We thank Salvatore A.E. Marras (Public Health Research Institute Center, Rutgers University) for the synthesis, labeling and purification of molecular beacons, and Daniel St Johnston (The Gurdon Institute, University of Cambridge) for the oskar-MS2/MCP-GFP transgenic fly stock. This work was supported by a National Science Foundation CAREER Award 1149738 and a Professional Staff Congress-CUNY Award to DPB.

1. Design of MBs for Live Cell Imaging
<ol>
	<li>Fold the target RNA sequence to predict the mRNA target&#8217;s secondary structure using the &#8220;RNA form&#8221; from the mfold server (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form).
	<ol>
		<li>Paste/upload the target sequence in FASTA format, select 5 or 10% sub-optimality (structures with a free energy of folding within 5 or 10% of the MFE value, respectively), and adjust the maximum number of computed foldings accordingly (e.g. larger for 10% sub-optimality). 
		Note: Inclusion of sub-optimal secondary structures when designing MBs allows for the identification of regions within the target mRNA that may be more flexible or more rigid than as predicted for the minimum free energy (MFE) structure alone, which improves the overall design of MBs suited for live cell imaging.</li>
		<li>Select an &#8220;immediate job&#8221; for mRNA targets of 800 nucleotides (nt), or a &#8220;batch job&#8221; for mRNA lengths between 801 and 8,000 nt. Save the &#8220;ss-count&#8221; file as simple text file.</li>
	</ol>
	</li>
	<li>Use the &#8220;ss-count&#8221; file obtained in step 1.1 as input for the PinMol program (https://bratulab.wordpress.com/software/) with the desired parameters, to design several MBs for the mRNA target (see tutorials describing usage of PinMol program24 at https://bratulab.wordpress.com/tutorial-pinmol-mac/).
	<ol>
		<li>Determine the specificity of selected MBs by performing BLAST analysis: use &#8220;blastn&#8221; with the appropriate database (e.g. for oskar mRNA-specific MBs use the &#8220;refseq-rna&#8221; database and the Drosophila melanogaster organism).</li>
		<li>Identify any tissue-specific expression of mRNA target (e.g. for oskar mRNA Flybase&#62; High-Throughput Expression Data&#62; FlyAtlas Anatomy Microarray or modENCODE Anatomy RNA-Seq; http://flybase.org/reports/FBgn0003015) and compare with any positive BLAST hits. Eliminate probes that show &#62;50% cross-homology with other mRNAs that are also expressed in the tissue/cell of interest.</li>
	</ol>
	</li>
	<li>Select the fluorophore and quencher pair appropriate for the microscopy set-up available to perform live cell imaging (e.g. Cy5/BHQ2)25.</li>
</ol>
2. MB Synthesis, Purification, and Characterization
<ol>
	<li>Use in-house synthesis and purification as previously described7, or services from commercial providers, to synthesize and purify one to five MBs (see above note), using the following labeling scheme: [5&#8217;(Fluorophore)-(C3 or C6 linker)-(2&#8217;-O-methyl MB sequence)-(Quencher)3&#8217;]. Purify MBs using reverse-phase HPLC, in house or using the services of the commercial provider. 
	NOTE: The phosphoramidites used for automated probe synthesis must have the 2&#8217;-O-methyl ribonucleotide modification. One can also use chimeras of alternating locked-nucleic acid (LNA) and 2&#8217;-O-methyl modifications to increase the stability of a hybrid between a shorter MB and its target mRNA26.</li>
	<li>Synthesize DNA oligonucleotides that match the sequence of the targeted RNA region and thus are complementary to the probe region of MBs, for use in in vitro characterization (see steps 2.3 to 2.5; above note). Maximize hybridization of the MB with the DNA-oligonucleotide target mimic, by including on each end of the DNA target four additional nucleotides, as found in the target mRNA sequence. 
	Note: A more rigorous characterization of the MB&#8217;s efficiency to detect the targeted sequence can be performed using in vitro synthesized RNA targets instead of complementary DNA oligonucleotides8.</li>
	<li>Perform thermal denaturation of the MB alone, measure its melting temperature (Tm), and confirm that the MB assumes the desired hairpin shape at physiological temperature. We have observed Tm values between 60 and 90 &#176;C.</li>
	<li>Perform thermal denaturation of the MB in the presence of the DNA oligonucleotide target and measure the MB:DNA target hybrid&#8217;s Tm, as previously described7. A Tm between 55 and 60 &#176;C is desired for the MB:DNA hybrid.</li>
	<li>Perform in vitro hybridization reactions with the corresponding DNA oligonucleotide target, and determine the efficiency of MB:DNA hybrid formation at physiological temperature, as previously described7. Fast hybridization kinetics with the DNA target mimic is desired, however MBs that do not show high hybridization efficiency with DNA targets may have a better performance with the target mRNA in vitro and/or in vivo.</li>
</ol>
3. Dissection and Preparation of Individual Egg Chambers for Microinjection
<ol>
	<li>Feed newly hatched, mated females for 2-3 days with fresh yeast paste.</li>
	<li>Anesthetize flies on a CO2 pad and, using fine tweezers (Dumont #5), transfer 1-2 females into a drop of Halocarbon oil 700 on a glass cover slip.</li>
	<li>Using a pair of tweezers, orient the fly with the dorsal side up under a stereomicroscope. Dissect the female abdomen by making a small incision at the posterior end and gently squeeze the pair of ovaries into the oil.</li>
	<li>Explant the ovaries onto an oil drop on a new coverslip. Gently hold one ovary with one tweezer while pinching off the youngest stages of the ovariole with the other tweezer. oskar mRNA is actively localized at and after mid-oogenesis (stages &#62; 7), and younger egg chambers ( stages &#60; 7) are more difficult to inject and do not survive as long. Slowly drag on the cover slip (with a downward movement) until individual ovarioles or egg chambers are isolated and aligned vertically. Further separate single egg chambers by displacing the unwanted stages from the ovariole egg chain. 
