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Xu X, Luo Q, Wang JX et al. Large-field objective lens for multi-wavelength microscopy at mesoscale and submicron resolution. Opto-Electron Adv 7, 230212 (2024). doi: 10.29026/oea.2024.230212
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Large-field objective lens for multi-wavelength microscopy at mesoscale and submicron resolution
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Abstract
Conventional microscopes designed for submicron resolution in biological research are hindered by a limited field of view, typically around 1 mm. This restriction poses a challenge when attempting to simultaneously analyze various parts of a sample, such as different brain areas. In addition, conventional objective lenses struggle to perform consistently across the required range of wavelengths for brain imaging in vivo. Here we present a novel mesoscopic objective lens with an impressive field of view of 8 mm, a numerical aperture of 0.5, and a working wavelength range from 400 to 1000 nm. We achieved a resolution of 0.74 μm in fluorescent beads imaging. The versatility of this lens was further demonstrated through high-quality images of mouse brain and kidney sections in a wide-field imaging system, a confocal laser scanning system, and a two-photon imaging system. This mesoscopic objective lens holds immense promise for advancing multi-wavelength imaging of large fields of view at high resolution. -
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References
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Xu X, Luo Q, Wang JX et al. Large-field objective lens for multi-wavelength microscopy at mesoscale and submicron resolution. Opto-Electron Adv 7, 230212 (2024). doi: 10.29026/oea.2024.230212
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Figure 1.
(a) Structure of the objective lens. (b) Aberration coefficients of the three lens groups and the sum of them at the image plane. SA: spherical aberration, AST: astigmatism, FC: field curvature, DIST: distortion, ACA: axial chromatic aberration, LCA: lateral chromatic aberration. (c) The wide-field imaging system. (d) The confocal laser scanning system.
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Figure 2.
(a) Strehl ratio of the lens. The center of the image plane lies at 0 mm, while the edge of the image plane in one direction lies at 16 mm. (b) Field curvature in the tangential plane (solid lines) and sagittal plane (dashed lines) over the entire image field at different wavelengths. (c) Distortion across the image field. (d) Focal shifts at different wavelengths. Data at (a)-(d) are obtained from the ray-tracing software (Opticstudio, Zemax). (e) Wide-field transmission imaging of a USAF 1951 target. (f) Zoomed-in of a custom-built negative resolution target. (g) Intensity spreading of red lines in panel (f). (h) Intensity spreading of blue lines in group 2 / element 1 in panel (e). (i) Imaging of a grid test target. (j) Intensity spreading of blue lines at the margin of the nominal FOV. (k) Intensity spreading of green lines in the center of the nominal FOV.
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Figure 3.
(a) Imaging of beads with a diameter of 500 nm across the entire FOV. (b) The FWHM results of microspheres in the central imaging field for the three objectives. (c) The FWHM results of microspheres in the right field area for each objective. (d) The FWHM results of microspheres in the left field area for each objective. (e) The FWHM results of microspheres in the upper field area for each objective. (f) The imaging results of microspheres in the lower field area for each objective. (g) The FWHM results for microspheres fluorescing at different wavelengths using our objective lens. (h) The FWHM results for microspheres fluorescing at different wavelengths using the 20× 0.5 NA objective. (i) The FWHM results for microspheres fluorescing at different wavelengths using the 4× 0.16 NA objectives. (j) The FWHM of microspheres in different field areas for the three objectives (mean ± SEM, n = 7 beads), with our lens achieving 0.74 ± 0.02 μm, 0.79 ± 0.042 μm, 0.79 ± 0.055 μm, 0.8 ± 0.036 μm, 0.81 ± 0.051 μm. The 20× 0.5 NA objective results were 0.69 ± 0.021 μm, 0.7 ± 0.018 μm, 0.71 ± 0.033 μm, 0.73 ± 0.036 μm, 0.75 ± 0.034 μm. The 4× 0.16 NA objective results were 2.1 ± 0.033 μm, 2.2 ± 0.032 μm, 2.3 ± 0.041 μm, 2.4 ± 0.037 μm, 2.3 ± 0.047 μm. (k) The FWHM results of microspheres at different wavelengths (mean ± SEM, n = 7 beads) for each objective. At 450 nm, 540 nm, 680 nm, and 800 nm imaging, our lens achieved 0.71 ± 0.032 μm, 0.79 ± 0.045 μm, 0.92 ± 0.048 μm, 1.21 ± 0.054 μm. The 20× 0.5 NA objective results were 0.68 ± 0.027 μm, 0.74 ± 0.032 μm, 0.91 ± 0.034 μm, 1.12 ± 0.051 μm. The 4× 0.16 NA objective results were 1.8 ± 0.038 μm, 2.2 ± 0.059 μm, 2.7 ± 0.036 μm, 3.1 ± 0.061 μm.
