The authors would like to thank Integrated Imaging Core facility at Johns Hopkins University for microscopes and data analysis software. We thank J. Snedeker and Q. E. Yu for proofreading and suggestions, and X.C. lab members for helpful discussions and suggestions. Supported by NIGMS/NIH R35GM127075, the Howard Hughes Medical Institute, the David and Lucile Packard Foundation, and Johns Hopkins University startup funds (X.C.).

1. Preparation of live cell imaging cocktail (live cell media)
<ol>
	<li>Supplement Schneider&#8217;s Drosophila medium with 15% fetal bovine serum (FBS) and 0.6x penicillin/streptomycin. Adjust the pH to approximately 7.0.</li>
	<li>Just before using the medium, add insulin to a final concentration of 200 &#181;g/mL. 
	NOTE: This media is critical for maintaining normal cell division and development of the Drosophila testis during time-lapse imaging10,<sup clas...

<ol>
	<li>Breedijk, R. M. P., 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+live-cell+super-resolution+technique+demonstrated+by+imaging+germinosomes+in+wild-type+bacterial+spores.">A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores.</a> Scientific Reports. 10 (1), 5312 (2020).</li><li>Maddox, P. S., 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=Imaging+the+mitotic+spindle.">Imaging the mitotic spindle.</a> Methods in Enzymology. 505, 81-103 (2012).</li><li>Frigault, M. M., Lacoste, J., Swift, J. L., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+microscopy+-+tips+and+tools.">Live-cell microscopy - tips and tools.</a> Journal of Cell Science. 122 (6), 753-767 (2009).</li><li>Bates, M., Jones, S. A., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+optical+reconstruction+microscopy+(STORM):+A+method+for+super+resolution+fluorescence+imaging.">Stochastic optical reconstruction microscopy (STORM): A method for super resolution fluorescence imaging.</a> Cold Spring Harbor Protocols. 2013 (6), 075143 (2013).</li><li>Shroff, H., Galbraith, C. G., Galbraith, J. A., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+photoactivated+localization+microscopy+of+nanoscale+adhesion+dynamics.">Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.</a> Nature Methods. 5 (5), 417-423 (2008).</li><li>Vicidomini, G., Bianchini, P., Diaspro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+super-resolved+microscopy.">STED super-resolved microscopy.</a> Nature Methods. 15 (3), 173-182 (2018).</li><li>Huff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Airyscan+detector+from+ZEISS:+confocal+imaging+with+improved+signal-to-noise+ratio+and+super-resolution.">The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution.</a> Nature Methods. 12 (12), (2015).</li><li>Huff, J., 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=The+new+2D+superresolution+mode+for+ZEISS+Airyscan.">The new 2D superresolution mode for ZEISS Airyscan.</a> Nature Methods. 14 (12), 1223 (2017).</li><li>Korobchevskaya, K., Lagerholm, B., Colin-York, H., Fritzsche, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+potential+of+Airyscan+microscopy+for+live+cell+imaging.">Exploring the potential of Airyscan microscopy for live cell imaging.</a> Photonics. 4 (4), 41 (2017).</li><li>Ranjan, R., Snedeker, J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+centromeres+differentially+coordinate+with+mitotic+machinery+to+ensure+biased+sister+chromatid+segregation+in+germline+stem+cells.">Asymmetric centromeres differentially coordinate with mitotic machinery to ensure biased sister chromatid segregation in germline stem cells.</a> Cell Stem Cell. 25 (5), 666-681 (2019).</li><li>Kahney, E. W., Ranjan, R., Gleason, R. J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetry+from+asymmetry+or+asymmetry+from+symmetry.">Symmetry from asymmetry or asymmetry from symmetry.</a> Cold Spring Harbor Symposia on Quantitative Biology. 82, 305-318 (2017).</li><li>Wooten, M., Ranjan, R., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+histone+inheritance+in+asymmetrically+dividing+stem+cells.">Asymmetric histone inheritance in asymmetrically dividing stem cells.</a> Trends in Genetics. 36 (1), 30-43 (2020).</li><li>Wooten, 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=Asymmetric+histone+inheritance+via+strand-specific+incorporation+and+biased+replication+fork+movement.">Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement.</a> Nature Structural &amp; Molecular Biology. 26 (8), 732-743 (2019).</li><li>Prasad, M., Jang, A. C. C., Starz-Gaiano, M., Melani, M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+culturing+Drosophila+melanogaster+stage+9+egg+chambers+for+live+imaging.">A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging.</a> Nature Protocols. 2 (10), 2467-2473 (2007).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> Journal of Cell Biology. 190 (2), 165-175 (2010).</li><li>Sivaguru, 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=Comparative+performance+of+airyscan+and+structured+illumination+superresolution+microscopy+in+the+study+of+the+surface+texture+and+3D+shape+of+pollen.">Comparative performance of airyscan and structured illumination superresolution microscopy in the study of the surface texture and 3D shape of pollen.</a> Microscopy Research and Technique. 81 (2), 101-114 (2018).</li></ol>

Super-resolution microscopy methods provide spatial resolution as high as 10s of nanometers4,5,6. The STORM and PALM microscopy methods allow resolution up to 20 to 50 nm (XY-resolution), while STED microscopy offers resolution of 20 to 100 nm (XY-resolution). The spatial resolution of SIM microscopy is limited to 100 to 130 nm15. However, due to its high-photon density and lengthy acquisition time, it is extremely ch...

