
</ol>
3. Dissections
<ol>
	<li>Place the PBS solution in a 50 mm watch glass so that there is enough solution to cover the insect&#39;s body. Place the watch glass under a stereoscope and position insect dissecting equipment (spring micro dissecting scissors, insect pins, fine point dissecting forceps) next to the stereoscope.</li>
	<li>After feeding, remove the insect from the tissue paper and hold it with wings closed. Remove the head, legs, and wings of the insect with the spring micro dissecting scissors and place the insect in the PBS solution in the watch glass (Figure 2).</li>
	<li>If needed, anesthetize insects before dissections. Use forceps to hold the insect by the cuticle near the distal region of the abdomen. With the dominant hand, use the spring micro dissecting scissors to cut the cuticle in an anterior direction along the lateral side of the abdomen, starting at the posterior end, until the thorax is reached. Take special care to ensure that only the cuticle is cut and that the alimentary canal inside the insect is not damaged (Figure 2).</li>
	<li>Make additional cuts of the cuticle with the dissecting scissors to open the abdomen to reveal the alimentary canal inside (Figure 2). Remove the abdominal cuticle, fat body, and other structures with the assistance of insect pins and relocate them outside of the watch glass for subsequent disposal, leaving only the thorax and alimentary canal in the watch glass. 
	NOTE: The dissection will reveal the crop, which is a sac-like structure (an extension of the alimentary canal) located near the juncture of the thorax and abdomen.</li>
	<li>If the crop is not exposed, make additional incisions into the thorax with the scissors until the crop is revealed. Once the crop is visible, cut away the remaining parts of the insect leaving only the alimentary canal with the crop in the watch glass (Figure 2). 
	NOTE: The lepidopteran crop is nearly transparent and cellophane-like in nature, which might be difficult to recognize if it is not filled with fluids and expanded or if it is cut during the dissection.</li>
	<li>Use fine point dissecting forceps to place the crop onto a coverslip (24 mm x 24 mm) for subsequent imaging (Figure 2).</li>
</ol>
4. Determination of Ingested Nanoparticles
<ol>
	<li>Position the crop on the coverslip with the fine point dissecting forceps using care to prevent rupturing the crop. Use the CY3 channel (or phase contrast) on an inverted confocal microscope for imaging at 20X magnification. Image the crop immediately after dissection to prevent it from drying out.</li>
	<li>Hold a magnetic stir bar (41.3 mm in length and 8 mm in diameter) in the hand that is not in control of the operating stage of the microscope. Wave the magnetic stir bar back and forth near the crop (approximately 10 mm distance from crop) so that each back and forth motion takes approximately one second (Figure 2).</li>
	<li>While the magnetic stir bar is waved, inspect the crop for the nanoparticles. Slowly move the operating stage back and forth while looking through the ocular lens of the microscope for nanoparticle movement inside the nearly transparent crop. If the nanoparticles are present in the crop, indicating positive feeding, they will respond and wave in unison with the magnetic stir bar (Figure 2).</li>
</ol>

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The authors have nothing to disclose.

Insect mouthpart functionality is historically inferred from studies of gross morphology (e.g., lepidopteran proboscis functionality related to a drinking straw25,26); however, recent studies that incorporate experimental evidence have resulted in a paradigm shift in our understanding of the complexities of insect mouthparts and structure-function relationships2,12,13...

