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    "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall">Confocal microscopy &#40;CM&#41; is an imaging technique that permits real-time visualization of skin structures with a resolution approaching that of conventional histology&#46;<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a> This technique has been applied in several skin diseases&#44; in particular&#44; tumors such as basal cell carcinoma &#40;BCC&#41; or squamous cell carcinoma&#46; With this technique&#44; the skin can be imaged directly &#40;<span class="elsevierStyleItalic">in vivo</span>&#41; or surgically removed pieces can be examined &#40;<span class="elsevierStyleItalic">ex vivo</span>&#41;&#46; In the latter case&#44; the use of fluorophores has improved the quality of the images obtained with a technique known as fluorescence CM &#40;FCM&#41;&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">Several different dyes have been used in FCM&#44; such as methylene blue or toluidine blue&#59; however&#44; acridine orange &#40;AO&#41; is the most widely used&#46;<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a> AO binds specifically to DNA or RNA of living cells&#44; acting as a fluorophore that is excited at a specific wavelength and then then emits fluorescence&#46; Nuclear cells can therefore be marked&#44; and they are observed as brilliant white structures in FCM mosaics&#46;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> Structures that do not have a nucleus&#44; such as collagen bundles that form part of the dermis&#44; would emit weak fluorescence or not at all&#46; AO can therefore increase image contrast 100-fold&#46;<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a> It is also important to point out that AO does not lead to sample degeneration and subsequent staining with traditional stains such as hematoxylin-eosin is still possible&#46;<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">To date&#44; in images of FCM published in the scientific literature&#44; fluorescence emitted by AO has been translated by microscopy to a final greyscale or dichromatic image&#46;<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a> Despite the excellent contrast between structures and the strong histopathologic correlation shown by these images&#44; the final greyscale mosaics are difficult to interpret for dermatologists and pathologists who are not expert in CM&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall">Recently&#44; our group has described a new technique for obtaining FCM images using a 3 color scale &#40;FCM-3CS&#41;&#44; through the joint use of AO and ethidium bromide &#40;EB&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a></p><p id="par0025" class="elsevierStylePara elsevierViewall">In this technique&#44; the resected pieces are immersed in liquid nitrogen&#44; thereby ensuring an almost instantaneous freezing&#46; They are then sectioned in a cryostat in rapid cuts measuring 20&#8211;30&#8239;&#956;m thick&#46; After sectioning&#44; the sample is stained by pouring on a mixture of AO 0&#46;1&#8239;mM and EB 0&#46;25&#8239;nM&#44; and leaving the solution to act for a minute&#46; After this brief processing&#44; the sample is placed under a commercially available confocal microscope&#44; Nikon A1R<span class="elsevierStyleSup">&#43;</span> &#40;Nikon Corporation&#174;&#44; Japan&#41;&#46; Once under the microscope&#44; the sample is excited simultaneously with 2 different wavelengths&#44; 405&#8239;nm and 488&#8239;nm&#46; The microscope collects fluorescence emitted by the sample after excitation to obtain images with a 3-color scale&#46; The process requires between 10 and 15&#8239;minutes to obtain the final color mosaics&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">These color images are the result of the joint action of the 2 fluorophores applied to the sample&#46; EB is a fluorophore that binds specifically to DNA in cells that have lost membrane integrity&#46;<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a> EB passes through the damaged nuclear membranes after freezing&#44; binding with high affinity to DNA and thus staining these cell nuclei&#46; In this way&#44; when the sample is excited with laser light at 405&#8239;nm&#44; EB emits red fluorescence&#44; marking the nuclei with high precision&#46; The dermis and anucleate structures emit green fluorescence from AO after excitation at 488&#8239;nm&#46; The weak blue fluorescence originates from intrinsic fluorescence of the tissues&#46; All this fluorescence is detected by the microscope&#44; and as a result&#44; the final images are obtained on a 3-color scale&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a> shows how a fragment of healthy skins looks with this technique&#46; Nucleated structures emit red fluorescence&#44; which