Name: reconstruction of single-cell innate fluorescence signatures by confocal microscopy
Uploaded: 2020-05-27T14:30:00
Description: Watch this Scientific Journal Video about Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy at JoVE.com

Este estudo foi apoiado em parte por uma subvenção em auxílio para pesquisas científicas do Ministério da Educação, Cultura, Esportes e Tecnologia do Japão (18K04843) para Y. Yawata, o JST ERATO (JPMJER1502) para N. Nomura.

1. Preparação da amostra
<ol><li>Coloque uma junta de silicone de 1 mm de espessura com poços em uma lâmina de vidro.</li>
	<li>Coloque uma laje de 1 mm de espessura de 0,8% (w/v) no poço da junta de silicone.</li>
	<li>Diluir a densidade celular de uma cultura celular microbiana arbitrária para uma densidade óptica a 600 nm (OD660) = 1,0.</li>
	<li>Coloque uma alíquota de 5 μL de suspensão celular na laje agarose.</li>
	<li>Cubra delicadamente com uma mancha de vidro.</li>
</ol><p class="jove_titl...

<ol>
	<li>Monici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+autofluorescence+research+and+diagnostic+applications.">Cell and tissue autofluorescence research and diagnostic applications.</a> Biotechnology Annual Review. 11, 227-256 (2005).</li><li>Tang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+metabolomics.">Microbial metabolomics.</a> Current Genomics. 12, 391-403 (2011).</li><li>Woo, P. C., Lau, S. K., Teng, J. L., Tse, H., Yuen, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Then+and+now:+use+of+16S+rDNA+gene+sequencing+for+bacterial+identification+and+discovery+of+novel+bacteria+in+clinical+microbiology+laboratories.">Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.</a> Clinical Microbiology and Infection. 14, 908-934 (2008).</li><li>Amman, R., Fuchs, 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=Single-cell+identification+in+microbial+communities+by+improved+fluorescence+in+situ+hybridization+techniques.">Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques.</a> Nature Reviews Microbiology. 6, 339-348 (2008).</li><li>Giana, H. E., Silveira, L., Z&#226;ngaro, R. A., Pacheco, M. T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+bacterial+species+by+fluorescence+spectroscopy+and+classification+through+principal+components+analysis.">Rapid identification of bacterial species by fluorescence spectroscopy and classification through principal components analysis.</a> Journal of Fluorescence. 13 (6), 489-493 (2003).</li><li>Leblanc, L., Dufour, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+identity+of+bacteria+using+their+intrinsic+fluorescence.">Monitoring the identity of bacteria using their intrinsic fluorescence.</a> FEMS Microbiology Letters. 211 (2), 147-153 (2002).</li><li>Hou, X., Liu, S., Feng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autofluorescence+characteristics+of+bacterial+intracellular+and+extracellular+substances+during+the+operation+of+anammox+reactor.">The autofluorescence characteristics of bacterial intracellular and extracellular substances during the operation of anammox reactor.</a> Scientific Reports. 7, 39289 (2017).</li><li>Ramanujam, N., 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=In+vivo+diagnosis+of+cervical+intraepithelial+neoplasia+using+337-nm-excited+laser-induced+fluorescence.">In vivo diagnosis of cervical intraepithelial neoplasia using 337-nm-excited laser-induced fluorescence.</a> Proceeding National Academy of Sciences of the United States of America. 91 (21), 10193-10197 (1994).</li><li>Zhang, J. 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=Innate+cellular+fluorescence+reflects+alterations+in+cellular+proliferation.">Innate cellular fluorescence reflects alterations in cellular proliferation.</a> Lasers in Surgery and Medicine. 20 (3), 319-331 (1997).</li><li>Gosnell, M. E., 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=Quantitative+non-invasive+cell+characterisation+and+discrimination+based+on+multispectral+autofluorescence+features.">Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features.</a> Scientific Reports. 6, 23453 (2016).</li><li>Yawata, Y., 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=Intra-+and+interspecies+variability+of+single-cell+innate+fluorescence+signature+of+microbial+cell.">Intra- and interspecies variability of single-cell innate fluorescence signature of microbial cell.</a> Applied and Environmental Microbiology. 85, 00608-00619 (2019).</li><li>Nelson, K. E., 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=Complete+genome+sequence+and+comparative+analysis+of+the+metabolically+versatile+Pseudomonas+putida+KT2440.">Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440.</a> Environmental Microbiology. 4 (12), 799-808 (2002).</li><li>Bagdasarian, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific-purpose+plasmid+cloning+vectors.+II.+Broad+host+range,+high+copy+number,+RSF1010-derived+vectors,+and+a+host-vector+system+for+gene+cloning+in+Pseudomonas.">Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.</a> Gene. 16 (1-3), 237-247 (1981).</li><li>Luo, Y., Vijaychander, S., Stile, J., Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+analysis+of+DNA-binding+proteins+by+yeast+one-hybrid+and+one-two-hybrid+systems.">Cloning and analysis of DNA-binding proteins by yeast one-hybrid and one-two-hybrid systems.</a> Biotechniques. 20 (4), 564-568 (1996).</li><li>Yawata, Y., 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=Monitoring+biofilm+development+in+a+microfluidic+device+using+modified+confocal+reflection+microscopy.">Monitoring biofilm development in a microfluidic device using modified confocal reflection microscopy.</a> Journal of Bioscience and Bioengineering. 110 (3), 377-380 (2010).</li><li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescence Spectroscopy. Third edition. , (2006).</li><li>Blacker, T. S., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+mitochondrial+redox+state+using+NADH+and+NADPH+autofluorescence.">Investigating mitochondrial redox state using NADH and NADPH autofluorescence.</a> Free Radical Biology &amp; Medicine. 100, 53-65 (2016).</li><li>Croce, A. 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Existem dois pontos críticos neste método que precisam ser acompanhados de perto para obter resultados reprodutíveis: 1) manter a saída de energia laser sob o objetivo do microscópio consistente através de comprimentos de onda e experimentos de excitação, e 2) realizar segmentação celular precisa.
O primeiro ponto é particularmente importante quando se compara a assinatura de fluorescência inata entre diferentes experimentos. Evite simplesmente aplicar as mesmas configurações de ...

