The authors acknowledge the funding support from Canada&#39;s New Frontiers in Research Fund (NFRF), Natural Sciences&#160;and Engineering Research Council of Canada (NSERC) Discovery program, and Fonds de Recherche du Qu&#233;bec Nature et Technologies (FRQNT) master&#39;s training scholarships. The authors also would like to thank Prof. Yaoyao Zhao&#39;s group at McGill for their technical support on 3D printing of some components.

The mosquito species used for this protocol is an uninfected adult female Aedes aegypti (A. aegypti), received frozen and stored in a -20 &#176;C degree freezer. The species was provided by the NIH/NIAID Filariasis Research Reagent Resource Center for distribution through BEI Resources, NIAID, NIH: Uninfected Aedes aegypti, Strain Black Eye Liverpool (Frozen), NR-48920. The reagents and equipment used for the study are listed in the Table of Materials.
1. Dissecting the labium from the proboscis
<ol>
	<li>Using tweezers, place a dead mosquito on a glass slide under the microscope and ensure there is a tapered tip at the end of the proboscis (Figure 2A).</li>
	<li>While keeping the mosquito on the glass slide, place a scalpel blade gently over the labium near the mosquito&#39;s head (Figure 2B).</li>
	<li>Proceed to make an incision across the entire upper half of the labium (a cut of approximately 80 &#181;m) with a shallow penetration depth through the thickness of the labium. Ensure to apply a light pressure on the blade as to cut only the labium but not the fascicle that lies beneath.</li>
	<li>With a pair of tweezers, firmly hold the head of the mosquito, and with another pair of precision tweezers, lightly pinch the labium at any position in between the tapered tip and the location of the incision (Figure 2B).
	<ol>
		<li>Pull the tweezers holding the labium in the direction of the tapered tip (Figure 2C). Continue pulling the tweezers until the labium has ripped free and has been entirely removed from the fascicle.</li>
	</ol>
	</li>
	<li>Place the mosquito under the microscope and verify if the labrum&#39;s tip is present. This can be identified by the presence of a tapered tip on the fascicle (Figure 2D).</li>
</ol>
2. Separating the labrum&#39;s tip from the other fascicle members
<ol>
	<li>Clamp and close the tips of a set of precision tweezers and place the tip of the tweezers right beside the labrum near its tip.</li>
	<li>Use the tweezer tip to apply a gentle force on the labrum in the direction perpendicular to the length of the fascicle (Figure 3A).</li>
	<li>Continue pushing the labrum across the glass slide until separation of the labrum from the other fascicle members is achieved.</li>
	<li>Inspect the sample under the microscope to verify proper separation between the labrum and other fascicle members has been achieved (Figure 3, left). If separation is unsuccessful, refer back to step 2.1.</li>
</ol>
3. Cutting the labrum&#39;s tip
<ol>
	<li>While the labrum is still on the glass slide, place a scalpel blade over the labrum at approximately ~200 &#181;m in distance from the labrum&#39;s tip (Figure 4A). Gently apply sufficient pressure and cut the tip of the labrum all the way through. While the labrum tip should ideally be as short as possible, ~200 &#181;m is the best the current approach can handle.</li>
	<li>Measure the length of the cut labrum to ensure it is no longer than 300 &#181;m (Figure 4B) using any digital measurement software. In this protocol, ImageJ was used14.</li>
</ol>
4. Grabbing the tip of the labrum
<ol>
	<li>With a pair of precision tweezers, locate and isolate the labrum&#39;s tip on the glass slide. Discard every part remaining on the glass slide other than the labrum&#39;s tip.</li>
	<li>With the same precision tweezers, slowly and lightly pinch the labrum so that the cut end is free and unobstructed by the tweezers. Also, ensure the orientation of the labrum is parallel to the direction of the tweezers&#39; length and the cut end of the labrum is pointing away from the tweezers&#39; body.</li>
