Background: Super-resolution microscopy techniques are often extremely susceptible to sample drift due to their high spatial resolution and the long time needed for data acquisition. While several techniques for stabilizing against drift exist, many require complicated additional hardware or intrusive sample preparations. We introduce a method that requires no additional sample preparation, is simple to implement and simultaneously corrects for x, y and z drift.
Results: We use bright-field images of the specimen itself to calculate drift in all three dimensions: x, y and z. Bright-field images are acquired on an inexpensive CCD. By correlating each acquired bright-field image with an in-focus and two out-of-focus reference images we determine and actively correct for drift at rates of a few Hertz. This method can maintain stability to within 10 nm for x and y and 20 nm for z over several minutes.
Conclusion: Our active drift stabilization system is capable of simultaneously compensating x, y and z drift through an image-based correlation method that requires no special sample treatment or extensive microscope modifications. While other techniques may provide better stability, especially for higher frequency drift, our method is easy to implement and widely applicable in terms of both sample type and microscopy technique.
{"title":"Active Microscope Stabilization in Three Dimensions Using Image Correlation.","authors":"Ryan McGorty, Daichi Kamiyama, Bo Huang","doi":"10.1186/2192-2853-2-3","DOIUrl":"https://doi.org/10.1186/2192-2853-2-3","url":null,"abstract":"<p><strong>Background: </strong>Super-resolution microscopy techniques are often extremely susceptible to sample drift due to their high spatial resolution and the long time needed for data acquisition. While several techniques for stabilizing against drift exist, many require complicated additional hardware or intrusive sample preparations. We introduce a method that requires no additional sample preparation, is simple to implement and simultaneously corrects for <i>x</i>, <i>y</i> and <i>z</i> drift.</p><p><strong>Results: </strong>We use bright-field images of the specimen itself to calculate drift in all three dimensions: <i>x</i>, <i>y</i> and <i>z</i>. Bright-field images are acquired on an inexpensive CCD. By correlating each acquired bright-field image with an in-focus and two out-of-focus reference images we determine and actively correct for drift at rates of a few Hertz. This method can maintain stability to within 10 nm for <i>x</i> and <i>y</i> and 20 nm for <i>z</i> over several minutes.</p><p><strong>Conclusion: </strong>Our active drift stabilization system is capable of simultaneously compensating <i>x</i>, <i>y</i> and <i>z</i> drift through an image-based correlation method that requires no special sample treatment or extensive microscope modifications. While other techniques may provide better stability, especially for higher frequency drift, our method is easy to implement and widely applicable in terms of both sample type and microscopy technique.</p>","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2013-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-2-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31992115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Maria Elena Gallina, Jianmin Xu, Thomas Dertinger, Adva Aizer, Yaron Shav-Tal, Shimon Weiss
Background: Multi-color super-resolution (SR) imaging microscopy techniques can resolve ultrastructura relationships between- and provide co-localization information of- different proteins inside the cell or even within organelles at a higher resolution than afforded by conventional diffraction-limited imaging. While still very challenging, important SR colocalization results have been reported in recent years using STED, PALM and STORM techniques.
Results: In this work, we demonstrate dual-color Super Resolution Optical Fluctuations Imaging (SOFI) using a standard far-field fluorescence microscope and different color blinking quantum dots. We define the spatial relationship between hDcp1a, a processing body (P-body, PB) protein, and the tubulin cytoskeletal network. Our finding could open up new perspectives on the role of the cytoskeleton in PB formation and assembly. Further insights into PB internal organization are also reported and discussed.
Conclusions: Our results demonstrate the suitability and facile use of multi-color SOFI for the investigation of intracellular ultrastructures.
{"title":"Resolving the spatial relationship between intracellular components by dual color super resolution optical fluctuations imaging (SOFI).","authors":"Maria Elena Gallina, Jianmin Xu, Thomas Dertinger, Adva Aizer, Yaron Shav-Tal, Shimon Weiss","doi":"10.1186/2192-2853-2-2","DOIUrl":"10.1186/2192-2853-2-2","url":null,"abstract":"<p><strong>Background: </strong>Multi-color super-resolution (SR) imaging microscopy techniques can resolve ultrastructura relationships between- and provide co-localization information of- different proteins inside the cell or even within organelles at a higher resolution than afforded by conventional diffraction-limited imaging. While still very challenging, important SR colocalization results have been reported in recent years using STED, PALM and STORM techniques.</p><p><strong>Results: </strong>In this work, we demonstrate dual-color Super Resolution Optical Fluctuations Imaging (SOFI) using a standard far-field fluorescence microscope and different color blinking quantum dots. We define the spatial relationship between hDcp1a, a processing body (P-body, PB) protein, and the tubulin cytoskeletal network. Our finding could open up new perspectives on the role of the cytoskeleton in PB formation and assembly. Further insights into PB internal organization are also reported and discussed.</p><p><strong>Conclusions: </strong>Our results demonstrate the suitability and facile use of multi-color SOFI for the investigation of intracellular ultrastructures.</p>","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"2 ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2013-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855418/pdf/nihms455154.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31943355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
E. Rees, M. Erdélyi, D. Pinotsi, A. Knight, D. Metcalf, C. Kaminski
{"title":"Blind assessment of localisation microscope image resolution","authors":"E. Rees, M. Erdélyi, D. Pinotsi, A. Knight, D. Metcalf, C. Kaminski","doi":"10.1186/2192-2853-1-12","DOIUrl":"https://doi.org/10.1186/2192-2853-1-12","url":null,"abstract":"","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"1 1","pages":"1-10"},"PeriodicalIF":0.0,"publicationDate":"2012-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-1-12","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"66111890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"GraspJ: an open source, real-time analysis package for super-resolution imaging","authors":"Norman Brede, M. Lakadamyali","doi":"10.1186/2192-2853-1-11","DOIUrl":"https://doi.org/10.1186/2192-2853-1-11","url":null,"abstract":"","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"1 1","pages":"1-7"},"PeriodicalIF":0.0,"publicationDate":"2012-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-1-11","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"66111873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Thomas Dertinger, Jianmin Xu, Omeed Foroutan Naini, Robert Vogel, Shimon Weiss
Background: Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, however, remains a challenge as its implementation by existing superresolution methods is non-trivial.