Diversas biomoléculas dentro de uma célula1 emitem sinais de autofluorescência, e a assinatura de fluorescência inata de uma célula consiste na montagem desses sinais. Essa fluorescência de assinatura reflete várias propriedades celulares e também diferenças no estado fisiológico. A análise da fluorescência inata é minimamente invasiva e pode complementar sondas microbiológicas tradicionais e mais invasivas que deixam uma gama de vestígios desde modificações metabólicas leves até destruição celular completa. Enquanto técnicas tradicionais como extração de DNA ou conteúdo celular2,3, hibridização fluorescente in situ4, e a introdução de genes repórteres fluorescentes ao genoma são eficazes na determinação do tipo celular ou status fisiológico, eles geralmente requerem manipulação das células ou marcação invasiva.
Estudos da fluorescência inata de várias colônias microbianas vivas e intactas, incluindo suspensões de cultura microbiana a granel5,6, lodo ativo7, tecidos mamíferos8,9 e células mamíferas1,10, mostraram que a análise de fluorescência inata facilita a análise livre de etiquetas de tipos celulares e status fisiológico. As assinaturas de fluorescência inata têm sido tradicionalmente analisadas no nível populacional e não no nível de célula única, e, portanto, necessitam de uma cultura clonal. Em contraste, a técnica de análise de fluorescência unicelular assistida por microscopia confocal (CRIF) descrita aqui reconstrói e cataloga a assinatura de fluorescência celular inata de cada célula microbiana viva individual. Além disso, o CRIF pode sistematicamente reunir a assinatura de fluorescência inata de uma única célula microbiana dentro de uma população que é distribuída em um espaço tridimensional (3D).

<table border="0" cellpadding="0" cellspacing="0" style="width:720px;" width="720">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Agarose</td>
			<td style="width:220px;">Wako Chemicals</td>
			<td style="width:119px;">312-01193</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:215px;">Beam splitters</td>
			<td style="width:220px;">Carl Zeiss, Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">MBSInVis405, MBS458, MBS488, MBS458/514, MBS488/543, or MBS 488/543/633 beam splitters (Carl Zeiss)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Confocal microscope</td>
			<td style="width:220px;">Carl Zeiss, Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model LSM 880 (Carl Zeiss), Model A1R (Nikon)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cover slips</td>
			<td style="width:220px;">Matsunami Glass</td>
			<td style="width:119px;">C024601</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass slides</td>
			<td style="width:220px;">Matsunami Glass</td>
			<td style="width:119px;">S011120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Half-reflection mirror</td>
			<td style="width:220px;">Carl Zeiss, Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">NT80/20</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Laser power meter</td>
			<td style="width:220px;">Thorlabs</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">PM400 (power meter console) and S175C (sensor)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">LB Broth</td>
			<td style="width:220px;">Nacalai tesque</td>
			<td style="width:119px;">20066-95</td>
			<td>For bacteria culture</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">Image analysis software</td>
			<td style="width:220px;">The MathWorks</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">MATLAB version 2019a or later, Image Processing Toolbox is needed</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Microscope objective</td>
			<td style="width:220px;">Carl Zeiss, Nikon</td>
			<td style="width:119px;">440762-9904</td>
			<td style="width:167px;">e.g. 63x plan Apochomat NA = 1.4 (Carl Zeiss)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Microscope software</td>
			<td style="width:220px;">Carl Zeiss, Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">ZEN (Carl Zeiss),NIS-elements (Nikon)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PBS(-)</td>
			<td style="width:220px;">Wako Chemicals</td>
			<td style="width:119px;">166-23555</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:215px;">Programming language</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Python and libraries, modules (numpy, scikit-learn, scikit-image, os, glob, matplotlib, tkinter) are rquired to run the supplied PCA script.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Silicone gasket</td>
			<td style="width:220px;">ThermoFisher Scientific</td>
			<td style="width:119px;">P24744</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">Workstation</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A high-performance workstation with discrete GPUs is recommended.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Yeast extract-peptone-dextrose (YPD) agar medium</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:119px;">Y1500-250G</td>
			<td>For yeast culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">YPD medium</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:119px;">Y1375-250G</td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstruction of single-cell innate fluorescence signatures by confocal microscopy