	<li>Once the specimen is firmly pinched, remove the clamping force holding the tweezers&#39; tips together. The labrum&#39;s tip will stick to one of the tweezers&#39; tips.</li>
	<li>Under a microscope, inspect the tweezers&#39; tips and ensure the labrum&#39;s tip is present on one of the tweezers&#39; tips (Figure 5). If the labrum&#39;s tip is not on the tweezers, refer back to step 4.2, and if the labrum&#39;s tip is not on the tweezers nor on the glass slide, refer back to step 1.</li>
</ol>
5. Applying epoxy on the tipless cantilever beam
<ol>
	<li>Place a drop (~0.05 mL) of epoxy on the edge of a new glass slide by directly drop-casting the adhesive from its original bottle/container. Place the epoxy-containing glass slide under the probe station and focus on it.</li>
	<li>Mount the tipless AFM cantilever to the clamp assembly by securing the base (i.e., the larger end), leaving the cantilever end free and suspended in space. Ensure that the bottom of the AFM cantilever is facing downward.</li>
	<li>Mount the manipulator onto the probe station.</li>
	<li>Raise the Z-axis of the manipulator to a position where the tipless cantilever is a few millimeters above the epoxy-containing glass slide.</li>
	<li>Manually move the manipulator such that the tipless cantilever is visible in the field view of the camera on the probe station.</li>
	<li>Using the manipulator, move the AFM cantilever along the X and Y directions until the tip of the cantilever is resting directly above the epoxy on the edge of the glass slide.</li>
	<li>Using the manipulator again, slowly lower the tipless cantilever in the Z direction over the edge of the glass slide.</li>
	<li>As the cantilever is lowered and approaches the glass slide, keep lowering the cantilever very slowly until it first touches the epoxy. Do not lower the cantilever any further.</li>
	<li>Cautiously, engage the manipulator to slowly move the cantilever in the X or Y direction and remove the cantilever from the pool of epoxy by continuously moving the cantilever in the selected direction until the cantilever is entirely separated from the epoxy on the glass slide. The tipless cantilever should have a miniature bubble of epoxy at its tip, visible under the probe station.</li>
	<li>Raise the cantilever in the Z-direction using the manipulator.</li>
</ol>
6. Bonding the labrum&#39;s tip to the tipless cantilever beam
<ol>
	<li>Rotate the manipulator about the cantilever&#39;s long axis by 90 degrees and rest the manipulator on the probe station on its side. In this configuration, the thicknesses along the length of the AFM cantilever is in the vertical direction.</li>
	<li>Position the precision tweezers that contain the labrum&#39;s tip under the probe station camera so that the entire length of the labrum&#39;s tip is visible on the computer monitor.</li>
	<li>Position the manipulator assembly holding the clamp and the tipless cantilever under the probe station camera so that the entire length of the tipless cantilever is visible on the computer monitor.</li>
	<li>Focus the probe station microscope on the labrum&#39;s tip and the tipless cantilever.</li>
	<li>Orient the cantilever perpendicularly to the labrum&#39;s tip by carefully and manually rotating the manipulator (Figure 6A).</li>
	<li>Using the degrees of freedom of the manipulator, slowly move the tipless cantilever in the XY directions such that the glue on the cantilever contacts the cut end of the labrum&#39;s tip (Figure 6B).</li>
	<li>Cure the epoxy, solidifying the intersection between the cantilever and mosquito labrum.</li>
	<li>Once the epoxy has cured, gently engage the manipulator in the XY directions and move the cantilever away from the tweezers, verifying that the labrum&#39;s tip is now standing on the tipless cantilever beam (Figure 6C).</li>
</ol>

