Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin – a protein located in the focal adhesion complex – and actin in human osteosarcoma cells.
- Award ID(s):
- 1847899
- NSF-PAR ID:
- 10319231
- Date Published:
- Journal Name:
- Entropy
- Volume:
- 23
- Issue:
- 5
- ISSN:
- 1099-4300
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
-
The main challenge of monocular 3D object detection is the accurate localization of 3D center. Motivated by a new and strong observation that this challenge can be remedied by a 3D-space local-grid search scheme in an ideal case, we propose a stage-wise approach, which combines the information flow from 2D-to-3D (3D bounding box proposal generation with a single 2D image) and 3D-to-2D (proposal verification by denoising with 3D-to-2D contexts) in a topdown manner. Specifically, we first obtain initial proposals from off-the-shelf backbone monocular 3D detectors. Then, we generate a 3D anchor space by local-grid sampling from the initial proposals. Finally, we perform 3D bounding box denoising at the 3D-to-2D proposal verification stage. To effectively learn discriminative features for denoising highly overlapped proposals, this paper presents a method of using the Perceiver I/O model [20] to fuse the 3D-to-2D geometric information and the 2D appearance information. With the encoded latent representation of a proposal, the verification head is implemented with a self-attention module. Our method, named as MonoXiver, is generic and can be easily adapted to any backbone monocular 3D detectors. Experimental results on the well-established KITTI dataset and the challenging large-scale Waymo dataset show that MonoXiver consistently achieves improvement with limited computation overhead.more » « less
-
By manipulating the spectral dispersion of detected photons, spectroscopic single-molecule localization microscopy (sSMLM) permits concurrent high-throughput single-molecular spectroscopic analysis and imaging. Despite its promising potential, using discrete optical components and managing the delicate balance between spectral dispersion and spatial localization compromise its performance, including non-uniform spectral dispersion, high transmission loss of grating, high optical alignment demands, and reduced precision. We designed a dual-wedge prism (DWP)-based monolithic imaging spectrometer to overcome these challenges. We optimized the DWP for spectrally dispersing focused beam without deviation and with minimal wavefront error. We integrated all components into a compact assembly, minimizing total transmission loss and significantly reducing optical alignment requirements. We show the feasibility of DWP using ray-tracing and numerical simulations. We validated our numerical simulations by experimentally imaging individual nanospheres and confirmed that DWP-sSMLM achieved much improved spatial and spectral precisions of grating-based sSMLM. We also demonstrated DWP-sSMLM in 3D multi-color imaging of cells.more » « less
-
more » « less
Abstract Spectroscopic single-molecule localization microscopy (sSMLM) was used to achieve simultaneous imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from a reduced photon budget because the photons from individual stochastic emissions are divided into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically dispersed sSMLM, or SDsSMLM, to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Compared with the existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multicolour imaging of fixed cells and three-dimensional single-particle tracking using SDsSMLM. SDsSMLM enables more precise spectroscopic single-molecule analysis in broader cell biology and material science applications.
-
more » « less
Summary Real‐time tracking of multiple particles is key for quantitative analysis of dynamic biophysical processes and materials science via time‐lapse microscopy image data, especially for single molecule biophysical techniques, such as magnetic tweezers and centrifugal force microscopy. However, real‐time multiple particle tracking with high resolution is limited by the current imaging processes or tracking algorithms. Here, we demonstrate 1 nm resolution in three dimensions in real‐time with a graphics‐processing unit (GPU) based on a compute unified device architecture (CUDA) parallel computing framework instead of only a central processing unit (CPU). We also explore the trade‐offs between processing speed and size of the utilized regions of interest and a maximum speedup of 137 is achieved with the GPU compared with the CPU. Moreover, we utilize this method with our recently self‐built centrifugal force microscope (CFM) in experiments that track multiple DNA‐tethered particles. Our approach paves the way for high‐throughput single molecule techniques with high resolution and efficiency.
Lay Description Particles are widely used as probes in life sciences through their motions. In single molecule techniques such as optical tweezers and magnetic tweezers, microbeads are used to study intermolecular or intramolecular interactions via beads tracking. Also tracking multiple beads’ motions could study cell–cell or cell–ECM interactions in traction force microscopy. Therefore, particle tracking is of key important during these researches. However, parallel 3D multiple particle tracking in real‐time with high resolution is a challenge either due to the algorithm or the program. Here, we combine the performance of CPU and CUDA‐based GPU to make a hybrid implementation for particle tracking. In this way, a speedup of 137 is obtained compared the program before only with CPU without loss of accuracy. Moreover, we improve and build a new centrifugal force microscope for multiple single molecule force spectroscopy research in parallel. Then we employed our program into centrifugal force microscope for DNA stretching study. Our results not only demonstrate the application of this program in single molecule techniques, also indicate the capability of multiple single molecule study with centrifugal force microscopy.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/e23050498
