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Microscopy|2 Article(s)
Woofer– tweeter adaptive optical structured illumination microscopy
Qinggele Li, Marc Reinig, Daich Kamiyama, Bo Huang, Xiaodong Tao, Alex Bardales, and Joel Kubby
A woofer–tweeter adaptive optical structured illumination microscope (AOSIM) is presented. By combining a low-spatial-frequency large-stroke deformable mirror (woofer) with a high-spatial-frequency low-stroke deformable mirror (tweeter), we are able to remove both large-amplitude and high-order aberrations. In addition, using the structured illumination method, as compared to widefield microscopy, the AOSIM can accomplish high-resolution imaging and possesses better sectioning capability. The AOSIM was tested by correcting a large aberration from a trial lens in the conjugate plane of the microscope objective aperture. The experimental results show that the AOSIM has a point spread function with an FWHM that is 140 nm wide (using a water immersion objective lens with NA=1.1) after correcting a large aberration (5.9 μm peak-to-valley wavefront error with 2.05 μm RMS aberration). After structured light illumination is applied, the results show that we are able to resolve two beads that are separated by 145 nm, 1.62× below the diffraction limit of 235 nm. Furthermore, we demonstrate the application of the AOSIM in the field of bioimaging. The sample under investigation was a green-fluorescent-protein-labeled Drosophila embryo. The aberrations from the refractive index mismatch between the microscope objective, the immersion fluid, the cover slip, and the sample itself are well corrected. Using AOSIM we were able to increase the SNR for our Drosophila embryo sample by 5×. A woofer–tweeter adaptive optical structured illumination microscope (AOSIM) is presented. By combining a low-spatial-frequency large-stroke deformable mirror (woofer) with a high-spatial-frequency low-stroke deformable mirror (tweeter), we are able to remove both large-amplitude and high-order aberrations. In addition, using the structured illumination method, as compared to widefield microscopy, the AOSIM can accomplish high-resolution imaging and possesses better sectioning capability. The AOSIM was tested by correcting a large aberration from a trial lens in the conjugate plane of the microscope objective aperture. The experimental results show that the AOSIM has a point spread function with an FWHM that is 140 nm wide (using a water immersion objective lens with NA=1.1) after correcting a large aberration (5.9 μm peak-to-valley wavefront error with 2.05 μm RMS aberration). After structured light illumination is applied, the results show that we are able to resolve two beads that are separated by 145 nm, 1.62× below the diffraction limit of 235 nm. Furthermore, we demonstrate the application of the AOSIM in the field of bioimaging. The sample under investigation was a green-fluorescent-protein-labeled Drosophila embryo. The aberrations from the refractive index mismatch between the microscope objective, the immersion fluid, the cover slip, and the sample itself are well corrected. Using AOSIM we were able to increase the SNR for our Drosophila embryo sample by 5×.
Photonics Research
- Publication Date: Jun. 29, 2017
- Vol. 5, Issue 4, 04000329 (2017)
Deep-UV fluorescence lifetime imaging microscopy
Christiaan J. de Jong, Alireza Lajevardipour, Mindaugas Gecevi?ius, Martynas Beresna, Gediminas Gervinskas, Peter G. Kazansky, Yves Bellouard, Andrew H. A. Clayton, and Saulius Juodkazis
A novel fluorescence lifetime imaging microscopy (FLIM) working with deep UV 240–280 nm wavelength excitations has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon annealing from femtosecond laser-induced modifications in fused silica. This FLIM technique can be used with microfluidic and biosamples to characterize temporal characteristics of fluorescence upon UV excitation, a capability easily added to a standardmicroscope-based FLIM. UV-FLIMwas tested to show annealing of the defects induced by silica structuringwith ultrashort laser pulses. Frequency-domain fluorescencemeasurementswere converted into the time domain to extract long fluorescence lifetimes from defects in silica. A novel fluorescence lifetime imaging microscopy (FLIM) working with deep UV 240–280 nm wavelength excitations has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon annealing from femtosecond laser-induced modifications in fused silica. This FLIM technique can be used with microfluidic and biosamples to characterize temporal characteristics of fluorescence upon UV excitation, a capability easily added to a standardmicroscope-based FLIM. UV-FLIMwas tested to show annealing of the defects induced by silica structuringwith ultrashort laser pulses. Frequency-domain fluorescencemeasurementswere converted into the time domain to extract long fluorescence lifetimes from defects in silica.
Photonics Research
- Publication Date: Sep. 28, 2015
- Vol. 3, Issue 5, 05000283 (2015)
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