	NOTE: Ensure that individually teased egg chambers do not float in the oil, and that they adhere to the cover slip. This is important for both successful microinjection and image acquisition.</li>
</ol>
4. Microinjection of MBs into the Nurse Cells of Egg Chambers
<ol>
	<li>Prepare the MB solution, using one molecular beacon (e.g. osk2216Cy5), or a mix of two MBs that target different mRNAs and which are labeled with spectrally distinct fluorophores (e.g. osk2216Cy5 and drongo1111Cy3). Use a concentration of 200-300 ng/&#181;L each MB in HybBuffer (50 mM Tris-HCl - pH 7.5, 1.5 mM MgCl2 and 100 mM NaCl). For a cocktail of four MBs labeled with the same fluorophore that are targeting the same mRNA at 200 ng/&#181;L each in HybBuffer (e.g. osk82, osk1236, osk2216). Spin down the MB solution immediately prior to loading the needle for microinjection.</li>
	<li>Select the objective. A 40x oil objective is recommended for finding an appropriate egg chamber and for performing microinjection.</li>
	<li>Mount the coverslip with dissected egg chamber onto the microscope stage. Bring up the objective in the focus position and identify an egg chamber at a mid-to-late developmental stage, that is properly oriented for microinjection (i.e., with the A&#224;P axis perpendicular to the needle tip to allow for easy injection within a nurse cell proximal to the oocyte).</li>
	<li>Load a needle (commercial or prepared in house27) with ~1 &#181;L MB solution (see step 4.1) and connect it to the microinjector. For microinjections in D. melanogaster egg chambers, orient the needle (see Table of Materials) at an angle &#60;45&#176; to the microscope stage (e.g. 30&#176;) to avoid puncturing several nurse cells.</li>
	<li>Set-up the injector with injection pressure of 500-1,000 hPa and compensation pressure of 100-250 hPa (see Table of Materials).</li>
	<li>Slowly move the stage to bring in the field of view an area of the oil drop void of egg chambers.</li>
	<li>Using the micromanipulator joystick, gently lower the needle into the oil drop and bring its tip into focus towards the periphery of the field of view.</li>
	<li>Perform a &#8216;clean&#8217; function to remove the air from the tip of needle and to ensure that there is flow from the needle.</li>
	<li>Bring the needle to the home position and focus on the egg chamber to be microinjected, then bring the needle back into focus and position it near the edge of the egg chamber.</li>
	<li>Perform a fine adjustment of the objective&#8217;s Z-position such that the membrane separating the follicle cells from nurse cells is in focus.</li>
	<li>Insert the needle into a nurse cell and perform injection for 2-5 s.</li>
	<li>Gently remove the needle and retract it to the home position.</li>
	<li>Change the objective to the desired magnification for image acquisition (60-63x or 100x), focus on the egg chamber, and begin acquisition.</li>
</ol>
5. Acquisition of Data Using a Spinning Disc Confocal Microscope Setup
NOTE: See Table of Materials for our specific setup.
<ol>
	<li>Set up acquisition protocol to record an XYZCt stack of 8-16-bit images (XYZ = volume, C = channel, t = time).</li>
	<li>Select laser lines for the desired channels (e.g. 641 nm laser for Cy5 and 491 nm for GFP) and acquire the channels sequentially: first the fluorescence signal in each channel and then change the Z position, to allow for proper colocalization analysis.</li>
	<li>Select the Z step (e.g. 0.3 &#181;m), and the top and bottom Z limits (e.g. -2 &#181;m to 2 &#181;m).</li>
	<li>Input the acquisition time and sampling rate (e.g. every 15-30 s for up to 1 h).</li>
	<li>Initiate acquisition.</li>
</ol>
6. Processing, Data Analysis to Obtain Tracking and Colocalization Information, and Preparation of Video Files
<ol>
	<li>Image Processing
	<ol>
		<li>Download, unpack, and open Icy, an open community platform for bioimage informatics (http://icy.bioimageanalysis.org/)</li>
		<li>Open the XYZCt stack acquired in step 5: Image/Sequence &#62;File&#62;Open.</li>
		<li>Convert stack to ImageJ: ImageJ&#62; Tools&#62; Convert to IJ, have Detached Mode ON.</li>
		<li>Make a substack (a selection of a range of Z steps and time points to be further analyzed): ImageJ&#62;Image&#62;Stacks&#62;Tools&#62;Make Substack&#8230;; select the desired channels, Z-steps and time points.</li>
		<li>Save substack as TIFF file: ImageJ&#62;File&#62;Save As&#62;Tiff&#8230;; use this file for subsequent steps.</li>
		<li>Split channels: ImageJ&#62;Image&#62;Color&#62;Split Channels.</li>
		<li>Subtract background either using a background stack: ImageJ&#62;Process&#62;Image Calculator&#8230;, or using the Rolling ball option: ImageJ&#62;Process&#62;Subtract Background&#8230;, select the Rolling ball radius. Preview the image for the radius selected before selecting &#8220;Accept&#8221;. 