Many insect groups have mouthparts (proboscises) adapted for feeding on fluids, such as nectar, rotting fruit, sap flows (e.g. Diptera1, Lepidoptera2, Hymenoptera3), xylem (Hemiptera4), tears (Lepidoptera5), and blood (Phthiraptera6, Siphonaptera7, Diptera7, Hemiptera8, Lepidoptera9). The ability of insects to feed on fluids is relevant to ecosystem health (e.g. pollination10), disease transmission4,11, biodiversification2,12, and studies of convergent evolution13. Despite the wide variety of food sources, a theme among some fluid-feeding insects is the ability to acquire small amounts of fluids, which could be confined to micro- or nano-sized droplets, liquid films, or porous surfaces.
Given the extensive diversity of fluid-feeding insects (more than 20% of all animal species14,15) and their ability to feed on a variety of food sources, understanding their feeding behaviors and fluid-uptake mechanisms is important in many fields. Insect mouthpart functionality, for instance, has played a role in the development of biomimetic technology, e.g., microfluidic devices that can perform tasks such as the acquisition of small amounts of fluids using methods similar to those employed by insects16. A fundamental problem in the studies of fluid uptake mechanisms, however, is determining not only how insects feed on fluids, but acquiring experimental evidence that supports the mechanism. Solely using behavior (e.g., probing with the proboscis12,17) as an indicator for feeding is insufficient because it does not confirm the successful uptake of fluids, nor does it provide a means to determine the route that fluids travel as they pass through the insect. In addition, performing experiments with small quantities of fluids better represents natural feeding scenarios where fluids are a limiting resource2,12.
X-ray phase contrast imaging was used with the Monarch butterfly (Danaus plexippus L.) to assess how butterflies feed on small amounts of fluids from porous surfaces12. Monarch butterflies use capillary action via spaces between cuticular projections (dorsal legulae) along the proboscis to bring fluids confined to small pores into the food canal. The incoming fluids form a film on the food canal wall that grows and collapses into a liquid bridge by Plateau instability12,18, which is then transported to the butterfly&#39;s gut by action of the sucking pump in the head. Although x-ray phase contrast imaging is an optimal tool for visualizing fluid flow inside insects12,19,20,21, the technique is not readily available and a more convenient method is needed for rapid assessment of an insect&#39;s ability to uptake fluids and ingest them.
To determine if the feeding mechanism for D. plexippus applies to other Lepidoptera and also to flies (Diptera) (both groups feed on liquids from porous surfaces), Lehnert et al.13 applied a technique for assessing an insect&#39;s ability to feed on small amounts of fluids from porous surfaces, which is reported in detail here. Although the protocol outlined here is for studies that use wetted and porous surfaces, the methodology can be altered for other studies, such as those addressing pool-feeding mechanisms. In addition, the applications extend to other fields, including microfluidics and bioinspired technology.

<table><tbody><tr><td>20% sucrose solution</td><td>Domino Sugar</td><td></td><td>Sugar needed to produce the sucrose solution with dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>P5493</td><td>10X concentration diluted to 1X in dH&lt;sub&gt;2&lt;/sub&gt;O for insect dissections</td></tr><tr><td>Single depression concave slide</td><td>AmScope</td><td>BS-C6</td><td>Slide is necessary for feeding stage setup</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY6004700 (60 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY4104700 (41 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY3004700 (30 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY2004700 (20 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY1104700 (11 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TCTP04700 (10 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TETP04700 (8 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TMTP04700 (5 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>RTTP04700 (1 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Iris microdissecting scissors</td><td>Carolina Biological Supply Company</td><td>623555</td><td>Scissors used for dissections</td></tr><tr><td>Insect pins (#1)</td><td>Bioquip Products</td><td>1208B1</td><td>Pins used during dissections and feeding trials</td></tr><tr><td>Extra-fine point dissecting forceps</td><td>Carolina Biological Supply Company</td><td>624684</td><td>Dissecting equipment</td></tr><tr><td>Leica M205 C Stereoscope</td><td>Leica Microsystems</td><td>M205 C</td><td>Stereoscope used for dissections</td></tr><tr><td>Inverted confocal microscope</td><td>Olympus</td><td>IX81</td><td>Fluorescent microscope used to detect magnetic nanoparticles</td></tr><tr><td>Fisherbrand PTFE Disposable Stir Bar</td><td>Fisherscientific</td><td>S68067</td><td>Magnet used to detect nanoparticles</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Kimberly-Clark Professional</td><td>34155</td><td>Tissues used to secure insects during feeding trials</td></tr><tr><td>House fly (Musca domestica) pupae</td><td>Mantisplace.com</td><td></td><td>insects for experiments</td></tr><tr><td>Blue bottle fly (Calliphora vomitoria) pupae</td><td>Mantisplace.com</td><td></td><td>insects for experiments</td></tr><tr><td>Cabbage butterfly (Pieris rapae) larvae</td><td>Carolina Biological Supply Company</td><td>144102</td><td>insects for experiments</td></tr><tr><td>Finnpipette F1&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>4641080N</td><td>micropipette for dispensing liquids</td></tr><tr><td>Finntip 250 pipette tips</td><td>ThermoFisher Scientific</td><td>9400250</td><td>micropipette tips</td></tr><tr><td>Microscope Glass cover slides (=coverslips) (24 x 24 mm)</td><td>AmScope</td><td>CS-S24-100</td><td>coverslips for viewing the insect&#039;s crop on confocal microscope</td></tr></tbody></table>

the ingestion of fluorescent, magnetic nanoparticles for determining fluid-uptake abilities in insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