Bio-Hybrid AFM Probe with Mosquito Labrum for Studying Biting Behaviors

<ol><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+vector+control+response+2017%E2%80%932030%5BTitle%5D)+AND+%22World+Health+Organization%22%5BJournal%5D)">Global vector control response 2017–2030.</a> World Health Organization. , (2017).</li>
<li>Gurera, D., Bhushan, B., Kumar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lessons+from+mosquitoes%E2%80%99+painless+piercing%5BTitle%5D)+AND+%22J+Mech+Behav+Biomed+Mater%22%5BJournal%5D)">Lessons from mosquitoes’ painless piercing.</a> J Mech Behav Biomed Mater. 84, 178-187 (2018).</li>
<li>Ma, G., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microneedle%2C+bio-microneedle+and+bio-inspired+microneedle%3A+A+review%5BTitle%5D)+AND+%22Journ+of+Contr+Relea%22%5BJournal%5D)">Microneedle, bio-microneedle and bio-inspired microneedle: A review.</a> Journ of Contr Relea. 251, 11-23 (2017).</li>
<li>Kong, X., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micronano+structure+and+mechanics+behavior+of+mosquito%E2%80%99s+proboscis+biomaterials+with+applications+to+microneedle+design%5BTitle%5D)+AND+%22Advan+Mater+Res%22%5BJournal%5D)">Micronano structure and mechanics behavior of mosquito’s proboscis biomaterials with applications to microneedle design.</a> Advan Mater Res. 299-300, 376-379 (2011).</li>
<li>Li, A. D. R., Putra, K. B., Chen, L., Montgomery, J. S., Shih, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mosquito+proboscis-inspired+needle+insertion+to+reduce+tissue+deformation+and+organ+displacement%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Mosquito proboscis-inspired needle insertion to reduce tissue deformation and organ displacement.</a> Sci Rep. 10 (1), 12248(2020).</li>
<li>Meyer, E., Hug, H. J., Bennewitz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introduction+to+Scanning+Probe+Microscopy%5BTitle%5D)+AND+%22Scanning+Probe+Microscopy%22%5BJournal%5D)">Introduction to Scanning Probe Microscopy.</a> Scanning Probe Microscopy. 1, 1-13 (2004).</li>
<li>García, R., Peréz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+atomic+force+microscopy+methods%5BTitle%5D)+AND+%22Surf+Sci+Rep%22%5BJournal%5D)">Dynamic atomic force microscopy methods.</a> Surf Sci Rep. 47 (6), 197-301 (2002).</li>
<li>Thurner, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic+force+microscopy+and+indentation+force+measurement+of+bone%5BTitle%5D)+AND+%22WIREs+Nanomed+and+Nanobio%22%5BJournal%5D)">Atomic force microscopy and indentation force measurement of bone.</a> WIREs Nanomed and Nanobio. 1 (6), 624-649 (2009).</li>
<li>Müller, D. J., Dufrêne, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic+force+microscopy+as+a+multifunctional+molecular+toolbox+in+nanobiotechnology%5BTitle%5D)+AND+%22Nat+Nanotech%22%5BJournal%5D)">Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology.</a> Nat Nanotech. 3 (5), 261-269 (2008).</li>
<li>Hussain, D., Ahmad, K., Song, J., Xie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+the+atomic+force+microscopy+for+-critical+dimension+metrology%5BTitle%5D)+AND+%22Meas+Sci+Technol%22%5BJournal%5D)">Advances in the atomic force microscopy for -critical dimension metrology.</a> Meas Sci Technol. 28 (1), 012001(2017).</li>
<li>Kong, X. Q., Wu, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mosquito+proboscis%3A+An+elegant+biomicroelectromechanical+system%5BTitle%5D)+AND+%22Phys+Rev+E%22%5BJournal%5D)">Mosquito proboscis: An elegant biomicroelectromechanical system.</a> Phys Rev E. 82 (1), 011910(2010).</li>
<li>Kong, X. Q., Wu, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+and+prediction+of+insertion+force+for+the+mosquito%5BTitle%5D)+AND+%22J+Bionic+Eng%22%5BJournal%5D)">Measurement and prediction of insertion force for the mosquito.</a> J Bionic Eng. 6 (2), 143-152 (2009).</li>
<li>Ramasubramanian, M. K., Barham, O. M., Swaminathan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanics+of+a+mosquito+bite+with+applications+to+microneedle+design%5BTitle%5D)+AND+%22Bioinspir+Biomim%22%5BJournal%5D)">Mechanics of a mosquito bite with applications to microneedle design.</a> Bioinspir Biomim. 3 (4), 046001(2008).</li>
<li>Ferreira, T., Rasband, W. ImageJ user guide. , Available from: 
 <a target="_blank" href="http://imagej.nih.gov/ij/docs/guide">http://imagej.nih.gov/ij/docs/guide</a> (2012).</li>
<li>Dai, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoscale+surface+measurements+at+sidewalls+of+nano-+and+micro-structures%5BTitle%5D)+AND+%22Measur+Sci+and+Technol%22%5BJournal%5D)">Nanoscale surface measurements at sidewalls of nano- and micro-structures.</a> Measur Sci and Technol. 18 (2), 334-341 (2007).</li>
</ol>