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Figure 4.
Wide-field imaging of mouse brain and kidney. (a) Brightfield imaging of mouse brain slice with our objective lens. Scale bar, 1.5 mm. Though the effective FOV with aberration optimization is 8 mm, FOVs slightly larger than 8 mm can also be imaged. Thus, the entire FOV is slightly larger than the nominal diameter of 8 mm. (b) Zoomed-in view of the yellow rectangle part of the panel (a). (c) Zoomed-in view of the green rectangle part of the panel (a). (d) Fluorescence imaging of a mouse kidney slice after simultaneous labeling with Alexa Fluor 488, Alexa Fluor 568, and DAPI, showing Alexa Fluor 488 labeling (excitation wavelength, 490 nm; emission wavelength, 515 nm). (e) Zoomed-in view of the red rectangle part of the panel (d), showing DAPI labeling (excitation, 380 nm; emission, 455 nm). (f) The same image zone as in panel (e), showing Alexa Fluor 488 labeling. (g) The same image zone as in panel (e), showing Alexa Fluor 568 labeling (excitation, 540 nm; emission, 600 nm).
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Figure 5.
Wide-field imaging comparison between our objective and commercial objectives. (a) Brightfield imaging of a mouse brain slice with our objective lens at a nominal FOV. (b) Brightfield imaging of a mouse brain slice with a commercial 20× 0.5 NA objective lens (UPLFLN20X, Olympus) at nominal FOV of about 1.2 mm diameter. (c) Brightfield imaging of a mouse brain slice with a commercial 4× 0.16 NA objective lens (UPLANSAPO4X, Olympus) at a nominal FOV of about 6 mm diameter. (d) Zoomed-in view of part of the panel (a). (e) Zoomed-in view of part of the panel (b). (f) Zoomed-in view of part of the panel (c). Scale bar: 30 μm in panels (d–f). (g) Fluorescence imaging of a mouse kidney slice with our lens at nominal FOV, showing Alexa Fluor 488 labeling. (h) Fluorescence imaging of mouse kidney slice with a commercial 20× 0.5 NA objective at nominal FOV. (i) Fluorescence imaging of a mouse kidney slice with a commercial 4× 0.16 NA objective at nominal FOV. (j) Zoomed-in view of part of the panel (g). (k) Zoomed-in view of part of the panel (h). (l) Zoomed-in view of part of the panel (i). Scale bar: 10 μm in panels (j–l). (m) The results of image clarity analysis for (d–f). (n) The results of image clarity analysis for (j–l).
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Figure 6.
Confocal laser scanning imaging results. (a) Imaging of mouse kidney slice. (b) Imaging of BPAE cells slice. The whole field images in (a) and (b) were scanned by all 3 scanners, these images are 5400 × 7000 pixels, and the pixel sizes are 1.1 μm × 1 μm. The zoomed-in images were scanned by resonant-x and galvo-Y scanners, they are 800 × 1000 pixels. The pixel size of the first zoomed-in image is 0.9 μm × 0.9 μm, and the further zoomed-in ones are 0.23 μm × 0.23 μm. (c) Scanning pattern of whole field imaging in (a) and (b). Resonant-x scans at 12 kHz. Galvo-Y scans at 2 Hz, and one cycle contains 6000 resonant-x cycles, and 10% of these cycles are at the scan-back duration of galvo-Y. Therefore, only 5400 resonant-x cycles are effective for imaging. One galvo-X scan cycle has only 7 positions and contains 7 galvo-Y cycles.
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Figure 7.
(a) The two-photon imaging results of a mouse kidney slice when excited with a 920 nm femtosecond laser using our objective lens. (b) The single-photon imaging results of a mouse kidney slice under the same system, except using a 488 mm continuous-wave laser for excitation. The whole field images in (a) and (b) are 5400 × 1000 pixels, with pixel sizes of 1.1 μm × 1 μm. The zoomed-in images are 900 × 1000 pixels, with pixel sizes of 0.36 μm × 0.36 μm. (c) The intensity distribution curves of the same glomerular region in both imaging results.
