Name: correlative light- and electron microscopy using quantum dot nanoparticles
Uploaded: 2016-08-07T14:00:00
Description: Watch this Scientific Journal Video about Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles at JoVE.com

The authors wish to acknowledge the support of Xiao Juan Wu (Immunohistochemistry Laboratory) and the Department of Anatomical Pathology, Sydney South West Pathology Service (SSWPS), NSW Health Pathology, Liverpool, New South Wales, Australia.

1. Tissue Dissection and Fixation
<ol>
	<li>Tissue Dissection
	<ol>
		<li>Dissect tissue pieces from a surgically resected tumor specimen or tissue biopsy. 
		Note: The tissue used in this study was routinely fixed in formalin but fresh tissue is also suitable. We selected an area confirmed and reported by an anatomical pathologist to contain somatostatinoma tumor after routine histological staining and anti-somatostatin immunostaining (not shown).</li>
		<li>Use tissue pieces no larger than appro...

<ol>
	<li>Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M., Briggs, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+fluorescence+and+3D+electron+microscopy+with+high+sensitivity+and+spatial+precision.">Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.</a> J Cell Biol. 192 (1), 111-119 (2011).</li><li>M&#252;ller-Reichert, T., Verkade, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+correlative+light+and+electron+microscopy.">Introduction to correlative light and electron microscopy.</a> Methods Cell Biol. , (2012).</li><li>De Boer, P., Hoogenboom, J. P., Giepmans, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+light+and+electron+microscopy:+ultrastructure+lights+up!.">Correlated light and electron microscopy: ultrastructure lights up!.</a> Nat Methods. 12 (6), 503-513 (2015).</li><li>Faas, F. 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=Localization+of+fluorescently+labeled+structures+in+frozen-hydrated+samples+using+integrated+light+electron+microscopy.">Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.</a> J Struct Biol. 181 (3), 283-290 (2013).</li><li>Redemann, S., M&#252;ller-Reichert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+light+and+electron+microscopy+for+the+analysis+of+cell+division.">Correlative light and electron microscopy for the analysis of cell division.</a> J Microsc. 251 (2), 109-112 (2013).</li><li>Spiegelhalter, 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=From+dynamic+live+cell+imaging+to+3D+ultrastructure:+novel+integrated+methods+for+high+pressure+freezing+and+correlative+light-electron+microscopy.">From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy.</a> PLoS One. 5 (2), e9014 (2010).</li><li>Killingsworth, M. C., Lai, K., Wu, X., Yong, J. L., Lee, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dot+immunocytochemical+localization+of+somatostatin+in+somatostatinoma+by+widefield+epifluorescence,+super-resolution+light,+and+immunoelectron+microscopy.">Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by widefield epifluorescence, super-resolution light, and immunoelectron microscopy.</a> J Histochem Cytochem. 60 (11), 832-843 (2012).