		Note: Background signal will mainly arise from improper quenching of the flurorophore. The signal:background ratio (S:B) is often used as an indicator for an MB&#8217;s &#8220;brightness&#8221;, and it is measured from in vitro hybridization experiments of the MB and DNA target oligonucleotide. For example, MBs osk1236 and osk2216 have an S:B of ~81 and ~120, respectively.</li>
		<li>Adjust the brightness and contrast for each channel: ImageJ&#62;Image&#62;Adjust&#62;Brightness/Contrast, select Apply.</li>
		<li>Save each channel as a separate TIFF file: ImageJ&#62;File&#62;Save As&#62;Tiff....</li>
		<li>Merge the two channels: ImageJ&#62;Image&#62;Color&#62;Merge Channels&#8230;; select the channels. Save the new stack as a new TIFF file (see step 6.1.8).</li>
	</ol>
	</li>
	<li>Spot Detection and Tracking
	<ol>
		<li>Convert back to Icy: ImageJ&#62;Tools&#62;Convert to Icy.</li>
		<li>A scale bar is automatically overlaid onto the stack upon conversion to Icy, if the scale bar plugin is installed [Search using Plugins&#62;Setup&#62;Online plugin]. If needed, edit the scale bar via Inspector window (right side of screen)&#62;Layer tab&#62;Name&#62;Scale bar.</li>
		<li>Deselect/inactivate the &#8216;eye&#8217; icon for Scale bar from the Layer tab&#62;Name to remove the scale bar from the original stack. It can be reactivated on the final stack.</li>
		<li>Save the newly processed stack by taking a screenshot using the &#8220;camera&#8221; icon from the Image Window&#8217;s menu bar, &#8220;Take a screenshot of current view&#8221; and File&#62;Save as&#62;Tiff&#8230;.</li>
		<li>Determine spot sensitivity, if spot sensitivity parameters have already been determined move onto step 6.2.7.</li>
		<li>Detect spots: select the window with the image or stack to be analyzed, Detection&#38;Tracking&#62;Detection&#62;Spot Detector, and fill in the Settings parameters:
		<ol>
			<li>For Input, select &#8220;currentSequenceInputDetection&#8221; (default).</li>
			<li>For Pre Processing, select &#8220;Channel 0&#8221; (default), or desired channel by cross-referencing the number in the Inspector window&#62;Sequence tab.</li>
			<li>For Detector, select &#8220;Detect bright spot over dark background;&#8221; use &#8220;Force use of 2D Wavelets for 3D&#8221; only if there are not enough Z-slices in the stacks to perform the analysis. Select &#8220;Scale(s)&#8221; and &#8220;Sensitivity&#8221; for each scale (add more scales for larger spots). The Scale and Sensitivity (the larger the number the more sensitive is the detection, a maximum of 140 is suggested by Icy) are trial and error variables, that must be visually checked afterwards and decided upon.</li>
			<li>For Region of Interest, use &#8220;ROIfromSequence&#8221; (default).</li>
			<li>For Filtering, use &#8220;NoFiltering&#8221; (default), or select &#8220;SizeFiltering&#8221; to define the &#8220;Range of accepted objects (in pixels)&#8221;.</li>
			<li>Output: select XLS or XML output setting (select XML format when using 2007 MS Excel or earlier and there are &#62;65,000 spots). If the spot detector results are used for the tracking analysis, also select &#8220;Export to SwimmingPool&#8221;.</li>
			<li>Repeat detection of spots using various scale/sensitivity values until all or most of the spots are detected. Record all of the final parameters.</li>
			<li>For colocalization analysis, repeat spot detection for the other channel.</li>
		</ol>
		</li>
		<li>To track spots, select Detection&#38;Tracking&#62;Tracking&#62;Spot Tracking&#62;Run the Spot Detector with parameters from step 6.2.6., or use &#8220;Select detection results here&#8221; pull-down menu to select an existing dataset (for this, keep Spot Detector window open from step 6.2.5). Press the &#8220;Estimate parameters&#8221; button and select the desired target motion in the Parameters estimation pop-up window (e.g. &#8220;is both diffusive and directed&#8221;). Press the &#8220;Run tracking&#8221; button.</li>
		<li>Repeat spot detection and tracking for other channels when tracking spots of multichannel stacks, following steps 6.2.6 and 6.2.7, beginning with the stack generated from step 6.2.7.</li>
		<li>To visualize tracks, select Detection&#38;Tracking&#62;Tracking&#62;Track Manager &#8211; this window opens automatically upon completion of a tracking run. For &#8220;Color Track Processor,&#8221; select &#8220;Enable&#8221; and choose the desired representation of color for the tracks. Relevant track processors can be accessed via the &#8220;add Track Processor&#8230;&#8221; pull-down menu (e.g. select &#8220;Track Processor Time Clip,&#8221; enable the &#8220;Track Clipper&#8221; window, and choose the desired number of detections to be displayed before and after the current time point.)</li>
		<li>Save tracks information as an XML track file: Detection&#38;Tracking&#62;Tracking&#62;Track Manager&#62;File&#62;Save as&#8230;.</li>
		<li>Save results by taking a screenshot using the &#8220;camera&#8221; icon from the Image Window&#8217;s menu bar, &#8220;Take a screenshot of current view&#8221;. Screenshots can be taken with the detected spots and/or the tracks simply by activating/deactivating the corresponding eye icon(s) found in Inspector window&#62;Layer tab&#62;Name&#62;Overlay wrapper.</li>
		<li>Install the TimeStamp Overlay plugin: Plugins&#62;Setup&#62;Online plugin&#62;TimeStamp Overlay&#62;Install.</li>
		<li>Add timestamp: Plugins&#62;TimeStamp Overlay (New). Follow instructions on the pop-up window (lower right corner of screen) for directions on placing and formatting the time stamp. The time interval can be added/changed in the Inspector window&#62;Sequence tab&#62;Sequence Properties&#62;Edit.</li>
