This work is supported by the Sheila and David Fuente Neuropathic Pain Research Program Graduate Fellowship (to J.M.B.), the Lois Pope LIFE Fellows Program (to C.L., Y.Z., and J.M.B.), the Snyder-Robinson Foundation Predoctoral Fellowship (to C.L.), the Dr. John T. Macdonald Foundation (to C.L.), contracts, grants from National Institutes of Health (NIH) HHSN268201300038C, R21GM119018, and R56NS095893 (to R.G.Z.), and by Taishan Scholar Project (Shandong Province, People&#39;s Republic of China) (to R.G.Z.).

1. Considerations and Preparations for Designing the Image Analysis Experiment
<ol>
	<li>Predetermine suitable anatomical, cellular, or subcellular markers to serve as landmarks for standardizing a region of interest (ROI) across different samples, for example 4&#39;,6-diamidino-2-phenylindole (DAPI), membrane markers, localized fluorescent protein, etc.</li>
	<li>Select a fluorescent marker that can delimit discrete puncta beyond background levels for the structure of interest. 
	NOTE: This protocol is o...

<ol>
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A Golgi analysis of wild-type structure.</a> Cell and tissue research. 258 (3), 441-475 (1989).</li><li>Ruan, K., Zhu, Y., Li, C., Brazill, J. M., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+Drosophila+Nmnat+functions+as+a+switch+to+enhance+neuroprotection+under+stress.">Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress.</a> Nature Communications. 6, 10057 (2015).</li><li>Zhai, R. G., 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=NAD+synthase+NMNAT+acts+as+a+chaperone+to+protect+against+neurodegeneration.">NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration.</a> Nature. 452 (7189), 887 (2008).</li><li>Li, C., 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=Spermine+synthase+deficiency+causes+lysosomal+dysfunction+and+oxidative+stress+in+models+of+Snyder-Robinson+syndrome.">Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome.</a> Nat Commun. 8 (1), 1257 (2017).</li><li>Prokop, A., Meinertzhagen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+structure+of+synaptic+contacts+in+Drosophila.">Development and structure of synaptic contacts in Drosophila.</a> Semin Cell Dev Biol. 17 (1), 20-30 (2006).</li><li>Maday, S., Holzbaur, E. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+lysosomal+dysfunction+causes+cargo-specific+deficits+of+axonal+transport+leading+to+Alzheimer-like+neuritic+dystrophy.">Primary lysosomal dysfunction causes cargo-specific deficits of axonal transport leading to Alzheimer-like neuritic dystrophy.</a> Autophagy. 7 (12), 1562-1563 (2011).</li><li>Vincent, L., Soille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watersheds+in+digital+spaces:+an+efficient+algorithm+based+on+immersion+simulations.">Watersheds in digital spaces: an efficient algorithm based on immersion simulations.</a> IEEE Transactions on Pattern Analysis &amp; Machine Intelligence. (6), 583-598 (1991).</li><li>Najman, L., Schmitt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geodesic+saliency+of+watershed+contours+and+hierarchical+segmentation.">Geodesic saliency of watershed contours and hierarchical segmentation.</a> IEEE Transactions on pattern analysis and machine intelligence. 18 (12), 1163-1173 (1996).</li><li>Shiber, A., Breuer, W., Ravid, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+quantification+and+characterization+of+intracellular+protein+aggregates+in+yeast.">Flow cytometric quantification and characterization of intracellular protein aggregates in yeast.</a> Prion. 8 (3), 276-284 (2014).</li><li>Mizushima, N., Yoshimori, T., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+mammalian+autophagy+research.">Methods in mammalian autophagy research.</a> Cell. 140 (3), 313-326 (2010).</li></ol>

The protocol outlined here can be used to robustly and reproducibly quantitate cell biological processes visualized by fluorescence-based imaging. Biological context and technical limitations need to be carefully considered to guide the experimental design. Fluorescent markers of subcellular structures of interest, whether immunohistochemical, dye-based, or genetically expressed, need to be distinguishable above background by morphology and intensity. The UAS/GAL4 system is widely used to drive targeted gene exp...