Descrito aqui é a análise de fluorescência unicelular assistida por microscopia de reflexão confocal (CRIF), um método minimamente invasivo para reconstruir a assinatura de fluorescência celular inata de cada célula viva individual em uma população distribuída em um espaço tridimensional (3D). A assinatura de fluorescência inata de uma célula é uma coleção de sinais de fluorescência emitidos por várias biomoléculas dentro da célula. Estudos anteriores estabeleceram que as assinaturas de fluorescência inata refletem várias propriedades celulares e diferenças no estado fisiológico e são uma rica fonte de informação para caracterização e identificação celular. As assinaturas de fluorescência inata têm sido tradicionalmente analisadas no nível populacional, necessitando de uma cultura clonal, mas não no nível de célula única. O CRIF é particularmente adequado para estudos que requerem resolução 3D e/ou extração seletiva de sinais de fluorescência de células individuais. Como a assinatura de fluorescência é uma propriedade inata de uma célula, o CRIF também é adequado para a previsão livre de tags do tipo e/ou status fisiológico de células intactas e únicas. Este método pode ser uma ferramenta poderosa para análise celular simplificada, onde o fenótipo de cada célula em uma população heterogênea pode ser diretamente avaliado por sua assinatura de autofluorescência sob um microscópio sem marcação celular.

A Figura 1A mostra a assinatura típica de fluorescência unicelular de uma célula bacteriana apresentada como uma trama de espectro tradicional (superior) e como um mapa de calor (meio). A Figura 1B mostra o resultado de uma segmentação de células 2D precisa sobreposta à imagem original de CRM de uma população de bactérias do solo (Pseudomonas putida KT2440)12. As assinaturas de fluorescência inata resultantes para a po...

Watch this Scientific Journal Video about Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy at JoVE.com

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

Aqui, é apresentado um protocolo para extração óptica e catalogação de assinaturas de fluorescência celular inata (ou seja, autofluorescência celular) de cada célula viva individual distribuída em um espaço tridimensional. Este método é adequado para estudar a assinatura de fluorescência inata de diversos sistemas biológicos em uma resolução unicelular, incluindo células de bactérias, fungos, leveduras, plantas e animais.

Reconstrução de assinaturas de fluorescência inata de células únicas por microscopia confocal

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing ...