		<li>Save results by taking another screenshot. Save image as 1) Tiff format, and 2) as AVI format; for AVI format first convert to RGB rendering (Image/Sequence&#62;Rendering&#62;RGB image).</li>
		<li>Rotate image to desired orientation: Inspector window&#62;Sequence tab&#62;Canvas&#62;Rotation.</li>
		<li>Save rotated image by &#8220;Take a screenshot of the current view&#8221;. Ensure the &#8220;eye&#8221; icon for the scale bar is deselected, as it will also rotate with the image.</li>
		<li>Choose and crop ROI: select Region Of Interest&#62;2D ROI&#62;Choose ROI shape and then create/draw ROI on the image; Image/Sequence&#62;Plane (XY)&#62;Fast crop.</li>
	</ol>
	</li>
	<li>Colocalization analysis
	<ol>
		<li>Prepare a colocalization protocol; several examples are provided on the Icy website (http://icy.bioimageanalysis.org/protocol/List) (see Supplemental Materials).</li>
		<li>Load colocalization protocol: Tools&#62;Scripting&#62;Protocols&#62;Load, and adjust parameters in the interacting blocks (e.g. in the &#8220;Wavelet Spot Detecting&#8221; block use parameters determined in step 6.2.6.).</li>
		<li>Measure the size of a particle in pixels, determine the colocalization distance and input it into &#8220;Colocalizer&#8221; block as &#8220;Max distance.&#8221; 
		NOTE: The size of the particle in pixels depends on the detection system. To measure size, zoom into a single particle and manually count the pixels that span the width of the signal across. Average the measurements from at least three particles. The maximum distance to be set for colocalization is the size of the particle in pixels (this represents the maximum sum of the radius of two particles touching).</li>
		<li>If desired, select one or more ROIs for colocalization analysis: Region Of Interest&#62;2D ROI&#62;Choose ROI shape&#62;Draw ROI on image.</li>
		<li>Crop ROI(s): Image/Sequence&#62;Plane (XY)&#62;Fast crop.</li>
		<li>Perform colocalization: Protocols editor window&#62;Chosen protocol tab&#62;Run. The final block in Protocols editor window will contain overall colocalization percentage based on spot detection, while information at each time point can be found in the Inspector window&#62; Output tab.</li>
		<li>Track colocalized and single particles by following step 6.2.7 (Track spots).</li>
		<li>Save as described in step 6.2.16.</li>
	</ol>
	</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+Oogenesis:+An+Overview.">Drosophila melanogaster Oogenesis: An Overview.</a> Methods in Molecular Biology. 1328, 1-20 (2015).</li><li>Bastock, R., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+oogenesis.">Drosophila oogenesis.</a> Current Biology. 18 (23), R1082-R1087 (2008).</li><li>Rongo, C., Gavis, E. R., Lehmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+oskar+RNA+regulates+oskar+translation+and+requires+Oskar+protein.">Localization of oskar RNA regulates oskar translation and requires Oskar protein.</a> Development. 121 (9), 2737-2746 (1995).</li><li>Mhlanga, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+colocalisation+of+oskar+mRNA+and+trans-acting+proteins+revealed+by+quantitative+imaging+of+the+Drosophila+oocyte.">In vivo colocalisation of oskar mRNA and trans-acting proteins revealed by quantitative imaging of the Drosophila oocyte.</a> PLoS One. 4 (7), e6241 (2009).</li><li>Bayer, L. V., Omar, O. S., Bratu, D. P., Catrina, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PinMol:+Python+application+for+designing+molecular+beacons+for+live+cell+imaging+of+endogenous+mRNAs.">PinMol: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs.</a> bioRxiv. , (2018).</li><li>Marras, S. A., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiencies+of+fluorescence+resonance+energy+transfer+and+contact-mediated+quenching+in+oligonucleotide+probes.">Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes.</a> Nucleic Acids Research. 30 (21), e122 (2002).</li><li>Bratu, D. P., Catrina, I. E., Marras, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiny+molecular+beacons+for+in+vivo+mRNA+detection.">Tiny molecular beacons for in vivo mRNA detection.</a> Methods in Molecular Biology. 714, 141-157 (2011).</li><li>Dean, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+(pulling)+of+needles+for+gene+delivery+by+microinjection.">Preparation (pulling) of needles for gene delivery by microinjection.</a> Cold Spring Harbor. 2006 (7), (2006).</li><li>Alami, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+TDP-43+mRNA+granules+is+impaired+by+ALS-causing+mutations.">Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations.</a> Neuron. 81 (3), 536-543 (2014).</li><li>Jackson, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Hairpin+DNA-Functionalized+Gold+Nanoparticles+for+Imaging+mRNA+in+Living+Cells.">Applications of Hairpin DNA-Functionalized Gold Nanoparticles for Imaging mRNA in Living Cells.</a> Methods in Enzymology. 572, 87-103 (2016).</li><li>Zimyanin, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+oskar+mRNA+transport+reveals+the+mechanism+of+posterior+localization.">In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization.</a> Cell. 134 (5), 843-853 (2008).</li><li>Catrina, I. E., Marras, S. A., Bratu, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiny+molecular+beacons:+LNA/2'-O-methyl+RNA+chimeric+probes+for+imaging+dynamic+mRNA+processes+in+living+cells.">Tiny molecular beacons: LNA/2'-O-methyl RNA chimeric probes for imaging dynamic mRNA processes in living cells.</a> ACS Chemical Biology. 7 (9), 1586-1595 (2012).</li><li>Chen, A. K., Behlke, M. A., Tsourkas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+cytosolic+delivery+of+molecular+beacon+conjugates+and+flow+cytometric+analysis+of+target+RNA.">Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA.</a> Nucleic Acids Research. 36 (12), e69 (2008).</li><li>Nitin, N., Santangelo, P. J., Kim, G., Nie, S., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-linked+molecular+beacons+for+efficient+delivery+and+rapid+mRNA+detection+in+living+cells.">Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells.</a> Nucleic Acids Research. 32 (6), e58 (2004).</li><li>Chen, A. K., Behlke, M. A., Tsourkas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+false-positive+signals+with+nuclease-vulnerable+molecular+beacons+in+single+living+cells.">Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.</a> Nucleic Acids Research. 35 (16), e105 (2007).</li><li>Bevilacqua, P. C., Ritchey, L. E., Su, Z., Assmann, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Analysis+of+RNA+Secondary+Structure.">Genome-Wide Analysis of RNA Secondary Structure.</a> Annual Review of Genetics. 50, 235-266 (2016).</li><li>Mhlanga, M. M., Vargas, D. Y., Fung, C. W., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tRNA-linked+molecular+beacons+for+imaging+mRNAs+in+the+cytoplasm+of+living+cells.">tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells.</a> Nucleic Acids Research. 33 (6), 1902-1912 (2005).</li><li>Eliceiri, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+imaging+software+tools.">Biological imaging software tools.</a> Nature Methods. 9 (7), 697-710 (2012).</li><li>Bolte, S., Cordelieres, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guided+tour+into+subcellular+colocalization+analysis+in+light+microscopy.">A guided tour into subcellular colocalization analysis in light microscopy.</a> Journal of Microscopy. 224 (Pt 3), 213-232 (2006).</li><li>Trcek, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+germ+granules+are+structured+and+contain+homotypic+mRNA+clusters.">Drosophila germ granules are structured and contain homotypic mRNA clusters.</a> Nature Commununications. 6, 7962 (2015).</li></ol>

The authors have no conflict of interest to disclose.

Live visualization of endogenous mRNA trafficking in Drosophila egg chambers relies on the use of specific, efficient, and nuclease-resistant MBs, which can now be easily designed with PinMol software. MBs are specific probes designed to detect unique sequences within a target mRNA (preferably regions free of secondary structure), making possible highly resolved detection of a transcript. The only limitation when adopting this technique/protocol for other tissues/cell types is the efficiency of MB delivery for the specimen of interest. While other approaches require genetic manipulation of the tissue to express an aptamer and an RNA-binding protein tagged with a fluorescent protein to visualize one target mRNA (e.g. MS2/MCP system), multiplexing is possible for, at most, two transcripts. MB technology stands alone for detecting endogenous mRNAs in living cells, and it is the only technique permitting the co-visualization of more than two mRNA species.
MBs can be labeled with a wide range of fluorescent moieties and are stable within the cellular environment when synthesized from modified nucleotides such as 2&#39;-O-methylribonucleotides or locked-nucleic acids26,31. These backbone modifications also increase MBs&#39; affinity for their target. Several MBs can be easily designed for target mRNAs of average length. However, some limitations may be encountered for short and/or highly structured targets. This can be overcome by adopting our tiny molecular beacons, for which the probe region is approximately half of the length of a classical MB probe31. Depending on the specimen type, MBs are delivered into cells via electroporation, linking to cell-penetrating peptides, lipofection, or microinjection23,32,33,34. An MB&#39;s performance efficiency in live cell imaging experiments leans on the capability of the probe sequence to hybridize to the corresponding complementary sequence within the mRNA target, which is determined by the target structure. The predicted MFE RNA secondary structure obtained using in vitro measured thermodynamics parameters is valuable in assessing target accessibility, but ultimately it is the in vivo target structure and the target interaction with other cellular factors that will determine the MB&#39;s suitability for live cell imaging. Genome-wide analysis of RNA secondary structure suggests that many RNAs are less structured in vivo than in vitro35. Although the efficiency of in vivo target detection using MBs is mainly dependent on the accessibility of the binding site, optimizing certain MB features will ensure an enhanced visualization of the mRNA target. Specifically, a careful selection of the following parameters should be performed: 1) the probe length can vary between 18 and 26, such that the probe&#39;s nucleotide composition is between 31 and 55% GC pairs in the target:MB hybrid, 2) the 5 bp stem sequence should be G/C rich, to maintain the hairpin shape in absence of mRNA target and to provide mismatch discrimination, 3) a modified backbone should be used for protection against nucleases of both MB and target:MB hybrid, 4) the fluorophore/quencher pair can offer an additional modest stability to the MB&#39;s stem, and 5) the fluorophore should be stable during long imaging time intervals. In addition, classical MBs usually generate a nuclear non-specific signal34, which in our case only moderately impacts data processing and analysis. However, for mRNA trafficking visualization at cellular level, this non-specific signal may become problematic. Several groups have proposed modifications or tags, such as tRNA, peptides and nanoparticles, which prevent the delivery of MBs into the nucleus and thus eliminate this possible non-specific signal33,36.