</li><li>Nisman, R., Dellaire, G., Ren, Y., Li, R., Bazett-Jones, 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=Application+of+quantum+dots+as+probes+for+correlative+fluorescence,+conventional,+and+energy-filtered+transmission+electron+microscopy.">Application of quantum dots as probes for correlative fluorescence, conventional, and energy-filtered transmission electron microscopy.</a> J Histochem Cytochem. 52 (1), 13-18 (2004).</li><li>Giepmans, B. N., Deerinck, T. J., Smarr, B. L., Jones, Y. Z., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+light+and+electron+microscopic+imaging+of+multiple+endogenous+proteins+using+quantum+dots.">Correlated light and electron microscopic imaging of multiple endogenous proteins using quantum dots.</a> Nat Methods. 2 (10), 743-749 (2005).</li><li>Kuipers, J., de Boer, P., Giepmans, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+EM+of+non-heavy+metal+stained+biosamples:+Large-field+of+view,+high+contrast+and+highly+efficient+immunolabeling.">Scanning EM of non-heavy metal stained biosamples: Large-field of view, high contrast and highly efficient immunolabeling.</a> Exp Cell Res. 337 (2), 202-207 (2015).</li><li>Barroso, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dots+in+cell+biology.">Quantum dots in cell biology.</a> J Histochem Cytochem. 59 (3), 237-251 (2011).</li><li>Michalet, X., 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=Quantum+dots+for+live+cells,+in+vivo+imaging,+and+diagnostics.">Quantum dots for live cells, in vivo imaging, and diagnostics.</a> Science. 5, 538-544 (2005).</li><li>Shu, X., 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+genetically+encoded+tag+for+correlated+light+and+electron+microscopy+of+intact+cells,+tissues+and+organisms.">A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues and organisms.</a> PLoS Biol. 9 (4), e1001041 (2011).</li><li>Thomas, J. M., Midgley, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+transmission+electron+microscopy:+the+ultimate+nanoanalytical+technique.">High-resolution transmission electron microscopy: the ultimate nanoanalytical technique.</a> Chem Commun. , 1253-1267 (2004).</li><li>Loussert Fonta, C., Humbel, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+microscopy.">Correlative microscopy.</a> Arch Biochem Biophys. 581, 98-110 (2015).</li><li>Marc, R. E., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamental+GABAergic+amacrine+cell+circuitries+in+the+retina:+nested+feedback,+concatenated+inhibition,+and+axosomatic+synapses.">Fundamental GABAergic amacrine cell circuitries in the retina: nested feedback, concatenated inhibition, and axosomatic synapses.</a> J Comp Neurol. 425 (4), 560-582 (2000).</li><li>Studer, D., Humbel, B. M., Chiquet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+high+pressure+frozen+samples:+bridging+the+gap+between+cellular+ultrastructure+and+atomic+resolution.">Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution.</a> Histochem Cell Biol. 130 (5), 877-889 (2008).</li><li>Tokuyasu, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemistry+on+ultrathin+frozen+sections.">Immunochemistry on ultrathin frozen sections.</a> Histochem J. 12 (4), 381-403 (1980).</li><li>Loussert Fonta, 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=Analysis+of+acute+brain+slices+by+electron+microscopy:+a+correlative+light-electron+microscopy+workflow+based+on+Tokuyasu+cryo-sectioning.">Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.</a> J Struct Biol. 189 (1), 53-61 (2015).</li></ol>