This technique offers a unique research to for proving your cell's identity or physiological characteristics at the single-cell level without the need for invasive tagging. The main advantages of this technique are that it facilitates single-cell level spatial resolution for your analysis, and that it allows the distinction between and background process. So this technique can potentially contribute to the identification and the phenotypic analysis of pathogenic microbes.This technique can also be applied to the study of the phenotypic heterogeneity, or the monitoring of the physiological status of microbial population of interests. Demonstrating the procedure will be Kyosuke Takabe, an assistant professor from my laboratory. The confocal reflection microscopy and multichannel confocal microspectroscopy setup.Connect the confocal microscope with descanned spectral channels to a photomultiplier tube. Equip the microscope with a high numerical aperture objective, with an adequate magnification. And equip the microscope with a half-reflection mirror to accommodate confocal reflection microscopy, which relies on the cellular scatter of incident light to visualize cell morphology.For multichannel confocal microspectroscopy, equip the microscope with dichroic mirrors and use a laser power meter to adjust the illumination intensity for each excitation wavelength. Then set the output under the microscope to be constant through the excitation wavelengths. To image the bacteria through the microscope, set the pinhole size to 1.0 area unit in the microscope software and set the pixel dwell time for each excitation wavelength.Set the scanning resolution. For small cells such as bacteria, use a scanning area of 1024 by 1024. Set the Z-Scanning range so that the region of interest is covered.And using a spectral window of eight to 10 nanometers, set the D-Scan detector to capture the visible wavelength range. Then acquire multichannel confocal microspectroscopy images in a sequence from longest to shortest excitation wavelengths to create Z-Stacks of fluorescence images and confocal reflection microscopy images, and save the images in 16-bit tiff format. To perform cell segmentation and the reconstruction of single cell innate fluorescent signatures, open an appropriate image analysis software program and double click to open one of the provided scripts.Under the editor tab, click run. A folder selection window will appear. Select the directory in which the Z-Stack images were saved and click open.A dialogue box prompting the input of the segmentation parameter will automatically appear. Set the threshold of image binarization to 0-1, the image binarization to 0.45, the upper threshold for a cell region to 200, the lower threshold for a cell region to 10, and the number of detectors to 32. A dialogue box requesting input for the number of excitation wavelengths will appear.Input the number of wavelengths used for the image acquisition and click okay. A dialogue box that is requesting input for the excitation wavelengths will appear. Enter the excitation wavelengths in sequence from shortest to longest and click okay.A new image window presenting a confocal reflection microscopy image will appear. Select an arbitrary background region to use for the background subtraction and draw a rectangle within the confocal reflection microscopy image. Double click within the selected region to confirm the selection and locate a new directory named, signature.To perform dimensional reduction techniques, create an empty directory and name the directory, parent_directory. Store the fluorescent signatures of each of the two cell populations into two separate directories and open the command line interface of the workstation. Enter the command.When select target directory is displayed, select the parent_directory. Then in the parent_directory folder, locate the PCA. png file, which will contain the resulting principle component analysis plot.Here, a typical single cell fluorescent signature of a bacterial cell presented as a traditional spectrum plot and as a heat map is shown. In this figure, inaccurate 2D cell segmentation superimposed over the original confocal reflection microscopy image of a population of soil bacteria can be observed. With the resulting innate fluorescent signatures for the population presented as a heat map.Note that the intra population variability was relatively minor following successful cell segmentation. Here, an example of inaccurate cell segmentation is shown super imposed onto the same population of P.putida as previously shown. The impact of the inaccurate cell segmentation on the innate fluorescent signatures of the population is readily apparent from the considerable number of outliers observed in the corresponding heat map.Inaccurate cell segmentation results in a looser cluster after principle component analysis, compared to the type cluster obtained following accurate cell segmentation. Despite the minor variabilities of innate fluorescent signature observed within individual bacterial strains, each population forms a distinct cluster on the principle component analysis plot. In this figure, inaccurate 3D cell segmentation superimposed over the original confocal reflection microscopy image of a population of budding yeast Saccharomyces cerevisiae YM4271, can be observed.Note the lack of outliers and the resulting innate fluorescent signatures for the population. It is essential to acquire as clean as possible, an image by confocal microscopy and to avoid a signal intensity saturation as noise can spoil accuracy of subsequent analysis. You can train machine learning models with the innate Western signature dataset for use in classification or prediction tasks, offering a tag-free population analysis and phenotypic prediction.

Research

JoVE Journal

Biology

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biologia

Time-lapse Imaging of Mitosis After siRNA Transfection

Lapso de tempo de imagens de Mitose Depois de Transfection siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Fluorescência baseada em Medição da loja operada entrada de cálcio em células vivas: a partir de células cancerosas cultivadas em fibra muscular esquelética

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Rastreamento de Stem hematopoiéticas e células progenitoras Marcado por cinco proteínas fluorescentes usando confocal e microscopia multifotônica

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Detecção de activados por ligandos L acoplado a proteína do receptor de internalização por Microscopia Confocal

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Quantificação da Competição Interbacteriana usando imagens de fluorescência unicelular

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Autofluorescence Imaging to Evaluate Red Algae Physiology

Imagem de autofluorescência para avaliar a fisiologia das algas vermelhas

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...

Isolation and Transcriptome Analysis of Plant Cell Types

Isolamento e Análise do Transcriptoma de Tipos Celulares Vegetais

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

Imagem de peróxido mitocondrial em células vivas de levedura usando mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Autor em destaque: Avançando na pesquisa mitocondrial - biossensor mtHyper7 para análise subcelular 

Imagem de mtHyPer7, um biossensor ratiométrico para peróxido mitocondrial, em células vivas de levedura

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...