Authors have no conflict of interest to declare.

Step 1 of the protocol is meant to clean the biological sample of the unwanted labium. To achieve this, an incision is made on the labium, but not on the fascicle, which rests directly beneath the labium (Figure 1). Because the fascicle and labium are not joined together at their interface (i.e., the labium is free to slide along the fascicle and is only kept in place by its attachment to the mosquito head), the performed incision is meant to separate part of the labium from the mosquito&#39...

World Health Organization (WHO) reported that vector-borne diseases (VBDs) account for over 17% of all infectious diseases, which cause more than 7,00,000 deaths per year globally. For example, as the deadliest animal in the world, mosquitoes spread numerous pathogens, such as dengue, malaria, and Zika, through blood-feeding arthropods, resulting in 700 million infections each year1. Explorations toward the development of effective measures to prevent VBDs are of crucial importance, including mimicking the penetration behaviors of mosquitoes to investigate their biting mechanisms and studies of potential barriers to prove their efficacy in preventing penetration. One key challenge is to develop proper approaches to perform such investigations. Efforts have been made in the literature, including the development of micro-scale needles that resemble the geometry of a mosquito stinger; however, many of the materials used to make these microneedles (i.e., viscoelastic materials2, silicon (Si), glass, ceramic3, etc.) have different mechanical properties than the biological material of the mosquito&#39;s proboscis. The engineered materials can be brittle and prone to fracture and buckling3,4, whereas the mosquito&#39;s proboscis can withstand fracture or buckling better4. The benefit of having a bio-hybrid probe using the labrum of a mosquito instead of engineered materials is that it can be a more accurate representation of the piercing mechanism of mosquitoes. Also, specialized tools must be integrated with micro needles to perform quantitative studies, such as the accurate measurement of force5, which is not easily achievable with customized setups using engineered microneedles.
Atomic force microscopy (AFM)-based approach is promising in that it operates by employing a cantilever with an ultra-fine tip that is carefully positioned close to a sample&#39;s surface. The tip can either scan across or be pressed towards/into a surface, experiencing varying attractive or repulsive forces due to its interactions with a sample6. These interactions lead to the cantilever&#39;s deflection, which is tracked by the reflection of a laser beam from the cantilever&#39;s top onto a photodetector6. The exceptional sensitivity to movement of the system enables AFM to conduct a diverse range of measurements, including but not limited to morphological mapping with picometer accuracy, force measurements ranging from piconewtons to micronewtons, and comprehensive multiphysics investigations7. For example, AFM indentations can be performed to precisely assess the response to the applied force of a sample and also to measure the hardness, elasticity, and other mechanical properties of a sample by coupling with appropriate analytical models8. The probe of the AFM is most commonly made of silicon (Si) or silicon nitride (Si3N4)8 with a length of 20-300 &#181;m9 and a tip radius on the order of several to tens of nanometers10. The nanometer scale tip radius can be ideal for applications such as high-resolution imaging; however, it does not possess the characteristics of biological stingers for studies that try to mimic penetration behaviors in terms of stiffness, radius, shape, and aspect ratio. For example, the microneedle structure of a mosquito is the fascicle, which has an aspect ratio of ~6011 (length ~1.5 mm to 2 mm; diameter ~30 &#181;m)12. While a conventional AFM probe can be assumed to resemble a biological stinger like a labrum, its distinct material properties and dimensions will not reflect the real situation during a bite.