The biggest drawback of this approach has been the manual design of MBs for live cell imaging. To address this, we have written a Python-based program (PinMol) that readily identifies accessible target sites within an mRNA by considering suboptimal secondary structures in addition to the MFE, as well as designs hairpin probes, which are best suited for detection of mRNAs in live cells24. PinMol uses structural information from secondary structures of the target RNA predicted via energy minimization approaches, and by including information from suboptimal structures, the flexibility or rigidity of specific targeted regions is assessed when designing MBs. It takes into account the accessibility of the targeted regions,as well as the inter- and intramolecular interactions of each selected probe. Additionally, highly regulated stretches of RNA (e.g. binding sites for microRNAs or RNA-binding proteins) should not be considered as target sites when selecting probes, as these regions may result in inefficient binding of the MBs. The user can evaluate and eliminate probes targeting these sites, or restrict the target region used by PinMol to design probes so it does not include such sites. The relative capability of PinMol was demonstrated by comparing the ranking of PinMol designed MBs with the experimental results of manually designed MBs24. Pinmol selected and designed MBs for similar target regions as well as identified new accessible sites on the mRNA. This is essential for the detection of low copy number transcripts where the fluorescent signal must be increased above background. By scaling up the MB numbers which effectively hybridize to several accessible sites on a target mRNA, signal amplification can be achieved. Therefore, this program facilitates a fast approach to design multiple MBs per target mRNA, and to simultaneously visualize numerous mRNAs in a live cell.
In order to achieve high quality 5D (XYZCt) acquisition data of transported mRNAs within the egg chamber, proper dissection of individual egg chambers and effective microinjection into nurse cells are critical. For studies during early stages of development, microinjection can be detrimental to the viability of the egg chamber and thus the length of a live cell imaging experiment is shortened (&#60;20 min). An increased success rate of the microinjection experiments can be ensured by using commercially available ultrafine needles. In addition, a quick set-up of the acquisition settings is important so that the early, post-injection time points can be captured. The quality of the spot detection and tracking data will only be as good as the quality of the images acquired.
Upon image acquisition, it is essential that subsequent analysis steps are also completed carefully and precisely. Although post-acquisition processing and analysis offer their own set of difficulties, they can be streamlined by choosing the appropriate software for one&#39;s particular experiment or sample. Current existing programs include Volocity (PerkinElmer), Imaris (Bitplane), ImageJ/Fiji and Icy37. Of the three, Icy offers several benefits, as it is an open community platform that allows for, both, processing (via ImageJ) and analysis of imaging data. Here we describe the steps necessary for efficient processing and analysis of RNA imaging data using Icy. Our results are representative of data obtained by co-visualizing two mRNA species in a whole egg chamber and by tracking an mRNA via the MB technology and the MS2/MCP system.
The post-acquisition processing with Icy software provides user flexibility in the image processing (e.g. brightness, contrast) and analysis (e.g. the thresholds/cut-offs settings for sensitivity of detection of the fluorescent particles as spots). Icy also enables the control relevant parameters within each block of the Protocols Editor run (e.g. determine the colocalization distance and use it as &#34;Max distance&#34; into the &#34;Colocalizer&#34; block). Icy software is updated at launch, and has reliable online support to troubleshoot any problems. The described protocol was designed for detecting oskar mRNA and the representative results were obtained using parameters optimized for the kind of mRNA particle to be detected and tracked. For example, oskar mRNA is an abundant transcript that is predominantly transported during mid stages of oogenesis, utilizing the microtubule network and dynamically associating with various protein factors throughout development of the oocyte. We previously reported that oskar mRNP undergoes extensive remodeling during transport from the nurse cells into the oocyte23. In addition, using MBs we characterized the temporal and spatial characteristics of endogenous oskar mRNA trafficking, and found that hundreds of oskar transcript copies can be incorporated to form large oskar mRNPs.
Post-acquisition analysis for colocalization and tracking can also be performed by using other software such as Imaris, ImageJ/Fiji and Volocity. Icy was selected for its capability to threshold and annotate fluorescent particles with high sensitivity and tracking capabilities. Here, we describe object-based colocalization, but colocalization, albeit without tracking, can also be quantified by determining the overlap and degree of colocalization using PCC(Costes) analysis using Icy and ImageJ plugins (Colocalization studio, JACoP)38,39.
In the future, an optimized, long-term imaging protocol is desired to ensure extended egg chamber survival. This would provide longer acquisition in order to analyze data pertaining to long-range mRNA trafficking studies.

Cell biology studies that visualize dynamic events with spatial and temporal resolution have been made possible by the development of fluorescence-based live cell imaging techniques. Presently, in vivo mRNA visualization is achieved via technologies that are based on RNA aptamer-protein interactions, RNA aptamer-induced fluorescence of organic dyes and nucleic acid probe annealing1,2,3. They all offer high specificity, sensitivity and signal-to-background ratio. However, RNA aptamer-centered approaches require extensive genetic manipulation, where a transgene is engineered to express an RNA with artificial structural motifs that are required for protein or organic dye binding. For example, the MS2/MCP system requires the co-expression of a transgene expressing an RNA construct containing multiple tandem repeats of the binding sequence for the bacteriophage MS2 coat protein (MCP), and another transgene encoding a fluorescent protein fused to MCP4,5. The addition of such secondary structural motifs to the RNA, along with a bulky fluorescently tagged protein, has raised concerns that native RNA processes may be affected6. A technology that addresses this concern and offers additional unique advantages is the nucleic acid-based approach, molecular beacons (MBs). MBs allow for the multiplex detection of endogenous mRNAs, discrimination of single nucleotide variations, and fast kinetics of hybridization with target mRNA7,8. MBs are oligonucleotide probes that remain in a quenched hairpin fold prior to undergoing a fluorogenic conformational change once they hybridize to their targets (Figure 1C)9. Several groups have had success in using MBs to detect both non-coding RNAs (microRNAs and lncRNAs)10,11,12,13, RNA retroviruses14 and dynamic DNA-protein interactions15. They have been successfully employed for imaging in various organisms and tissues, such as zebrafish embryos16, neurons13, tumor tissue17, differentiating cardiomyocytes18, and Salmonella19.