This study has demonstrated the potential utility of QDs as universal probes for CLEM studies. The 585 nm QD nanoparticles used showed bright and stable fluorescence when viewed by widefield light microscopy and were readily observed by TEM. A previous study by one of the present authors has shown QDs also to be suitable for super-resolution light microscopy7. Their photostability was particularly useful for extended viewing periods and long imaging exposures. QDs can also be used for multiplex immunohistochem...

Correlative light- and electron microscopy (CLEM) is a powerful approach for the analysis of transient dynamic events1, rare events2, 3 and complex systems4. There are many different technical permutations available5 depending on the question being asked however a common requirement is that the same structure in a single sample6 is imaged by multiple microscopy modalities. Our particular approach to CLEM was developed for the study of archival human pathology tissue and the case used here has been well characterized and published previously7. The aim was firstly, to maximize the analytical data from a single biopsy or surgical sample and secondly, to use fluorescence light microscopy to help clarify the context of the immunocytochemical labeling pattern seen at the ultrastructural level.
Quantum dot nanocrystals (QDs) offer the potential of a universal marker system able to be viewed by both, fluorescence light microscopy and electron microscopy8, 9, 10. Their crystalline core structure allows QDs of different sizes to generate a wide range of fluorescence emission peaks when excited by light at wavelengths far from their emission spectra11. Their atomic weight is sufficient to yield electron density that is detectable by transmission electron microscopy, scanning transmission electron microscopy (STEM) or field emission scanning electron microscopy. They are particularly suited to immunocytochemical studies as even single QDs may be observed giving an ultimate sensitivity of one QD per target molecule12. Furthermore, depending on the QD used they can possess an individual elemental signature suitable for mapping.
Human pathology samples offer significant benefits for translational biomedical research. Surgical tissue and biopsy samples are routinely submitted for biobanking and with appropriate ethics clearances can be accessed for research studies. Human tissue does not have issues of relevance or interpretation that can occur in animal or in vitro models of disease. However, specimen preparation of pathology samples often is not optimal. There can be delay in tissue being placed in fixative, inappropriate fixative used such as formalin rather than glutaraldehyde for TEM and inappropriate sampling. CLEM methods have the potential to optimize the diagnostic and prognostic information available from a single human sample. However, some newly developed correlative approaches such as those employing mini Singlet Oxygen Generator (miniSOG) are not available for use in pathology due to the need for the tag to be genetically encoded into the cell of interest13. For this reason we have explored the utility of QD labeling of routinely prepared TEM tissue for correlative immunocytochemical studies. QDs applied to etched epoxy or acrylic resin sections from lightly aldehyde fixed biopsy and tissue samples offer the possibility of obtaining correlative fluorescence light microscopy and TEM data from a single sample.

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			<td height="21" style="height:21px;width:255px;">Sodium cacodylate</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C0205</td>
			<td style="width:167px;">Harmful chemical</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Osmium tetroxide</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C010</td>
			<td>Use only in fume hood</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Uranyl acetate</td>
			<td style="width:117px;">Univar-Ajax</td>
			<td style="width:119px;">569</td>
			<td>Hazardous chemical</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ethanol 100%</td>
			<td style="width:117px;">Fronine</td>
			<td style="width:119px;">JJ008</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Acetone 100%</td>
			<td style="width:117px;">Fronine</td>
			<td style="width:119px;">JJ006</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ERL 4221</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C056</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DER 732</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C047</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NSA</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C059</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMAE</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">C050</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sodium metaperiodate</td>
			<td style="width:117px;">Analar BDH</td>
			<td style="width:119px;">10259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-somatostatin antibody</td>
			<td style="width:117px;">Dako</td>
			<td style="width:119px;">A0566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibody diluent</td>
			<td style="width:117px;">Dako</td>
			<td style="width:119px;">S3022</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Qdot 585 Streptavidin Conjugate&#160;</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">Q10113MP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Biotinylated goat anti-rabbit IgG antibody</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">B7389-1ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glutaraldehyde 50%</td>
			<td style="width:117px;">EMS</td>
			<td style="width:119px;">16320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Normal goat serum</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">PCN5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PBS &#34;Dulbecco A&#34;</td>
			<td style="width:117px;">Oxoid&#160;</td>
			<td style="width:119px;">BR0014G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BSAc (10%)</td>
			<td style="width:117px;">Aurion</td>
			<td style="width:119px;">900.022</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Parafilm</td>
			<td style="width:117px;">Pechiney PP</td>
			<td style="width:119px;">M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH indicator strips (pH 2.0 - 9.0)</td>
			<td style="width:117px;">Merck</td>
			<td style="width:119px;">1.09584.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micromoulds</td>
			<td style="width:117px;">Proscitech</td>
			<td style="width:119px;">RL063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Diamond knife</td>
			<td style="width:117px;">Diatome</td>
			<td style="width:119px;">Ultra 45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Transmission electron microscope</td>
			<td style="width:117px;">FEI</td>
			<td style="width:119px;">Morgagni 268D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fluorescence light microscope</td>
			<td style="width:117px;">Carl Zeiss</td>
			<td style="width:119px;">Axioscope A1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Grids 300 mesh nickel (thin bar)</td>
			<td style="width:117px;">Agar Scientific</td>
			<td style="width:119px;">G2740N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ultramicrotome</td>
			<td style="width:117px;">RMC</td>
			<td style="width:119px;">Powertome</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">TEM camera control software</td>
			<td style="width:117px;">Soft Imaging System</td>
			<td style="width:119px;">AnalySIS</td>
			<td>Version 3.0</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Image processing software</td>
			<td style="width:117px;">Adobe Systems Incorporated</td>
			<td style="width:119px;">Photoshop CS2</td>
			<td></td>
		</tr>
	</tbody>
</table>

correlative light- and electron microscopy using quantum dot nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

