INTRODUCTION
RESULTS AND DISCUSSION
Characterization of ImTLSM performance
1 Calibration of ImTLSM illumination profile. A Schematic diagram showing the illumination geometry of ImTLSM. The effective field of view (eFOV) is defined as the range inside which the center focal plane is effectively illuminated by the light sheet (inside the beam waist of light sheet). B Calibration method. To calibrate the light sheet profile, we changed the offset direction of illumination objective such that the light sheet tilted angle was the surplus of θ. Light sheet profile images were taken using EMCCD directly under weak 560 nm laser illumination, with detection objective scan along z-axis (500 nm step size). FWHM and the position of fitting center were calculated from these images. C Positions of fitting centers as a function of the distance from light sheet focus. The estimated focal plane under different iris open conditions was plotted as the zero point. The slopes of fitting curves for each iris diameter were nearly identical, showing approximately 18 degrees of inclination in each case. D FWHM of light sheet as a function of the distance from light sheet focus |
2 Background removal performance of ImTLSM. A Comparison between epi-illumination (Epi) and light-sheet illumination (ImTLSM) at different z-axis positions throughout a cell nucleolus. B Immuno-fluorescence labelled NCL protein distribution. Images were taken under different illumination modes (left: ImTLSM (iris open), right: epi-illumination, scale bar: 2 μm). C Intensity profile from dashed lines on B (orange solid line: ImTLSM, blue solid line: epi-illumination). D Alternatingly collected ImTLSM and epi-illumination images of a HeLa cell expressing YAP–HaloTag with 30 ms time resolution (iris open. Scale bar: 2 μm). E SBR statistics under different imaging modes from D (*** stands for p-value < 0.001) |
Deep learning training performance using high-throughput imaging of ImTLSM
4 Deep learning prediction performance. A Comparison between epi-illumination results, ImTLSM results and deep learning prediction results (scale bar: 1 μm). B Structural similarity (SSIM) index and normalized root mean square error (NRMSE) statistics for Conv-ImTLSM image pairs and Predict-ImTLSM image pairs (***p < 0.001). C Performance of generalization to large field (left: large-field homogeneous illumination image, right: deep learning prediction result). Scale bar: 10 μm for large images and 2 μm for small images |
Application on intra-nuclear super-resolution imaging
5 Performance enhancement in SMLM experiments. A Comparison between large-field homogeneous illumination SMLM image (left, scale bar: 10 μm) and deep learning predicted SMLM image (right). B SBR statistics for high localization precision fittings in A. C Performance comparison between conventional background removal method and PN-ImTLSM method in single molecule identification. Single-molecule localization was performed using ThunderSTORM with peak intensity threshold of 4*std (Wave.F1). Orange circles indicate missing localizations near the edge while blue circles indicate missing localizations in other positions. PN-ImTLSM demonstrates much better performance in areas where background changed rapidly, such as the edges of nucleus or nucleolus (scale bar: 2 μm). D Comparison between the conventional background removal method and PN-ImTLSM background removal method. The threshold in ThunderSTORM was set to 4*std to investigate the image reconstruction performance under high localization precision filtering. The enlarged image areas indicated that PN-ImTLSM background removal method performed well near edges where background level changed rapidly, while ImageJ plugin background removal method failed to remove such uneven background (scale bar: 2 μm for large images and 1 μm for small images) |
DISCUSSION AND CONCLUSION
MATERIALS AND METHODS
Immersion tilted light sheet microscopy (ImTLSM)
6 Principle and mechanical design of ImTLSM. A Schematic diagram showing the illumination mode of ImTLSM. The dashed circle emphasizes the excitation effect under ImTLSM illumination mode. The red dots indicate the fluorophores that were excited while the gray ones were not. B Photograph of the ImTLSM illumination part. C Schematic diagram of ImTLSM illumination principle. The Gaussian beam was formed through a laser combination system and a single-mode fiber, and was pre-collimated by a lens before directed into the illumination system. A cylindrical lens (focus length = 150 mm) was used to form a sheet pattern on the back-focal-plane of the illumination objective, where an adjustable iris was used for adjustment of the final FWHM of light sheet. D Light sheet profile along vertical direction. The image was taken by directing the light sheet imaged on the EMCCD (560 nm, 60× magnification in total) |
Large-field homogeneous illumination
Sample preparation
Cell culture and manipulations for H2B-mMaple3 stable cell line
Immunostaining
YAP-HaloTag labeling
Data acquisition
Calibration of ImTLSM illumination profile
Fluorescence imaging of cellular structures (NCL, Lamin B1, Nup133)
Single-molecule tracking
Collection of deep learning datasets
Large-field homogeneous illumination imaging
SMLM imaging
Modified PReNet structure and training
Data preparation
Model architecture
Optimization
1 Parameters used in Adam optimizer |
Name | Value |
Lr | |
Beta1 | 0.9 |
Beta2 | 0.999 |
Epsilon | |
Batch size | 128 |