Methods: Here we demonstrate a facile and straightforward 3D superresolution imaging and sectioning of the cytoskeletal network of a fixed cell using superresolution optical fluctuation imaging (SOFI) performed on a conventional lamp-based widefield microscope.
Results and conclusion: SOFI's inherent sectioning capability effectively transforms a conventional widefield microscope into a superresolution 'confocal widefield' microscope.
{"title":"SOFI-based 3D superresolution sectioning with a widefield microscope.","authors":"Thomas Dertinger, Jianmin Xu, Omeed Foroutan Naini, Robert Vogel, Shimon Weiss","doi":"10.1186/2192-2853-1-2","DOIUrl":"https://doi.org/10.1186/2192-2853-1-2","url":null,"abstract":"<p><strong>Background: </strong>Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, however, remains a challenge as its implementation by existing superresolution methods is non-trivial.</p><p><strong>Methods: </strong>Here we demonstrate a facile and straightforward 3D superresolution imaging and sectioning of the cytoskeletal network of a fixed cell using superresolution optical fluctuation imaging (SOFI) performed on a conventional lamp-based widefield microscope.</p><p><strong>Results and conclusion: </strong>SOFI's inherent sectioning capability effectively transforms a conventional widefield microscope into a superresolution 'confocal widefield' microscope.</p>","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"1 2","pages":"2"},"PeriodicalIF":0.0,"publicationDate":"2012-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-1-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40270801","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Bartels, M. Priebe, R. Wilke, S. P. Krüger, K. Giewekemeyer, S. Kalbfleisch, C. Olendrowitz, M. Sprung, T. Salditt
{"title":"Low-dose three-dimensional hard x-ray imaging of bacterial cells","authors":"M. Bartels, M. Priebe, R. Wilke, S. P. Krüger, K. Giewekemeyer, S. Kalbfleisch, C. Olendrowitz, M. Sprung, T. Salditt","doi":"10.1186/2192-2853-1-10","DOIUrl":"https://doi.org/10.1186/2192-2853-1-10","url":null,"abstract":"","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"1 1","pages":"1-7"},"PeriodicalIF":0.0,"publicationDate":"2012-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-1-10","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"66111840","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
C. Wurm, K. Kolmakov, F. Göttfert, Haisen Ta, Mariano Bossi, H. Schill, Sebastian Berning, S. Jakobs, Gerald Donnert, V. Belov, S. Hell
{"title":"Novel red fluorophores with superior performance in STED microscopy","authors":"C. Wurm, K. Kolmakov, F. Göttfert, Haisen Ta, Mariano Bossi, H. Schill, Sebastian Berning, S. Jakobs, Gerald Donnert, V. Belov, S. Hell","doi":"10.1186/2192-2853-1-7","DOIUrl":"https://doi.org/10.1186/2192-2853-1-7","url":null,"abstract":"","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"1 1","pages":"1-7"},"PeriodicalIF":0.0,"publicationDate":"2012-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-1-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"66111715","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Dynamic fluorescence imaging of the free radical products of X-ray absorption in live cells","authors":"C. Rappole, K. Mitra, H. Wen","doi":"10.1186/2192-2853-1-5","DOIUrl":"https://doi.org/10.1186/2192-2853-1-5","url":null,"abstract":"","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"1 1","pages":"1-9"},"PeriodicalIF":0.0,"publicationDate":"2012-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-1-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"66111662","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
B. Harke, J. Chacko, H. Haschke, C. Canale, A. Diaspro
{"title":"A novel nanoscopic tool by combining AFM with STED microscopy","authors":"B. Harke, J. Chacko, H. Haschke, C. Canale, A. Diaspro","doi":"10.1186/2192-2853-1-3","DOIUrl":"https://doi.org/10.1186/2192-2853-1-3","url":null,"abstract":"","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"1 1","pages":"1-6"},"PeriodicalIF":0.0,"publicationDate":"2012-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-1-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"66111918","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
S. Geissbuehler, N. Bocchio, Claudio Dellagiacoma, C. Berclaz, M. Leutenegger, T. Lasser
{"title":"Mapping molecular statistics with balanced super-resolution optical fluctuation imaging (bSOFI)","authors":"S. Geissbuehler, N. Bocchio, Claudio Dellagiacoma, C. Berclaz, M. Leutenegger, T. Lasser","doi":"10.1186/2192-2853-1-4","DOIUrl":"https://doi.org/10.1186/2192-2853-1-4","url":null,"abstract":"","PeriodicalId":90036,"journal":{"name":"Optical nanoscopy","volume":"1 1","pages":"1-7"},"PeriodicalIF":0.0,"publicationDate":"2012-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/2192-2853-1-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"66111576","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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