Visualizing subcellular structures and protein dynamics in live cells with resolution beyond the diffraction limit of light is typically very challenging1-3. While multiple super-resolution techniques such as Stochastic-Optical-Reconstruction-Microscopy (STORM), Photo-Activated-Localization-Microscopy (PALM) and Stimulated-Emission-Depletion (STED)4,5,6 microscopy have been developed, complications in specimen preparation as well as the need to maintain viability and activity ex vivo, limit the use of conventional super-resolution microscopy for imaging live samples. Conventional confocal microscopy cannot reach spatial resolution beyond ~230 nm XY-resolution and is often insufficient to observe intricate cellular substructures5,6. However, a recent development in confocal microscopy, Airyscan super-resolution imaging, is able to achieve approximately 140 nm (XY-resolution)7,8 and has a relatively simple sample preparation that is compatible with live imaging. Since this imaging detection system requires a long acquisition time, its high spatial resolution does come at the cost of temporal resolution9. Therefore, a method is needed to ensure that live cell imaging is extended with high spatial resolution.
Here, we developed a method for observing live cells in intact tissue at its optimal resolution to decipher subcellular structures with detailed spatial information. This method is designed as such so that samples can be mounted stably for a long period of time (~ 10 h) without moving or degenerating. The live cell media used in this technique can support cellular function and avoid photobleaching for up to 10 hours under a super-resolution microscope. Finally, this protocol minimizes most stresses caused by the constant illumination of lasers over extended periods of time such as hypoxia, changes in humidity and temperature, as well as nutrient exhaustion.
Using this protocol to image Drosophila male germline stem cells (GSCs), we were able to observe how the asymmetric activity of microtubules allows for preferential interaction with epigenetically distinct sister chromatids10,11,12,13. These types of cellular events are highly dynamic and are very difficult to visualize in live cells using other super-resolution imaging methods such as STORM, PALM, or STED. We anticipate that this method will become highly useful for cell biologists as they aim to understand the dynamic subcellular structures of live cells residing in tissues. There are many areas in which this method can be applied to, such as studying the dynamics of proteins; understanding the movement of cells; and lineage tracing and cellular differentiation processes, among other possible applications.

<table>
	<tbody>
		<tr>
			<td>Adobe Illustrator CS6 (figure making software)</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis membrane</td>
			<td>Spectra/Por 1-4 Standard RC Dialysis Membrane</td>
			<td>Cat No. 08-67121</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat. no. 26140079</td>
			<td>15% (V/V)</td>
		</tr>
		<tr>
			<td>Fiji (analysis software)</td>
			<td>NIH</td>
			<td>N/A</td>
			<td>Image fluorescence intensity quantification</td>
		</tr>
		<tr>
			<td>Glass bottom cell culture dishes (FluroDish)</td>
			<td>World Precision Instrument, Inc.</td>
			<td>FD35PDL-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris (image reconstruction software)</td>
			<td>Bitplane</td>
			<td>N/A</td>
			<td>3D image reconstruction</td>
		</tr>
		<tr>
			<td>Imerssion oil</td>
			<td>Zeiss</td>
			<td>Immersol 518F/30 &#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>Cat. No. 15550</td>
			<td>200 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>Cat No. - 15140-122</td>
			<td>0.6x</td>
		</tr>
		<tr>
			<td>Schneider Drosophila media</td>
			<td>Invitrogen</td>
			<td>Cat No. - 11720-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc confocal microscope</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>equipped with an evolve camera (Photometrics), using a 63x Zeiss objective (1.4 NA).</td>
		</tr>
		<tr>
			<td>Tissue paper</td>
			<td>Kimwipe</td>
			<td>N/A</td>
			<td>Wet to form humid chamber</td>
		</tr>
		<tr>
			<td>LSM 800 confocal microscope with AiryScan super-resolution module</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>equipped with highly sensitive GaAsP (Gallium Arsenide Phosphide) detectors using a 63x Zeiss objective (1.4 NA)</td>
		</tr>
		<tr>
			<td>ZEN (imaging software)</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

super-resolution live cell imaging of subcellular structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Live cell imaging beyond the diffraction limit in Drosophila tissue, especially for GSCs, provides an opportunity to investigate the dynamics of subcellular events in the context of cell cycle progression. Recently, a study utilizing this protocol has shown that microtubule activities at the mother centrosome versus the daughter centrosome are temporally asymmetric in GSCs10. The mother centrosome emanates microtubules approximately 4 hours prior to the mitotic entry, whereas the daughter...