contrasts significantly with green fluorescence&#44; yielding images that are very intuitive and easy to interpret&#44; with a very high resolution&#46; This resolution enables the skin tumor margins to be delimited with high precision&#44; for example in the case of BCC shown in <a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>A&#46; Note the complete correlation with classic hematoxylin-eosin staining &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>b&#41;&#46; After processing&#44; in relation to images H&#8211;E&#44; we do not observe significant changes in image quality&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0040" class="elsevierStylePara elsevierViewall">The main application of <span class="elsevierStyleItalic">ex vivo</span> CM currently is in oncological surgical processes&#44; such as Mohs surgery&#46; It can be expected that in coming years&#44; the cost of devices needed for CM will come down&#44; making it a cost-effective technique that could be incorporated progressively into everyday clinical practice&#46;</p><p id="par0045" class="elsevierStylePara elsevierViewall">In conclusion&#44; images with color FCM images are significantly simpler to interpret than greyscale images for dermatologists and pathologists who are not expert in CM&#46; Furthermore&#44; thanks to processing by sample freezing&#44; the sample is completely flat&#46; Therefore&#44; complete sample images can be obtained&#44; without losing mosaics due to sample folding&#46; These are all important advantages compared with traditional images obtained with CM&#46; Nevertheless&#44; more studies are needed to validate this new technique&#46;</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0005">Funding</span><p id="par0050" class="elsevierStylePara elsevierViewall">This study did not receive any funding&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Conflicts of interest</span><p id="par0055" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest&#46;</p></span></span>"
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Case and Research Letters
Ex vivo fluorescence confocal microscopy on a 3-color scale: A new imaging technique
Microscopía confocal de fluorescencia ex vivo en escala de tres colores (mcf-3cs): una nueva técnica de imagen
M. Sendín-Martína,
Corresponding author
mercedessendin@gmail.com

Corresponding author.
, J.J. Domínguez-Cruza, K.-L. Levitskyb, J. Conejo-Mir Sáncheza
a Unidad de Gestión Clínica de Dermatología, Hospital Universitario Virgen del Rocío, Sevilla, Spain
b Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain
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    "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall">Confocal microscopy &#40;CM&#41; is an imaging technique that permits real-time visualization of skin structures with a resolution approaching that of conventional histology&#46;<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a> This technique has been applied in several skin diseases&#44; in particular&#44; tumors such as basal cell carcinoma &#40;BCC&#41; or squamous cell carcinoma&#46; With this technique&#44; the skin can be imaged directly &#40;<span class="elsevierStyleItalic">in vivo</span>&#41; or surgically removed pieces can be examined &#40;<span class="elsevierStyleItalic">ex vivo</span>&#41;&#46; In the latter case&#44; the use of fluorophores has improved the quality of the images obtained with a technique known as fluorescence CM &#40;FCM&#41;&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">Several different dyes have been used in FCM&#44; such as methylene blue or toluidine blue&#59; however&#44; acridine orange &#40;AO&#41; is the most widely used&#46;<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a> AO binds specifically to DNA or RNA of living cells&#44; acting as a fluorophore that is excited at a specific wavelength and then then emits fluorescence&#46; Nuclear cells can therefore be marked&#44; and they are observed as brilliant white structures in FCM mosaics&#46;<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a> Structures that do not have a nucleus&#44; such as collagen bundles that form part of the dermis&#44; would emit weak fluorescence or not at all&#46; AO can therefore increase image contrast 100-fold&#46;<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a> It is also important to point out that AO does not lead to sample degeneration and subsequent staining with traditional stains such as hematoxylin-eosin is still possible&#46;<a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">To date&#44; in images of FCM published in the scientific literature&#44; fluorescence emitted by AO has been translated by microscopy to a final greyscale or dichromatic image&#46;<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a> Despite the excellent contrast between structures