Aberration correction in stimulated emission depletion microscopy to increase imaging depth in living brain tissue
Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberratio...
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| Vydané v: | Neurophotonics (Print) Ročník 8; číslo 3; s. 035001 |
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| Hlavní autori: | , , , |
| Médium: | Journal Article |
| Jazyk: | English |
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Bellingham
Society of Photo-Optical Instrumentation Engineers
01.07.2021
S P I E - International Society for Society of Photo-optical Instrumentation Engineers (SPIE) |
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| ISSN: | 2329-423X, 2329-4248 |
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| Abstract | Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue.
Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth.
Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices.
Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of 90 μm inside the biological tissue and obtain a 60% signal increase after correction.
Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue. |
|---|---|
| AbstractList | Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue. Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth. Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices. Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of 90 μ m inside the biological tissue and obtain a 60% signal increase after correction. Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue.Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue. Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth. Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices. Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of 90 μ m inside the biological tissue and obtain a 60% signal increase after correction. Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue. Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue. Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth. Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices. Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of 90 μm inside the biological tissue and obtain a 60% signal increase after correction. Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue. Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue.Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth.Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices.Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of 90 μm inside the biological tissue and obtain a 60% signal increase after correction.Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue. Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue. Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth. Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices. Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of 90 μm inside the biological tissue and obtain a 60% signal increase after correction. Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue. We demonstrate an approach based on adaptive optics to improve the spatial resolution of STED microscopy inside thick biological tissue by a priori correction of spherical aberrations as a function of imaging depth. We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. Using a spatial light modulator to apply corrective phase shifts, we imaged neurons in living brain slices and show that the corrections can substantially increase image quality. Specifically, we could measure structures as small as 80 nm at a depth of 90 μ m inside the biological tissue, and obtain a 60% signal increase after correction. |
| Author | Mercier, Luc Murana, Emanuele Bancelin, Stéphane Nägerl, U. Valentin |
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| Snippet | Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that... We demonstrate an approach based on adaptive optics to improve the spatial resolution of STED microscopy inside thick biological tissue by a priori correction... |
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| Title | Aberration correction in stimulated emission depletion microscopy to increase imaging depth in living brain tissue |
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