Here we describe the design, delivery and detection approach for endogenous mRNAs in living D. melanogaster egg chambers coupled with a microscopy set-up that is fast enough to capture the dynamic range of active molecular transport. The D. melanogaster egg chamber has served as an ideal multicellular model system for a wide range of developmental studies, from early germline stem cell division and maternal gene expression to the generation of segmental body plan20,21. Egg chambers are easily isolated, large and translucent, and able to withstand hours of ex vivo analysis, making them highly amenable to imaging experiments. Much work has focused on the asymmetric localization of maternal transcripts to discrete subcellular regions prior to being actively translated. In particular, oskar mRNA localization and its subsequent translation at the oocyte&#39;s posterior pole must occur in a tightly regulated manner to avoid a lethal bicaudal embryo phenotype22. oskar mRNA is transcribed in the 15 germline cells, called nurse cells, and actively transported through cytoplasmic bridges, called ring canals, into the oocyte, the germline cell that becomes the mature egg and is ultimately fertilized (Figure 1A). The considerable amount of information already available regarding the dynamic recruitment and exchange of protein factors to and from oskar mRNP, along with its long-range intracellular travel, make oskar a preferred candidate to study the many processes of the mRNA life cycle. MBs have been instrumental in revealing details about the process of mRNA localization and deciphering the regulation and function of protein factors that control mRNA transport during Drosophila oogenesis. In particular, by microinjecting MBs into nurse cells and performing live cell imaging experiments, the tracking of endogenous mRNAs is possible8,23.
The roadmap presented here offers the steps of a complete process, from carrying out a live cell imaging experiment using MBs, acquiring imaging data, to performing data analysis to track endogenous mRNA in its native cellular environment. The steps can be modified and further optimized to meet the needs of researchers working with other tissues/cell types within their own lab setting.

<table>
	<tbody>
		<tr>
			<td>Spectrofluorometer</td>
			<td>Fluoromax-4 Horiba-Jobin Yvon</td>
			<td>n/a</td>
			<td>Photon counting spectrofluorometer</td>
		</tr>
		<tr>
			<td>Quartz cuvette</td>
			<td>Fireflysci (former Precision Cells Inc.)</td>
			<td>701MFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 tweezer</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td>Thin tweezers are very important to separate out the individual egg chambers</td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma-Aldrich</td>
			<td>H8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip No.1 22 x 40mm</td>
			<td>VWR</td>
			<td>48393-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica MZ6 Leica Microsystems Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 fruit fly anesthesia pad</td>
			<td>Genesee Scienific</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL pH7.5</td>
			<td>Sigma-Aldrich</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc confocal microscope</td>
			<td>Leica DMI-4000B inverted microscope equipped with Yokogawa CSU 10 spinning disc Leica Microsystems Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu C9100-13 ImagEM EMCCD camera</td>
			<td>Hamamatsu</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>PatchMan NP 2 Micromanipulator</td>
			<td>Eppendorf Inc.</td>
			<td>920000037</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet Microinjector</td>
			<td>Eppendorf Inc.</td>
			<td>920010504</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle: Femtotips II</td>
			<td>Eppendorf Inc.</td>
			<td>930000043</td>
			<td></td>
		</tr>
		<tr>
			<td>Loading tip: 20ul Microloader</td>
			<td>Eppendorf Inc.</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover glasses no. 1 or 1.5, 22x40mm</td>
			<td>VWR</td>
			<td>48393-026; 48393-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry yeast</td>
			<td>Any grocery store</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer, &#62; 20 GB RAM</td>
			<td></td>
			<td></td>
			<td>Although processing can be carried out on most computers, higher capabilities will increase the speed of the processing</td>
		</tr>
	</tbody>
</table>

visualizing and tracking endogenous mrnas in live drosophila melanogaster egg chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Using PinMol, several MBs can be designed for one mRNA target (Figure 1B-C). After synthesis and purification, the selected MBs are characterized and compared using in vitro analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58545/58545fig1.jpg" /> 
Figure 1: Technique and tissue description for live cell imaging of endogenous mRNAs. (A) Depiction of a mid-stage Drosophila egg chamber used for microinjection. The microinjection needle (green) delivers a cocktail of molecular beacons specific for oskar mRNA. A quick injection into a nurse cell enables detection of mRNAs in transit to the oocyte, as well as visualization of already localized mRNA at the posterior cortex. (B) PinMol software output of molecular beacon ranking for targeting oskar mRNA (C) Secondary structure region within oskar mRNA targeted by a molecular beacon. (C&#39;) Sequence and folding of oskar-specific molecular beacon, osk2216. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58545/58545fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After optimum performance of MBs is confirmed via in vitro characterization, the probes are used for live visualization of endogenous target mRNA(s). It is possible to visualize the patterns of oskar mRNA transport and localization at various stages of oogenesis, and in particular at and after mid-oogenesis (7-10) (Figure 2A-B). Due to their small size, it is difficult to inject egg chambers at very early stages (1-4). When individually injected in the same stage egg chambers, oskar-specific MBs present the same patterns of localization (Figure 2, osk1236 vs osk2216).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58545/58545fig2.jpg" /> 