The somatostatinoma tumor specimen used for this study comprised tumor cells forming ductal structures mixed with collagenous stomal tissue. By fluorescence light microscopy, individual tumor cells that contained abundant secretory granules showed positive labeling for the somatostatin hormone. Nuclei appeared as dark holes with minimal non-specific labeling detectable (Figure 1). At low magnifications, variably intense granular orange fluorescence was seen in the cytopla...

Watch this Scientific Journal Video about Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles at JoVE.com

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

The overall goal of this method is to obtain correlative light and electron microscopy, or CLEM data, by combining fluorescence immunocytochemical information with ultra-structural morphology from transmission electron microscopy in a single image. This method can help answer key questions in the field of immunocytochemistry and pathology, such as what subcellular structure is a particular protein of interest associated with. The main advantage of this technique is that it uses the same quantum dot nanoparticle probe to obtain both the live microscopy signal from the target protein and the electron microscopy structural localization.After fixing, embedding and sectioning human somatostatinoma tumor tissue according to the text protocol, prepare a section adhesive solution by placing 20 centimeters of clear adhesive tape into a five milliliter bottle containing 2.0 milliliters of acetone and let it stand for 10 minutes. Remove the tape and dip a nickel grid into the solution, then remove the grid and allow it to air dry. Once dry, use the dull side of the grid to pick up an ultra thin section.It's critical to establish the correct etching time for the chosen resin formulation. 30 minutes works well for this formulation, but it may need to be recalculated for harder or softer formulations. Prepare one milliliter of fresh saturated sodium metaperiodate etching solution in distilled water and place droplets of the solution on a clean lab film surface.Place completely dry grids with sections onto the droplets and incubate at room temperature for 30 minutes. Then, transfer the grids to distilled water droplets for 60 seconds to wash them off. To carry out antibody and quantum dot, or QD labeling, on a clean lab film surface, pipette drops of 0.05 molar glycene and PBS.Place the grids on the droplets and incubate for 10 minutes to block residual aldehyde on the sections. To remove the aldehyde block, briefly blot the edge of the grid. Then place the grid on a droplet of one percent normal goat serum, or NGS, and one percent BSAC in PBS for 10 minutes.Prepare one milliliter of blocking solution by adding 10 microliters of NGS and 100 microliters of BSAC to 890 microliters of PBS. Then, condition the sections by placing them on a droplet of antibody diluent for 10 minutes. Next, use antibody diluent to dilute anti-somatostatin polyclonal antibody one to 10, and place the grids on droplets of the solution.Incubate in a moist chamber at room temperature for one hour. Following the incubation, remove the antibody reagent by blotting the edge of the grid. Then use antibody diluent to wash the sections two times for five minutes each.After diluting secondary antibody one to 10 and preparing drops, incubate the grids on the droplets at room temperature for one hour. Then, remove the antibody solution and wash the sections two times. Dilute streptavidin conjugated QDs one to 10 and incubate the samples on drops of the solution in the dark, at room temperature for one hour.Then use antibody diluent to wash the grids twice, each time for five minutes, and blot the edge of the grids. Next, wash the grids in distilled water for two minutes before blotting dry the edges. Place the immunostained grid section in a droplet of water on a glass slide and use a glass cover slip to cover it.To carry out fluorescence light microscopy, insert a filter cube with the following characteristics and use 365 nanometer LED illumination. Place the immunostained section on the immunofluorescence microscope stage. Use light microscopy to evaluate the labeling pattern, the level of any non-specific labeling and to establish that negative controls are clear.Next, identify ROIs in relation to grid bars. Then set a full colored digital camera to FL auto color and set white balance to 3, 200 K.Set the camera to automatic exposure mode with a gamma setting