To enable quantitative investigations of penetration behaviors mimicking biological bites of insects or other animals with stingers, here, a process to fabricate bio-hybrid AFM cantilevers with a biological stinger as its tip is developed. As a case study, an AFM cantilever with a mosquito labrum&#39;s tip attached was successfully demonstrated. Harnessing existing information from literature on the typical insertion forces that a mosquito uses to pierce through a victim&#39;s skin12,13, this bio-hybrid AFM cantilever can potentially allow near-real mimicry of mosquito bites under a regular AFM. The protocol of leveraging micro biological stingers to fabricate bio-hybrid AFM cantilevers can also be applied to the development of other sharp stinger-based biohybrid AFM cantilevers for quantitative investigations of a variety of biting mechanisms.
Terminologies 
A schematic of a proboscis and its components of interest are shown in Figure 1, and their definitions are (1) Proboscis: a body part from the mouth of a mosquito that allows the mosquito to feed itself, with a core-shell structure composed of the fascicle(core) and the labium(shell), (2) Labium: the dark and blunt outer cover of a proboscis2, (3) Fascicle: a group of slender needles contained inside of the labium, including two maxillae, two mandibles, a hypopharynx, and a labrum2, (4) Hypopharynx: responsible for secreting saliva into the host&#39;s bloodstream2, (5) Maxillae: serrated member assisting in the feeding mechanism2, (5) Mandibles: similar to the maxilla, they help the mosquito in the feeding mechanism and have a sharp tip2, (6) Labrum: the main member for penetrating the skin of a victim, which is much bigger than the maxillae, mandibles and hypopharynx. It also has sensory structures that allow it to find blood vessels and internal channels under the skin2, (7) Manipulator: an assembly with three degrees of freedom and micron-scale accuracy for positioning, allowing movement in XYZ directions, (8) Clamp assembly: a custom-made 2-part clamp mounted to the manipulator used to clamp the tipless AFM cantilever during the experiment.

<table><tbody><tr><td>&amp;nbsp;5-SA-SE Straight Tapered Ultra Fine-Pointed Tweezers</td><td>Excelta</td><td>N/A</td><td>For manipulating/dissecting the proboscis.</td></tr><tr><td>C-4D Probe station</td><td>Everbeing Int&amp;rsquo;l Corp&amp;nbsp;</td><td>N/A</td><td>Used for AFM assembly.</td></tr><tr><td>Tipless Tapping Mode Cantilever</td><td>NanoAndMore USA</td><td>TL-NCH</td><td>AFM cantilever used for mounting the labrum.&lt;br/&gt; Specs are shown here:&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Shape&lt;/strong&gt;: Beam&lt;br/&gt; &lt;strong&gt;Force Constant&lt;/strong&gt;: 42 N/m (10 - 130 N/m)&lt;br/&gt; &lt;strong&gt;Resonance Frequency&lt;/strong&gt;: 330 kHz (204 - 497 kHz)&lt;br/&gt; &lt;strong&gt;Length&lt;/strong&gt;: 125 &amp;micro;m (115 - 135 &amp;micro;m)&lt;br/&gt; &lt;strong&gt;Width&lt;/strong&gt;: 30 &amp;micro;m (22.5 - 37.5 &amp;micro;m)&lt;br/&gt; &lt;strong&gt;Thickness&lt;/strong&gt;: 4 &amp;micro;m ( 3 - 5 &amp;micro;m)</td></tr><tr><td>UV Expoxy</td><td>Let&#039;s resin</td><td>ALR00146</td><td>For stinger attachment.</td></tr></tbody></table>

