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.

<table border="0" cellpadding="0" cellspacing="0" width="657">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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.

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Research

JoVE Journal

Bioengineering

9.6K vistas.  University of New South Wales Australia. 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 quant...