Neurodegenerative diseases affect millions of people each year and the incidence is increasing with an aging population1. While each neurodegenerative disease has a unique etiology, aggregation of misfolded proteins and breakdown of the proteostasis network are common pathological hallmarks of many of these diseases. Elucidating how disruption of these fundamental and interrelated processes goes awry to contribute to neuronal dysfunction and cell death is critical for understanding neurodegenerative diseases as well as guiding therapeutic interventions. Fluorescence-based imaging allows for investigation of these complex and dynamic processes in neurons and has greatly contributed to our understanding of neuronal cell biology. Analysis of fluorescently tagged proteins is challenging, particularly when experiments are carried out in vivo, due to highly compact tissues, diverse cell types, and morphological heterogeneity. Manually assisted quantification is affordable and straightforward, but is often time-consuming and subject to human bias. Therefore, there is a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies.
We have outlined a simple and adaptable workflow using Fiji/ImageJ, a powerful and freely accessible image processing software2,3, to extract quantitative data from fluorescence imaging studies in experimental models of neurodegeneration using Drosophila. By following this protocol to quantify protein aggregation and autophagic flux &#8212; two cell biological features that are highly relevant to neurodegenerative disease pathology &#8212; we demonstrated the sensitivity and reproducibility of this approach. Analysis of fluorescently tagged mutant huntingtin (Htt) proteins in the Drosophila optic lobe revealed the number, size, and intensity of protein aggregates. We visualized a tandem fluorescent reporter of autophagic flux within the Drosophila visual system, which displays different emission signals depending on the compartmental environment4. Ratiometric-based analysis of the tandem reporter allowed for a quantitative and comprehensive view of autophagy-lysosome flux from autophagosome formation, maturation, and transport to degradation in the lysosome, and additionally highlighted vulnerable compartments disrupted in neurodegenerative conditions. Importantly, in both analyses we implemented semi-automated thresholding and segmentation steps in our protocol to minimize unconscious bias, increase sampling power, and provide a standard to facilitate comparisons between similar studies. The straightforward workflow is intended to make powerful Fiji/ImageJ plugins (developed by computer scientists based on mathematic algorithms) more accessible to neurobiologists and the life sciences community at large.

<table border="0" cellpadding="0" cellspacing="0" width="1102">
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">SYLGARD(R) 184 Silicone Elastomer Kit</td>
			<td style="width:164px;">Dow Corning Corporation</td>
			<td style="width:243px;">PF184250</td>
			<td style="width:349px;">Dissection dish</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">Falcon 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish</td>
			<td style="width:164px;">Corning</td>
			<td style="width:243px;">351008</td>
			<td style="width:349px;">Dissection dish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dumont #5 Forceps</td>
			<td style="width:164px;">Fine Science Tools</td>
			<td style="width:243px;">11251-20</td>
			<td style="width:349px;">Dissection tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Sodium Chloride</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">S3014</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Sodium Phosphate Dibasic&#160;</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">S5136</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Potassium Phosphate Monobasic</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">P5655</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Triton&#160;X-100</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">T9284</td>
			<td style="width:349px;">Washing and antibody incubaton solution.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">37% Formaldehyde</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">10790-710</td>
			<td style="width:349px;">Fixation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Disposable Microcentrifuge Tubes (0.5mL, blue)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">89000-022</td>
			<td style="width:349px;">Fixation, washing, and antibody incubaton.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Plain and Frosted Micro Slides (25&#215;75mm)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">48312-004</td>
			<td style="width:349px;">Slides for confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Micro Cover Glasses, rectangular (22&#215;40mm)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">48393-172</td>
			<td style="width:349px;">Slides for confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Rubber Cement</td>
			<td style="width:164px;">Slime</td>
			<td style="width:243px;">1051-A</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">VECTASHIELD Antifade Mounting Medium</td>
			<td style="width:164px;">Vector Laboratories, Inc.</td>
			<td style="width:243px;">H-1000</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Scotch Magic 810 Invisible Tape (19mm&#215;25.4m)</td>
			<td style="width:164px;">3M Company</td>
			<td style="width:243px;">810</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">Normal Goat Serum</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:243px;">PCN5000</td>
			<td style="width:349px;">Antibody incubaton</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:347px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)&#160;</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:243px;">D1306</td>
			<td style="width:349px;">Nucleic acid staining. Dissolve in deionized water to make a 5 mg/mL stock solution and store at -80&#176;C. Dilute to a working concentration of 10-20 &#956;g/mL in PBTx.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">3.5X-90X Stereo Zoom Inspection Industrial Microscope</td>
			<td style="width:164px;">AmScope</td>
			<td style="width:243px;">SM-1BNZ</td>
			<td style="width:349px;">Dissection scope. Equipped with 6W LED Dual Gooseneck Illuminator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">ImageJ/Fiji</td>
			<td style="width:164px;">NIH</td>
			<td style="width:243px;">v1.51u</td>
			<td style="width:349px;">With SCF_MPI_CBG plugins (version 1.1.2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">FV1000-IX81 Confocal-laser Scanning Microscope</td>
			<td style="width:164px;">Olympus</td>
			<td style="width:243px;"></td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Construct</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td style="width:243px;">BL37749</td>
			<td style="width:349px;"></td>
		</tr>
	</tbody>
</table>

quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantification of the number, area, and intensity of fluorescently tagged mutant Htt aggregates in the Drosophila optic lobe
To investigate misfolded protein aggregation in the central nervous system of a Drosophila model of Huntington&#39;s disease, RFP-tagged mutant human Htt with a non-pathological (UAS-RFP-hHttQ15) or pathological expansion (UAS-RFP-hHttQ138) and membrane GFP (UAS-mCD8::G...

Watch this Scientific Journal Video about Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ at JoVE.com

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

We developed this method to extract quantitative data from fluorescence-based imaging studies to extract complex and dynamic cell biological processes in Drosophila models of neurodegeneration. The main advantage of this technique is its an adaptable, semi-automated, image analysis workflow for minimizing selection bias, maximizing sampling power, and achieving reproducibility across the field. This approach can be extended for the analysis of other proteinaceous puncta and membrane-bound compartments implicated in neurodegeneration that ultimately will enhance our mechanistic understanding of neurodegenerative diseases.To keep the lamina intact during the dissection under a stereo microscope slide two forceps under the retina and gently tear through the middle of the eye to remove the retina. Keeping the forceps parallel to the lamina surface pull away any remaining retinal tissue attached to the lamina. Dissect three to five brains for each group and transfer the brains to a microcentrifuge tube containing an appropriate fixative for 15 minutes at room temperature with gentle rocking.After three 15-minute washes with PBS plus Triton X-100, or PBTx, replace the last wash with the primary antibody of interest diluted in PBTx with 5%normal goat serum for an overnight incubation in an aliquot mixer at four degrees Celsius. The next morning remove the excess antibody with three 15-minute washes in PBTx and incubate the brains in an appropriate secondary antibody diluted in PBTx with 5%normal goat serum for one hour at room temperature. At the end of the incubation wash the brains three times as demonstrated and place a drop of mounting medium between two pieces of clear tape in the center of one microscopy slide per tissue sample.Place the brains in the mounting media for one to two minutes. When the tissues become transparent use a pipette to carefully remove the mounting medium from each side. To image the stained tissues apply a drop of fresh mounting medium onto the brains and carefully overlay the samples with the a cover glass.Seal the cover glass edges with rubber cement and air dry the slides for 20 minutes at room temperature. Next view the specimen on the microscope and adjust the image detector controls on the fluorescence microscope to capture the highest signal-to-noise ratio in the highest a dynamic range of the specimen. Once the settings have been optimized view a specimen from another group to confirm the settings will be appropriate across the experiment.The first specimen can then be imaged taking care to save the image as a file type that can preserve metadata such as acquisition parameters and spatial calibration. To analyze the images open an image in Fiji select View Stack with Hyperstack from the Bio-formats Import Options dialog box and set the color mode to Grayscale. Identify an area based on the marker channels that can be used as a standardized region of interest across specimens using the C scroll bar to view the channels and the Z scroll bar to move through the focal planes.To simplify the image analysis generate a new image containing the channels and focal planes and select Image and Duplicate. Set the desired channels and slices and change the file name to reflect the selection. Manually select a standardized region of interest using a Selection Tool based on the previously determined selection criteria.Click Analyze, Tools, Region of Interest Manager, and Add to add the region of interest to the Region of Interest Manager. Then click More