Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.
Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point...
Saved in:
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2012-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043942&type=printable |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849726050590785536 |
|---|---|
| author | Franck P Martial Nicholas A Hartell |
| author_facet | Franck P Martial Nicholas A Hartell |
| author_sort | Franck P Martial |
| collection | DOAJ |
| description | Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor. |
| format | Article |
| id | doaj-art-a784e54705f14536876ac79971e36621 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-a784e54705f14536876ac79971e366212025-08-20T03:10:18ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0178e4394210.1371/journal.pone.0043942Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.Franck P MartialNicholas A HartellConfocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043942&type=printable |
| spellingShingle | Franck P Martial Nicholas A Hartell Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror. PLoS ONE |
| title | Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror. |
| title_full | Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror. |
| title_fullStr | Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror. |
| title_full_unstemmed | Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror. |
| title_short | Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror. |
| title_sort | programmable illumination and high speed multi wavelength confocal microscopy using a digital micromirror |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043942&type=printable |
| work_keys_str_mv | AT franckpmartial programmableilluminationandhighspeedmultiwavelengthconfocalmicroscopyusingadigitalmicromirror AT nicholasahartell programmableilluminationandhighspeedmultiwavelengthconfocalmicroscopyusingadigitalmicromirror |