4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.
<h4>Background</h4>Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing t...
Guardado en:
| Autores principales: | , , , , , , , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Public Library of Science (PLoS)
2011
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/275e5a23c994488d9b10d4bad1c78623 |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| id |
oai:doaj.org-article:275e5a23c994488d9b10d4bad1c78623 |
|---|---|
| record_format |
dspace |
| spelling |
oai:doaj.org-article:275e5a23c994488d9b10d4bad1c786232021-11-18T06:52:57Z4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.1932-620310.1371/journal.pone.0020645https://doaj.org/article/275e5a23c994488d9b10d4bad1c786232011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21655189/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.<h4>Methodology/principal findings</h4>We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.<h4>Conclusions/significance</h4>Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.David BaddeleyDavid CrossmanSabrina RossbergerJuliette E CheyneJohanna M MontgomeryIsuru D JayasingheChristoph CremerMark B CannellChristian SoellerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 5, p e20645 (2011) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
Medicine R Science Q |
| spellingShingle |
Medicine R Science Q David Baddeley David Crossman Sabrina Rossberger Juliette E Cheyne Johanna M Montgomery Isuru D Jayasinghe Christoph Cremer Mark B Cannell Christian Soeller 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. |
| description |
<h4>Background</h4>Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.<h4>Methodology/principal findings</h4>We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.<h4>Conclusions/significance</h4>Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories. |
| format |
article |
| author |
David Baddeley David Crossman Sabrina Rossberger Juliette E Cheyne Johanna M Montgomery Isuru D Jayasinghe Christoph Cremer Mark B Cannell Christian Soeller |
| author_facet |
David Baddeley David Crossman Sabrina Rossberger Juliette E Cheyne Johanna M Montgomery Isuru D Jayasinghe Christoph Cremer Mark B Cannell Christian Soeller |
| author_sort |
David Baddeley |
| title |
4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. |
| title_short |
4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. |
| title_full |
4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. |
| title_fullStr |
4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. |
| title_full_unstemmed |
4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. |
| title_sort |
4d super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2011 |
| url |
https://doaj.org/article/275e5a23c994488d9b10d4bad1c78623 |
| work_keys_str_mv |
AT davidbaddeley 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues AT davidcrossman 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues AT sabrinarossberger 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues AT julietteecheyne 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues AT johannammontgomery 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues AT isurudjayasinghe 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues AT christophcremer 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues AT markbcannell 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues AT christiansoeller 4dsuperresolutionmicroscopywithconventionalfluorophoresandsinglewavelengthexcitationinopticallythickcellsandtissues |
| _version_ |
1718424217032065024 |