We have designed and implemented an approach for three-dimensional (3D) structured illumination (SI) microscopy (SIM) based on a quasi-monochromatic extended source illuminating a Wollaston prism to improve robustness, light efficiency and flexibility over our previous design. We show through analytical and experimental verification of the presented theoretical framework for our proposed tunable structured illumination microscopy (TSIM) system, that a simple and accurate determination of the axial modulation of the SI pattern is achieved, enabling a realistic characterization of the system’s effective optical transfer function (OTF). System performance as a function of the extended source size is investigated with simulations. Results from a comparative performance analysis of the proposed TSIM system and traditional SIM systems show some advantages over the traditional two-wave and three-wave interference SIM systems. We show that by controlling the source size and thereby the axial modulation of the 3D SI pattern, the TSIM scheme offers increased OTF compact support and improved optical sectioning capability, quantified by the integrated intensity, under certain conditions, which may be desirable when imaging optically thick samples. The additional tunability of the 3D SI pattern, provides a unique opportunity for OTF engineering in our TSIM system.
Optical sectioning structured illumination microscopy (OS-SIM) provides optical sectioning capability in wide-field microscopy. The required illumination patterns have traditionally been generated using spatial light modulators (SLM), laser interference patterns, or digital micromirror devices (DMDs) which are too complex to implement in miniscope systems. MicroLEDs have emerged as an alternative light source for patterned illumination due to their extreme brightness capability and small emitter sizes. This paper presents a directly addressable striped microLED microdisplay with 100 rows on a flexible cable (70 cm long) for use as an OS-SIM light source in a benchtop setup. The overall design of the microdisplay is described in detail with luminance-current-voltage characterization. OS-SIM implementation with a benchtop setup shows the optical sectioning capability of the system by imaging within a 500 µm thick fixed brain slice from a transgenic mouse where oligodendrocytes are labeled with a green fluorescent protein (GFP). Results show improved contrast in reconstructed optically sectioned images of 86.92% (OS-SIM) compared with 44.31% (pseudo-widefield). MicroLED based OS-SIM therefore offers a new capability for deep tissue widefield imaging.
more » « less- Award ID(s):
- 1926747
- NSF-PAR ID:
- 10411418
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Optics Express
- Volume:
- 31
- Issue:
- 10
- ISSN:
- 1094-4087; OPEXFF
- Page Range / eLocation ID:
- Article No. 16709
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Background Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.more » « less
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Summary Structured‐illumination microscopy allows widefield fluorescence imaging with resolution beyond the classical diffraction limit. Its linear form extends resolution by a factor of two, and its nonlinear form by an in‐principle infinite factor, the effective resolution in practice being determined by noise. In this paper, we analyse the noise properties and achievable resolution of linear and nonlinear 1D and 2D patterned SIM from a frequency–space perspective. We develop an analytical theory for a general case of linear or nonlinear fluorescent imaging, and verify the analytical calculations with numerical simulation for a special case where nonlinearity is produced by photoswitching of fluorescent labels. We compare the performance of two alternative implementations, using either two‐dimensional (2D) illumination patterns or sequentially rotated one‐dimensional (ID) patterns. We show that 1D patterns are advantageous in the linear case, and that in the nonlinear case 2D patterns provide a slight signal‐to‐noise advantage under idealised conditions, but perform worse than 1D patterns in the presence of nonswitchable fluorescent background.
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