development of a bio-hybrid mosquito stinger-based atomic force microscopy probe

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The primary prevention strategy currently in use involves chemical repellents, which often prove ineffective as mosquitoes rapidly develop resistance. Consequently, the invention of new preventive methods is crucial. Such development hinges on a thorough understanding of mosquito biting behaviors, necessitating an experimental setup that accurately replicates actual biting scenarios with controllable testing parameters and quantitative measurements. To bridge this gap, a bio-hybrid atomic force microscopy (AFM) probe was engineered, featuring a biological stinger - specifically, a mosquito labrum - as its tip. This bio-hybrid probe, compatible with standard AFM systems, enables a near-authentic simulation of mosquito penetration behaviors. This method marks a step forward in the quantitative study of biting mechanisms, potentially leading to the creation of effective barriers against vector-borne diseases (VBDs) and opening new avenues in the fight against mosquito-transmitted illnesses.

Scanning electron microscopy (SEM) images of the fabricated bio-hybrid AFM probe can be found in Figure 7. The end of the labrum was successfully glued to the tipless cantilever beam. Due to the natural curvature of mosquito stingers and the manual operation of the presented protocol, it is extremely difficult to obtain a cantilever with a stinger tip perfectly perpendicular to the cantilever. The off-center angle between the stinger and an imaginary center line perpendicular to the cantilev...

Watch this Scientific Journal Video about Author Spotlight: Development of Bio-Hybrid AFM Cantilevers for Quantitative Analysis of Mosquito Biting Mechanisms at JoVE.com

Author Spotlight: Development of Bio-Hybrid AFM Cantilevers for Quantitative Analysis of Mosquito Biting Mechanisms

Quantitative and controlled investigations into insect biting behaviors are crucial for devising effective strategies to combat vector-borne diseases. In this context, a method for fabricating a bio-hybrid atomic force microscopy (AFM) probe is introduced.

Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The ...

development-bio-hybrid-mosquito-stinger-based-atomic-force-microscopy

Research

JoVE Journal

Engineering

795 Views.  McGill University. Quantitative and controlled investigations into insect biting behaviors are crucial for devising effective strategies to combat vector-borne diseases. In this context, a method for fabricating a bio-hybrid atomic force microscopy (AFM) probe is introduced.

Video: Author Spotlight: Development of Bio-Hybrid AFM Cantilevers for Quantitative Analysis of Mosquito Biting Mechanisms

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

Behavior

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Bioengineering

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Immunology and Infection

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Parallel Active Cantilever Arrays in AFMS to Enable High-Throughput Inspections

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. However, due to their limited imaging throughput, AFMs have not been widely adopted for large-scale inspection purposes. Researchers have developed high-speed AFM systems to record dynamic process videos in chemical and biological reactions at tens of frames per second, at the cost of a small imaging area of up to several square micrometers. In contrast, inspecting large-scale nanofabricated structures, such as semiconductor wafers, requires nanoscale spatial resolution imaging of a static sample over hundreds of square centimeters with high productivity. Conventional AFMs use a single passive cantilever probe with an optical beam deflection system, which can only collect one pixel at a time during AFM imaging, resulting in low imaging throughput. This work utilizes an array of active cantilevers with embedded piezoresistive sensors and thermomechanical actuators, which allows simultaneous multi-cantilever operation in parallel operation for increased imaging throughput. When combined with large-range nano-positioners and proper control algorithms, each cantilever can be individually controlled to capture multiple AFM images. With data-driven post-processing algorithms, the images can be stitched together, and defect detection can be performed by comparing them to the desired geometry. This paper introduces principles of the custom AFM using the active cantilever arrays, followed by a discussion on practical experiment considerations for inspection applications. Selected example images of silicon calibration grating, highly-oriented pyrolytic graphite, and extreme ultraviolet lithography masks are captured using an array of four active cantilevers (&#34;Quattro&#34;) with a 125 &#181;m tip separation distance. With more engineering integration, this high-throughput, large-scale imaging tool can provide 3D metrological data for extreme ultraviolet (EUV) masks, chemical mechanical planarization (CMP) inspection, failure analysis, displays, thin-film step measurements, roughness measurement dies, and laser-engraved dry gas seal grooves.