Watch this Scientific Journal Video about Super-Resolution Live Cell Imaging of Subcellular Structures at JoVE.com

Super-Resolution Live Cell Imaging of Subcellular Structures

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Our protocol uses regular fluorophore probes and simple specimen preparation techniques that maintain the imaged cell in physiological condition to study highly dynamic cellular process and detailed sub-cellular structures. This technique allows us to investigate cellular processes at a super resolution and under physiological conditions for an extended period without sacrificing either the special or temporary resolution. Begin by cutting a 12 to 14 kilodalton molecular weight cutoff dialysis membrane into small pieces and soaking the pieces with 100 microliters of live cell medium for about five minutes.While the membranes are soaking, cut the outer ring of a 50 milliliter tube into small pieces and sterilize the pieces in 70%ethanol. For testes dissection and mounting, transfer ten two to three day old anesthetized male flies into a dissection dish under a dissecting microscope and use fine forceps to remove the testes. When all of the testes have been collected, wash the testes two times in live cell medium and use a pipette tip to spread 100 to 150 microliters of live cell medium to the prepared glass bottom dish.Use fine forceps to transfer the fly testes to the center of the dish and remove all but about 10 microliters of the medium. Quickly, place the pre-wet membrane onto the testes and place two to three small plastic weights onto the membrane. Immediately, add 100 to 150 microliters of fresh live cell medium and place the ring back onto the elevated side of the dish.Place the cover slip back onto the ring and swirl a piece of tissue paper in water before placing the paper onto the cover slip. Then, cover the dish with a lid and secure the dish on the stage of a super resolution microscope. For germline stem cell imaging, open the imaging software and turn on the transmitted light.Use the 63X objective to focus on the testes tissue and turn on the lasers. Click Live"to locate germinal stem cells. Adjust the focus and click Frame Size"to set the frame size to between 512 by 512 and 1024 by 1024 pixels, and Averaging"to set the frame average to one or two.Click Master Gain"to set the electron multiplying gain to less than 800, and Lasers"to set the laser power to 1%to 2%Zoom in to the region or cell of interest to reduce the image acquisition time and to reduce photo bleaching, and confirm that the image has been optimally configured before clicking Start Experiment"to start the time lapse image capture. If Airyscan Acquisition is not configured optimally"is displayed, click Optimal"in the frame size, Z-Stack, and Scan Area"sections, and optimize the time interval, number of Z slices and duration of the time-lapse imaging, according to the experimental design and type of specimen. After imaging, select Processing, Batch"and Airyscan Processing, and select the images to be processed.Then click Run Process"to obtain the super resolution images. Live cell imaging of drosophila testes expressing alpha tubulin GFP in early stage germ cells using a spinning disc confocal microscope, allows visualization of the asymmetric intensity of GFP signals at two centrosomes, as a brighter signal at the mother centrosome and a relatively weaker signal at the daughter centrosomes. The difference in brightness is likely reflected by the temporal asymmetry of the microtubule nucleation, but the detailed morphology and quantity of the microtubules can not be resolved using spinning disc confocal microscopy.In contrast, live cell super resolution imaging allows the visualization and quantification of microtubule morphology and numbers. This improved resolution also reveals patterns of asymmetric microtubule nucleation, elongation and increased interaction with the nuclear membrane. Live cell imaging also allows the visualization of asymmetric nuclear membrane invagination, but not of individual microtubules that directly enter the nucleus.In contrast, this live cell super resolution technique allows the direct observation of these events by simultaneous imaging of both microtubules and the nuclear lamina. During metaphase, both sister centromeres can be detected as one signal using spinning disc microscopy, although the microtubule central attachment can not be observed. In contrast, super resolution live imaging allows visualization of the microtubule centromere attachment in early prophase.Be sure to place a membrane over the testes to ensure that they do not move or float during imaging and to optimize the microscope settings to avoid photo bleaching and photo toxicity. There are many ways that this method can be applied, such as to investigate protein dynamics, linear stressing, or cellular defenses and processes.

super-resolution-live-cell-imaging-of-subcellular-structures

Research

JoVE Journal

Biology

4.5K visualizações.  The Johns Hopkins University. Nosso protocolo usa sondas fluoróforas regulares e técnicas simples de preparação de espécimes que mantêm a célula imagem em condições fisiológicas para estudar processos celulares altamente dinâmicos e estruturas subcelulares detalhadas. Essa técnica nos permite investigar processos celulares em uma super resolução e em condições fisiológicas por um período prolongado sem sacrificar a resolução especial ou temporária. Comece cortando uma membrana de diálise de corte de ...