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>

<ol>
	<li>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=Imaging+intracellular+RNA+distribution+and+dynamics+in+living+cells.">Imaging intracellular RNA distribution and dynamics in living cells.</a> Nature Methods. 6 (5), 331-338 (2009).</li><li>Bao, G., Rhee, W. J., 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=Fluorescent+probes+for+live-cell+RNA+detection.">Fluorescent probes for live-cell RNA detection.</a> Annual Reviews of Biomedical Engineering. 11, 25-47 (2009).</li><li>Mannack, L. V., Eising, S., Rentmeister, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+techniques+for+visualizing+RNA+in+cells.">Current techniques for visualizing RNA in cells.</a> F1000Research. 5, (2016).</li><li>Larson, D. R., Zenklusen, D., Wu, B., Chao, J. A., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+observation+of+transcription+initiation+and+elongation+on+an+endogenous+yeast+gene.">Real-time observation of transcription initiation and elongation on an endogenous yeast gene.</a> Science. 332 (6028), 475-478 (2011).</li><li>Bertrand, E., 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=Localization+of+ASH1+mRNA+particles+in+living+yeast.">Localization of ASH1 mRNA particles in living yeast.</a> Molecular Cell. 2 (4), 437-445 (1998).</li><li>Garcia, J. F., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MS2+coat+proteins+bound+to+yeast+mRNAs+block+5'+to+3'+degradation+and+trap+mRNA+decay+products:+implications+for+the+localization+of+mRNAs+by+MS2-MCP+system.">MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system.</a> RNA. 21 (8), 1393-1395 (2015).</li><li>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=Molecular+beacons:+Fluorescent+probes+for+detection+of+endogenous+mRNAs+in+living+cells.">Molecular beacons: Fluorescent probes for detection of endogenous mRNAs in living cells.</a> Methods in Molecular Biology. 319, 1-14 (2006).</li><li>Bratu, D. P., Cha, B. J., Mhlanga, M. M., 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=Visualizing+the+distribution+and+transport+of+mRNAs+in+living+cells.">Visualizing the distribution and transport of mRNAs in living cells.</a> Proceedings of the National Academy of Sciences of the Unites States of America. 100 (23), 13308-13313 (2003).</li><li>Tyagi, S., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+beacons:+probes+that+fluoresce+upon+hybridization.">Molecular beacons: probes that fluoresce upon hybridization.</a> Nature Biotechnology. 14 (3), 303-308 (1996).</li><li>Chen, 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=A+molecular+beacon-based+approach+for+live-cell+imaging+of+RNA+transcripts+with+minimal+target+engineering+at+the+single-molecule+level.">A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level.</a> Scientific Reports. 7 (1), 1550 (2017).</li><li>Liu, Y., 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=Multiplex+detection+of+microRNAs+by+combining+molecular+beacon+probes+with+T7+exonuclease-assisted+cyclic+amplification+reaction.">Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.</a> Analytical and Bioanalytical Chemistry. 409 (1), 107-114 (2017).</li><li>Baker, M. B., Bao, G., Searles, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+quantification+of+specific+microRNA+using+molecular+beacons.">In vitro quantification of specific microRNA using molecular beacons.</a> Nucleic Acids Research. 40 (2), e13 (2012).</li><li>Ko, H. Y., 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=A+color-tunable+molecular+beacon+to+sense+miRNA-9+expression+during+neurogenesis.">A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis.</a> Scientific Reports. 4, 4626 (2014).</li><li>Vet, J. A., 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=Multiplex+detection+of+four+pathogenic+retroviruses+using+molecular+beacons.">Multiplex detection of four pathogenic retroviruses using molecular beacons.</a> Proceedings of the National Academy of Sciences of the Unites States of America. 96 (11), 6394-6399 (1999).</li><li>Li, J., Cao, Z. C., Tang, Z., Wang, K., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+beacons+for+protein-DNA+interaction+studies.">Molecular beacons for protein-DNA interaction studies.</a> Methods in Molecular Biology. 429, 209-224 (2008).</li><li>Li, W. M., Chan, C. M., Miller, A. L., Lee, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Functional+Roles+of+Molecular+Beacon+as+a+MicroRNA+Detector+and+Inhibitor.">Dual Functional Roles of Molecular Beacon as a MicroRNA Detector and Inhibitor.</a> Journal of Biological Chemistry. 292 (9), 3568-3580 (2017).</li><li>Kuang, T., Chang, L., Peng, X., Hu, X., Gallego-Perez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Beacon+Nano-Sensors+for+Probing+Living+Cancer+Cells.">Molecular Beacon Nano-Sensors for Probing Living Cancer Cells.</a> Trends in Biotechnology. 35 (4), 347-359 (2017).</li><li>Ban, K., 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=Non-genetic+Purification+of+Ventricular+Cardiomyocytes+from+Differentiating+Embryonic+Stem+Cells+through+Molecular+Beacons+Targeting+IRX-4.">Non-genetic Purification of Ventricular Cardiomyocytes from Differentiating Embryonic Stem Cells through Molecular Beacons Targeting IRX-4.</a> Stem Cell Reports. 5 (6), 1239-1249 (2015).</li><li>Hadjinicolaou, A. V., Demetriou, V. L., Emmanuel, M. A., Kakoyiannis, C. K., Kostrikis, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+beacon-based+real-time+PCR+detection+of+primary+isolates+of+Salmonella+Typhimurium+and+Salmonella+Enteritidis+in+environmental+and+clinical+samples.">Molecular beacon-based real-time PCR detection of primary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and clinical samples.</a> BMC Microbiology. 9, 97 (2009).</li><li>McLaughlin, J. M., 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=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 deliv...

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 mm x 40 mm</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>CO&lt;sub&gt;2&lt;/sub&gt; fruit fly anesthesia pad</td><td>Genesee Scienific</td><td>59-114</td><td></td></tr><tr><td>Tris-HCL pH 7.5</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: 20 &amp;mu;L Microloader</td><td>Eppendorf Inc.</td><td>930001007</td><td></td></tr><tr><td>Micro Cover glasses no. 1 or 1.5, 22 mm x 40 mm</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, &gt; 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. ...

Watch this Scientific Journal Video about Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers at JoVE.com

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 ...

visualizing-tracking-endogenous-mrnas-live-drosophila-melanogaster

Research

Education

JoVE Core

JoVE Journal

Cell Biology

Biology

Unit 1

Control of Gene Expression

SCIENTISTS IN ACTION

7.4K Views.  City University of New York. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het RNA-biologieveld over het mechanisme en de functie van gepolariseerde RNA-lokalisatie. Het belangrijkste voordeel van deze techniek is dat het zorgt voor de real-time visualisatie van endogene RNA-handel in levende weefsels. De implicaties van deze techniek uit te breiden tot de therapie van RNA-gerelateerde ziekten, omdat het een potentieel heeft om nieuwe therapeutische doelen te ontdekken.Hoewel deze methode ...

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

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

Developmental Biology

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

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 ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

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, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Samenvatting

Meer video's verkennen