Author Spotlight: Introduction to Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays

Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections.

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Biology

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

Improved Imaging of DNA Star Motifs by PORT High-Speed Atomic Force Microscopy

High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.

Here, we show the imaging protocol for observing biomolecular interactions with photothermal off-resonance tapping (PORT), where we optimized imaging parameters, identified system limits, and investigated potential improvements in imaging three-point-star DNA motif assembly.

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due ...

Chemosensory Gene Expression Mapping in Mosquito Olfactory Appendages

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory appendages. Understanding how chemosensory receptors are spatially organized in the peripheral olfactory appendages can offer insights into how odor is encoded in the mosquito olfactory system and inform new ways to combat the spread of mosquito-borne diseases. The emergence of third-generation hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) allows for spatial mapping and simultaneous expression profiling of multiple chemosensory genes. Here, we describe a stepwise approach for performing HCR RNA WM-FISH on the Anopheles mosquito antenna and maxillary palp. We investigated the sensitivity of this technique by examining the expression profile of ionotropic olfactory receptors. We asked if the HCR WM-FISH technique described was suitable for multiplexed studies by tethering RNA probes to three spectrally distinct fluorophores. Results provided evidence that HCR RNA WM-FISH is robustly sensitive to simultaneously detect multiple chemosensory genes in the antenna and maxillary palp olfactory appendages. Further investigations attest to the suitability of HCR WM-FISH for co-expression profiling of double and triple RNA targets. This technique, when applied with modifications, could be adaptable to localize genes of interest in the olfactory tissues of other insect species or in other appendages.

Author Spotlight: Visualizing Olfactory Receptor Expression in Mosquitoes 

The article describes the methods and reagents necessary to perform hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) to reveal insights into the spatial and cellular resolution of chemosensory receptor genes in the mosquito antenna and maxillary palp.

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory ...

Neuroscience

Non-invasive Electropenetrography to Study Mosquito Probing Behaviors

AC-DC Electropenetrography for the Study of Probing and Ingestion Behaviors of Culex tarsalis Mosquitoes on Human Hands

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with hosts and pathogens could be exploited to develop new targets for controlling mosquito-borne diseases. Unfortunately, probing (i.e., biting) behaviors of mosquitoes are poorly understood because they occur inside host tissues. Here, a non-invasive procedure is described for using AC-DC electropenetrography (EPG) to indirectly visualize and quantify mosquito feeding behaviors by recording changes in electrical signals generated when probing and ingesting on human hands. Thin gold wires are attached to the mosquitoes using conductive silver glue and connected to the EPG instrument. The human host holds a substrate voltage probe in their hand. Probing of the host by the wired mosquito completes the electrical circuit, and electrical signals are recorded on a computer as &#34;waveforms&#34; that can then be measured and enumerated for analysis. This methodology has been used to characterize the probing and ingestion behaviors of Aedes aegypti and Culex tarsalis mosquitoes and can be applied to other mosquito species. EPG can be used to study the effects of pathogens, insecticides, and other factors on mosquito feeding behaviors.

Procedures are described for studying the probing and ingestion behaviors of Culex tarsalis&#160;mosquitoes on human hands using AC-DC electropenetrography.

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with ...

Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe

Summary

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Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

AC-DC Electropenetrography for the Study of Probing and Ingestion Behaviors of Culex tarsalis Mosquitoes on Human Hands