Figure 2: Time sequence of oskar mRNA in wild type egg chamber at t = 0, 10, and 30 min time points, after initiation of acquisition. Nurse cell injections of two oskar-specific molecular beacons (osk1236 and osk2216) in (A) stage 6-7 egg chambers and (B) stage 9 egg chambers. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58545/58545fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Different mRNA targets can be co-visualized by using spectrally distinct fluorescently labeled MBs (Figure 3). The MB solution can be microinjected into a nurse cell (Figure 3A) or into the oocyte (Figure 3B). MBs injected into a nurse cell&#39;s cytoplasm will freely diffuse into the other nurse cells as well as into the oocyte, and thus are able to find their target and generate fluorescence signal at other sites than the microinjection site. For example, when performing microinjections in a nurse cell of a late oogenesis stage (9-10) egg chamber, most of the fluorescence signal visualized within the oocyte is generated by the already localized oskar mRNA, and less by actively transported transcripts, which are more prevalent in earlier stages (7-8). Note that classical MBs will give rise to non-specific signal within the nuclei, therefore limiting the analysis to the cytoplasmic regions of the egg chamber. Additional modifications, such as NeutrAvidin or gold nanoparticles, have been employed to reduce or eliminate this non-specific signal28,29. In spite of this nuclear non-specific signal, the specificity of MBs for in vivo detection of oskar mRNA has been established using a FRET approach8, and MB co-injection with in vitro transcribed oskar mRNA labeled with a fluorophore spectrally distinct from the MB&#39;s label23.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58545/58545fig3.jpg" /> 
Figure 3: Co-visualization of two mRNA species in live egg chambers. Co-injections of oskar- and drongo-specific molecular beacons in the (A) nurse cell and (B) oocyte of stage 8-9 egg chambers. oskar (red) and drongo (green) mRNAs colocalize at the posterior end of the oocyte (asterix), and drongo also shows dorso-anterior accumulation (arrowhead). Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58545/58545fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For mRNA targets that show low expression levels, the fluorescence signal per mRNA molecule is increased by injecting a cocktail solution containing at least two MBs, each binding to different target regions (Figures 4 and 5).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58545/58545fig4.jpg" /> 
Figure 4: Visualization of oskar mRNA with both MBs and MS2-GFP system. Microinjections of a MB cocktail solution (MB) in an egg chamber expressing osk-MS2::MCP-GFP30 (GFP). After the microinjection of a nurse cell, images were acquired every 30 s for 20 min. Two regions of interest were selected, one ROI in a nurse cell and one in the oocyte. Different sensitivity was used for each ROI to detect the spots. ROI nurse cell: Scale 2, Sensitivity 100 for GFP and MB. ROI oocyte: Scale 2, Sensitivity 50 for GFP and 110 for MB. Colocalization distance is 4 pixels for both ROIs. The whole egg chamber and the zoom in on the nurse cell are shown at the 12 min time point, and the zoom in on the oocyte is shown at the 14 min time point. MB spots (red circles) and GFP spots (green circles) identify oskar mRNA particles detected by each approach, and the colocalized particles (yellow) indicate where MB and GFP spots are at most 4 pixels apart. XY-projections of 14 Z optical slices at 0.3 &#181;m steps. Acquired as 16-bit data with an 63x objective (oil, NA = 1.4), XY = 0.24 &#181;m, exposure time 500 ms, at 5.23 and 5.39 mW laser power for the 641 nm and 491 nm laser, respectively. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58545/58545fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58545/58545fig5.jpg" /> 
Figure 5: Tracking analysis in the oocyte, after nurse cell microinjection with oskar mRNA-specific MBs. MB particles were detected at Scale 2 with Sensitivity 110, and 8 time points are shown before/after the current time frame. MB spots (red circles) are tracked in the volume of the oocyte; tracks represent detection information from 8 time points before/after the shown 12 min time point. Each color represents an individual track. XY-projections of 14 Z optical slices at 0.3 &#181;m steps. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58545/58545fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
When comparing trafficking of oskar mRNA as detected with MBs vs MS2/MCP, the fluorescence signal generated by oskar-specific MBs faithfully documents on the transport and localization of transgenic oskar mRNA labeled with 10 GFP molecules via the MS2/MCP system (Figure 4). In oskar-MS2/MCP-GFP transgenic egg chambers at mid oogenesis, acquisition data analysis showed extensive colocalization between fluorescent signals of genetically engineered oskar-MS2 mRNA detected using MBs and GFP-tagging. At 12 and 14 min post-injection, 57% (7 MB-objects and 13 GFP-objects, with 4 colocalized objects) and 93% (30 MB-objects and 51 GFP-objects, with 28 colocalized objects) of detected MB particles colocalized with GFP particles in the nurse cells and oocyte, respectively. Our analysis yields 31% and 55% colocalization percentages of oskar-MS2 mRNA with oskar mRNA detected with MBs within the cytoplasm of a nurse cell and the oocyte, respectively. Moreover, 5D-stacks can be further analyzed to determine oskar mRNA trajectories for long-distance transport in both the nurse cell and oocyte cytoplasm (Figure 5).

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Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

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7.4K Views.  City University of New York. Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Video: Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection z larw CNS na Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...