between 0.45 and 1.00 to optimize the appearance of the images, and analog gain set to one X.Click the camera icon in the ZEN Two Light software graphical interface to live. Then focus on the image and click Snap in the ZEN Two Light software graphical interface to capture the image to the frame store.Save the images as tf files to avoid compression and pixelation. Prepare a print showing the position of the ROI in relation to the grid bars or other significant tissue landmarks. Then remove the grid from the slide, wash in distilled water, and gently blot the edges dry.To carry out transmission electron microscopy, transfer the grid to the microscope for examination using an accelerating voltage of 100 kilovolts. Begin with low magnification of approximately 1, 400X to navigate around the grid and find the ROIs with those found with light microscopy. Observe individual QDs at 70, 000X magnification or above.They will appear as irregular crystalline structures with moderate electron density. Then, using a digital camera with a monochrome 1, 392 by 1, 040 pixel sensor in the imaging software's graphical interface, click the camera icon to on and acquire images. Move the camera icon to the off position to store the image in the frame store.To carry out CLEM imaging, use a print from the light microscopy imaging to locate corresponding ROIs by TEM. Tissue architectural features are useful for navigation around the section. Capture an image of the corresponding ROI by TEM.Next, to prepare a CLEM image, ensure that the TEM image is correctly oriented and enlarged to the same size and resolution as the light microscope image, in this case, 300 pixels per inch. Extend the size of the light microscopy image canvas to double its size. Then copy and paste the TEM image beside the fluorescence image.To create a fluorescence overlay in Photoshop CS2, click on the rectangular marquee tool, select the fluorescent image, and create a duplicate layer from it. Adjust the layer style blending options to 30 to 40%transparency. Then click on the move tool and drag the fluorescence overlay layer over the corresponding TEM image, and align the images.After aligning the images, flatten the image to produce the final overlay. Then use the rectangular marquee tool to select the new overlay image and copy it to a new canvas. Finally, save the image as a tf file.In this fluorescence microscopy image, individual somatostatinoma tumor cells contain abundant secretory granules and were positively labeled for the somatostatin hormone. The nuclei appeared as dark holes and at low magnification, variably intense granular orange QD fluorescence was seen in the cytoplasm. The ROI chosen by light microscopy was recognized and imaged using TEM by referring to a 20X light microscopy image as indicated here.At higher magnification, the cell demonstrates bright fluorescence and intense QD labeling in cytoplasmic granules. The overlaying of fluorescence data on TEM images, as in this example, suggests strong positive labeling corresponds to granules containing moderately electron dense material within the vesicle limiting membrane. Finally, as seen here at higher magnification, the moderately electron dense granules showed intense QD nanocrystal immunolabeling.Once mastered, this technique can be done in eight hours if performed properly. While attempting the procedure, it's important to remember to handle the grids carefully, by the edges only. Touching the section while picking up the grid may detach the section from the grid.Following this procedure, other methods like multiplex immunolabeling can be performed in order to answer additional questions, such as does your protein of interest co-localize with another protein. After its development, this technique paved the way for researchers in the field of pathology to explore protein localization in archival human tissue process for electron microscopy. After watching this video, you should have a good understanding of how to combine immunocytochemical information from fluorescence light microscopy with ultra structure from transmission electron microscopy.Don't forget that working with osmium tetroxide can be extremely hazardous, and precautions such as wearing protective equipment, working in a fume hood and proper waste disposal should be taken while performing this procedure.

correlative-light-electron-microscopy-using-quantum-dot

Research

JoVE Journal

Bioengineering

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...