and the strong histopathologic correlation shown by these images&#44; the final greyscale mosaics are difficult to interpret for dermatologists and pathologists who are not expert in CM&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall">Recently&#44; our group has described a new technique for obtaining FCM images using a 3 color scale &#40;FCM-3CS&#41;&#44; through the joint use of AO and ethidium bromide &#40;EB&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a></p><p id="par0025" class="elsevierStylePara elsevierViewall">In this technique&#44; the resected pieces are immersed in liquid nitrogen&#44; thereby ensuring an almost instantaneous freezing&#46; They are then sectioned in a cryostat in rapid cuts measuring 20&#8211;30&#8239;&#956;m thick&#46; After sectioning&#44; the sample is stained by pouring on a mixture of AO 0&#46;1&#8239;mM and EB 0&#46;25&#8239;nM&#44; and leaving the solution to act for a minute&#46; After this brief processing&#44; the sample is placed under a commercially available confocal microscope&#44; Nikon A1R<span class="elsevierStyleSup">&#43;</span> &#40;Nikon Corporation&#174;&#44; Japan&#41;&#46; Once under the microscope&#44; the sample is excited simultaneously with 2 different wavelengths&#44; 405&#8239;nm and 488&#8239;nm&#46; The microscope collects fluorescence emitted by the sample after excitation to obtain images with a 3-color scale&#46; The process requires between 10 and 15&#8239;minutes to obtain the final color mosaics&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">These color images are the result of the joint action of the 2 fluorophores applied to the sample&#46; EB is a fluorophore that binds specifically to DNA in cells that have lost membrane integrity&#46;<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a> EB passes through the damaged nuclear membranes after freezing&#44; binding with high affinity to DNA and thus staining these cell nuclei&#46; In this way&#44; when the sample is excited with laser light at 405&#8239;nm&#44; EB emits red fluorescence&#44; marking the nuclei with high precision&#46; The dermis and anucleate structures emit green fluorescence from AO after excitation at 488&#8239;nm&#46; The weak blue fluorescence originates from intrinsic fluorescence of the tissues&#46; All this fluorescence is detected by the microscope&#44; and as a result&#44; the final images are obtained on a 3-color scale&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a> shows how a fragment of healthy skins looks with this technique&#46; Nucleated structures emit red fluorescence&#44; which contrasts significantly with green fluorescence&#44; yielding images that are very intuitive and easy to interpret&#44; with a very high resolution&#46; This resolution enables the skin tumor margins to be delimited with high precision&#44; for example in the case of BCC shown in <a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>A&#46; Note the complete correlation with classic hematoxylin-eosin staining &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>b&#41;&#46; After processing&#44; in relation to images H&#8211;E&#44; we do not observe significant changes in image quality&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0040" class="elsevierStylePara elsevierViewall">The main application of <span class="elsevierStyleItalic">ex vivo</span> CM currently is in oncological surgical processes&#44; such as Mohs surgery&#46; It can be expected that in coming years&#44; the cost of devices needed for CM will come down&#44; making it a cost-effective technique that could be incorporated progressively into everyday clinical practice&#46;</p><p id="par0045" class="elsevierStylePara elsevierViewall">In conclusion&#44; images with color FCM images are significantly simpler to interpret than greyscale images for dermatologists and pathologists who are not expert in CM&#46; Furthermore&#44; thanks to processing by sample freezing&#44; the sample is completely flat&#46; Therefore&#44; complete sample images can be obtained&#44; without losing mosaics due to sample folding&#46; These are all important advantages compared with traditional images obtained with CM&#46; Nevertheless&#44; more studies are needed to validate this new technique&#46;</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0005">Funding</span><p id="par0050" class="elsevierStylePara elsevierViewall">This study did not receive any funding&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Conflicts of interest</span><p id="par0055" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest&#46;</p></span></span>"
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Article information
ISSN: 15782190
Original language: English
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Idiomas
Actas Dermo-Sifiliográficas
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