The study of patterns in fluid-uptake abilities among fluid-feeding insects requires determination of when feeding occurs. The protocol outlined here is used to test the limiting pore size hypothesis among Lepidoptera and Diptera13. The limiting pore size hypothesis states that fluid-feeding insects cannot feed from liquid-filled pores if the pore size diameter is smaller than the diameter of the feeding conduits12. Incoming fluids from the ...

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The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

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Research

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Biology

6.3K Views.  Kent State University at Stark. Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

Video: The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Bioengineering

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Developmental Biology

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Behavior

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Neuroscience

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Immunology and Infection

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

Source: Hern&#225;ndez-Ortiz, J. A., et al. <a href="https://app.jove.com/v/63899">Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays.</a> J. Vis. Exp. (2022).
This video demonstrates a microfluidic system for a magnetic nanoparticle-based immunoassay. The detection system utilizes a microfluidic device incorporating a magnetic trap within its channels. Pre-formed immunocomplexes, comprised of antigen-coated magnetic nanoparticles bound to primary and peroxidase-conjugated secondary antibodies, are introduced into the channels. The immunocomplexes become captured in the porous magnetic trap upon applying an external magnetic field. Using a fluorogenic substrate emitting fluorescence upon catalysis by the peroxidases, the captured immunocomplexes are detected under a microscope.

1. Device preparation

	Fill the channels with distilled water using a syringe. Make sure there are no leaks or resistance to flow. Immerse the device in ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

An In Vivo Assay to Quantify Bacterial Phagocytosis in Adult Drosophila Flies

Use a micrometer to hold the needle under a microscope, and use number 5 fine-point stainless steel tweezers to break the tip to a 100-micrometer tip diameter. To measure the volume of liquid that will be injected into each fly, load a capillary needle with sterile 5% food coloring in PBS, and expel the liquid onto a drop of mineral oil on a 0.01-millimeter stage micrometer.
Dispense 10 microliters of 1.6mg/ml particles onto a small square of parafilm, and pull the liquid into the needle. Mount the needle into the injector nozzle, and line the anesthetized flies along their designated area on the fly pad, ventral side up, with the heads oriented toward the front of the pad.
Place the vials and corresponding areas on the bench, and inject the flies at the upper corner of the abdomen with five 100-millisecond pumps of liquid to deliver about 10 nanoliters of particles total. Transfer each fly into the appropriate vial as it is injected, noting the time on the vial.
Next, load a new needle with 0.4% trypan blue solution, and set the pneumatic injector to &#34;Gated&#34;, to allow a constant flow of air to push the liquid out of the needle. 30 minutes after the initial injection, inject each fly abdomen with trypan blue until the abdomens are full and distended. Mount the flys on microscope slides with electrical tape, ventral side down, pushing the wings to the side of the fly to secure them to the tape. Then, gently push the head into the tape to ensure that the fly will not move.
Immediately after all of the flies have been secured, image the insects, one at a time, at a 25 or 32 times magnification on an inverted fluorescence microscope attached to a digital camera and computer, focusing on the dorsal vessel of each fly using the computer software for the digital camera. Then, record the exposure time and magnification between experiments.
To quantify the fluorescence, open an appropriate imaging analysis program, and open one image. To measure the fluorescence intensity of the dorsal vessel, draw a polygon around the dorsal vessel, and select &#34;Measure&#34; to record the fluorescence intensity inside the polygon.
To determine the background fluorescence intensity, copy the first polygon, and move it to an area adjacent to the dorsal vessel of each fly. Then, select &#34;Measure&#34;, and record the fluorescence intensity of the background area.
To normalize the dorsal vessel fluorescence by the background fluorescence, after measuring the fluorescence intensities of the rest of the flies, divide the dorsal vessel fluorescence by the background fluorescence, and calculate the average normalized dorsal vessel fluorescence intensity of all the flies in one strain.

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Summary

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Chapters in this video

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

An In Vivo Assay to Quantify Bacterial Phagocytosis in Adult Drosophila Flies