An official website of the United States government
Single-shot 3D optical microscopy that can capture high-resolution information over a large volume has broad applications in biology. Existing 3D imaging methods using point-spread-function (PSF) engineering often have limited depth of field (DOF) or require custom and often complex design of phase masks. We propose a new, to the best of our knowledge, PSF approach that is easy to implement and offers a large DOF. The PSF appears to be axially V-shaped, engineered by replacing the conventional tube lens with a pair of axicon lenses behind the objective lens of a wide-field microscope. The 3D information can be reconstructed from a single-shot image using a deep neural network. Simulations in a 10× magnification wide-field microscope show the V-shaped PSF offers excellent 3D resolution (<2.5 µm lateral and ∼15 µm axial) over a ∼350 µm DOF at a 550 nm wavelength. Compared to other popular PSFs designed for 3D imaging, the V-shaped PSF is simple to deploy and provides high 3D reconstruction quality over an extended DOF.
more »
« less
Towards optimal point spread function design for resolving closely spaced emitters in three dimensions
The past decade has brought many innovations in optical design for 3D super-resolution imaging of point-like emitters, but these methods often focus on single-emitter localization precision as a performance metric. Here, we propose a simple heuristic for designing a point spread function (PSF) that allows for precise measurement of the distance between two emitters. We discover that there are two types of PSFs that achieve high performance for resolving emitters in 3D, as quantified by the Cramér-Rao bounds for estimating the separation between two closely spaced emitters. One PSF is very similar to the existing Tetrapod PSFs; the other is a rotating single-spot PSF, which we call the crescent PSF. The latter exhibits excellent performance for localizing single emitters throughout a 1-µm focal volume (localization precisions of 7.3 nm inx, 7.7 nm iny, and 18.3 nm inzusing 1000 detected photons), and it distinguishes between one and two closely spaced emitters with superior accuracy (25-53% lower error rates than the best-performing Tetrapod PSF, averaged throughout a 1-µm focal volume). Our study provides additional insights into optimal strategies for encoding 3D spatial information into optical PSFs.
more »
« less
- Award ID(s):
- 1653777
- PAR ID:
- 10372096
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Optics Express
- Volume:
- 30
- Issue:
- 20
- ISSN:
- 1094-4087; OPEXFF
- Format(s):
- Medium: X Size: Article No. 37154
- Size(s):
- Article No. 37154
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Simultaneous measurements of single-molecule positions and orientations provide critical insight into a variety of biological and chemical processes. Various engineered point spread functions (PSFs) have been introduced for measuring the orientation and rotational diffusion of dipole-like emitters, but the widely used Cramér-Rao bound (CRB) only evaluates performance for one specific orientation at a time. Here, we report a performance metric, termed variance upper bound (VUB), that yields a global maximum CRB for all possible molecular orientations, thereby enabling the measurement performance of any PSF to be computed efficiently ( faster than calculating average CRB). Our VUB reveals that the simple polarized standard PSF provides robust and precise orientation measurements if emitters are near a refractive index interface. Using this PSF, we measure the orientations and positions of Nile red (NR) molecules transiently bound to amyloid aggregates. Our super-resolved images reveal the main binding mode of NR on amyloid fiber surfaces, as well as structural heterogeneities along amyloid fibrillar networks, that cannot be resolved by single-molecule localization alone.more » « less
-
Within the family of super-resolution (SR) fluorescence microscopy, single-molecule localization microscopies (PALM[1], STORM[2] and their derivatives) afford among the highest spatial resolution (approximately 5 to 10 nm), but often with moderate temporal resolution. The high spatial resolution relies on the adequate accumulation of precise localizations, which requires a relatively low density of bright fluorophores. Several methods have demonstrated localization at higher densities in both two dimensions (2D)[3, 4] and three dimensions (3D)[5-7]. Additionally, with further advancements, such as functional super-resolution[8, 9] and point spread function (PSF) engineering with[8-11] or without[12] multi-channel observations, extra information (spectra, dipole orientation) can be encoded and recovered at the single molecule level. However, such advancements are not fully extended for high-density conditions in 3D. In this work, we adopt sparse recovery using simple matrix/vector operations, and propose a systematic progressive refinement method (dubbed as PRIS) for 3D high-density condition. We also generalized the method for PSF engineering, multichannel and multi-species observations using different forms of matrix concatenations. Specifically, we demonstrate reconstructions with both double-helix and astigmatic PSFs, for both single and biplane settings. We also demonstrate the recovery capability for a mixture of two different color species.more » « less
-
null (Ed.)Lensless imaging is a new, emerging modality where image sensors utilize optical elements in front of the sensor to perform multiplexed imaging. There have been several recent papers to reconstruct images from lensless imagers, including methods that utilize deep learning for state-of-the-art performance. However, many of these methods require explicit knowledge of the optical element, such as the point spread function, or learn the reconstruction mapping for a single fixed PSF. In this paper, we explore a neural network architecture that performs joint image reconstruction and PSF estimation to robustly recover images captured with multiple PSFs from different cameras. Using adversarial learning, this approach achieves improved reconstruction results that do not require explicit knowledge of the PSF at test-time and shows an added improvement in the reconstruction model’s ability to generalize to variations in the camera’s PSF. This allows lensless cameras to be utilized in a wider range of applications that require multiple cameras without the need to explicitly train a separate model for each new camera.more » « less
-
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.more » « less