and Save and name the file to reflect the region of interest. For pre processing the images click Process in Filters and select an appropriate filter.Here select filter settings that reduce noise while preserving edges in order to aid in detection of the structures of interest during segmentation. With the pre processed image active in Fiji click SCF, Labeling, and Interactive H_Watershed. From the control panel adjust the Seed dynamics, Intensity threshold, and Peak flooding to optimize the segmentation.When the segmentation results have been optimized select Export Regions Mask and Export to generate a binary image of the watershed results. Open the saved standardized region of interest. From the Region of Interest Manager select the region of interest identifier to limit the feature extraction to the standardized region of interest in the image.With the region of interest active on the binary watershed mask select Analyze, Analyze Particles, and Add to Manager to extract the features to add a particle identifier for each structure delineated during the segmentation to the Region of Interest Manager. Then save the added particles taking care that the identifiers are deselected and that all of the particles are saved in a zip file. Now open the pre processed raw image and scroll to the channel used for capturing the structures of interest.Open the particles obtained from the feature extraction and select Show All in the Region of Interest Manager to overlay an outline of each particle onto the image. Click Analyze and Set Measurements and set the appropriate experimental measurements. Then from the Region of Interest Manager select Measure and copy and paste the results into an appropriate spreadsheet program for compilation and further calculations.Quantification of the number of semi-automatically segmented RFP huntingtin aggregates from standardized focal planes through the optic lobe of Drosophila reveals a diffuse expression of the non-pathogenic Huntington Q15 expansion in the accumulation of protein aggregates by the disease-causing Huntington Q138 expansion. Size and intensity profiles of all the aggregates from a standardized focal plane of five different optic lobes expressing Huntington Q138 illustrate the robustness and reproducibility of this workflow. When aggregates are over-exposed during image acquisition quantification of the size and number of aggregates is comparable to aggregates captured with ideal exposure.However the intensity of the over-exposed aggregates becomes a function of size as the saturated pixels exhibit the maximum intensity value. Visualization of mCherry and GFP intensity across several autophagy-related or Atg8 positive structures reveals differences in the reporter where the high intensity of both fluorophores indicates autophagosomes. High mCherry and low GFP indicates fusion with lysosome as GFP is quenched in this acidic compartment.And finally low mCherry reflects degradation within the lysosome. A scatterplot generated from the mean fluorophore intensity of each semi-automatically segmented mCherry Atg8 positive particle illustrates flux through the autophagy lysosome pathway that can be separated into discrete steps by quadrant analysis. While attempting this procedure it's important to remember that the user-defined parameters in image analysis steps are highly application dependent.So be careful to document the workflow to ensure consistency and comparability across specimens.

quantitative-cell-biology-neurodegeneration-drosophila-through

Research

JoVE Journal

Neuroscience

8.9K 조회수.  University of Miami Miller School of Medicine. We developed this method to extract quantitative data from fluorescence-based imaging studies to extract complex and dynamic cell biological processes in Drosophila models of neurodegeneration. The main advantage of this technique is its an adaptable, semi-automated, image analysis workflow for minimizing selection bias, maximizing sampling power, and achieving reproducibility across the field. This approach can be extended for the analysis of other proteinaceous puncta and membrane-bound com...