Volumetric structured illumination microscopy enabled by tunable focus lens (Conference Presentation)

Structured illumination microscopy (SIM) is a widely adopted super-resolution imaging technique. Conventional 3D-SIM requires at least 15 exposures at each z-plane to achieve ∼2 × improved lateral and axial resolution. However, this requirement for a large number of exposures for “super-resolution” exacerbates photobleaching and slows imaging speed, thus significantly limiting its application in volumetric biological imaging. Here, we introduce multi-scan SIM (MS-SIM) that integrates a simple beam splitter for simultaneously imaging three different focal planes and a deformable mirror that enables rapid z-scanning over three contiguous sub-volumes. We demonstrate the MS-SIM system through high-quality live whole cell SIM imaging at ∼1 Hz. The high efficiency and flexibility of MS-SIM can significantly impact 3D super-resolution imaging of biological and dense colloidal systems.

We present the use of a commercially available electrically tunable lens to achieve axial scanning in a reflectance confocal microscope. Over a 255 μm axial scan range, the lateral and axial resolutions varied from 1-2 μm and 4-14 μm, respectively, dependent on the variable focal length of the tunable lens. Confocal imaging was performed on normal human biopsies from the oral cavity ex vivo. Sub-cellular morphologic features were seen throughout the depth of the epithelium while axially scanning using the focus tunable lens.

Structured illumination microscopy (SIM) is a valuable tool for three-dimensional microscopy and has numerous applications in bioscience. Its success has been limited to static objects, though, as three sequential image acquisitions are required per final processed, focused image. To overcome this problem we have developed a multicolored grid which when used in tandem with a color camera is capable of performing SIM with just a single exposure. Images and movies demonstrating optical sectioning of three-dimensional objects are presented, and results of applying color SIM for wide-field focused imaging are compared to those of SIM. From computer modeling and analytical calculations a theoretical estimate of the maximum observable object velocity in both the lateral and axial directions is available, implying that the new method will be capable of imaging a variety of live objects. Sample images of the technique applied to lens paper and a pigeon feather are included to show both advantages and disadvantages of CSIM.

.Significance: Confocal laser scanning enables optical sectioning in clinical fiber bundle endomicroscopes, but lower-cost, simplified endomicroscopes use widefield incoherent illumination instead. Optical sectioning can be introduced in these simple systems using structured illumination microscopy (SIM), a multiframe digital subtraction process. However, SIM results in artifacts when the probe is in motion, making the technique difficult to use in vivo and preventing the use of mosaicking to synthesize a larger effective field of view (FOV).Aim: We report and validate an automatic motion compensation technique to overcome motion artifacts and allow generation of mosaics in SIM endomicroscopy.Approach: Motion compensation is achieved using image registration and real-time pattern orientation correction via a digital micromirror device. We quantify the similarity of moving probe reconstructions to those acquired with a stationary probe using the relative mean of the absolute differences (MAD). We further demonstrate mosaicking with a moving probe in mechanical and freehand operation.Results: Reconstructed SIM images show an improvement in the MAD from 0.85 to 0.13 for lens paper and from 0.27 to 0.12 for bovine tissue. Mosaics also show vastly reduced artifacts.Conclusion: The reduction in motion artifacts in individual SIM reconstructions leads to mosaics that more faithfully represent the morphology of tissue, giving clinicians a larger effective FOV than the probe itself can provide.

Improved extended depth of focus full field spectral domain Optical Coherence Tomography

Structured illumination microscopy (SIM) is a super-resolution fluorescence microscopy with a 2-fold higher lateral resolution than conventional wide-field fluorescence (WF) microscopy. Confocal fluorescence (CF) microscopy has approximately the same optical cutoff frequency as SIM; however, the maximum theoretical increase in lateral resolution over that of WF is 1.4-fold with an infinitesimal pinhole diameter. Quantitative comparisons based on an analytical imaging formula revealed that modulation transfer functions (MTFs) of SIM reconstructed images before postprocessing are nearly identical to those of CF images recorded with an infinitesimal pinhole diameter. Here, we propose a new method using an adequate pinhole diameter combined with the use of an apodized Fourier inverse filter to increase the lateral resolution of CF images to as much as that SIM images without significant noise degradation in practice. Furthermore, the proposed method does not require a posteriori parameterization and has reproducibility. This approach can be easily applied to conventional laser scanning CF, spinning disk CF, and multiphoton microscopies.

Biological samples are three dimensional and, therefore, optical sectioning is mandatory for microscopic images to precisely show the localization or function of structures within biological samples. Today, researchers can choose from a variety of methods to obtain optical sections. This article focuses on structured illumination microscopy, which is a group of techniques utilizing a combination of optics and mathematics to obtain optical sections: A structure is imaged onto the sample by optical means and the additional information thereby encoded in the image is used to calculate an optical section from several acquired images. Different methods of structured illumination microscopy (mainly grid projection and aperture correlation) are discussed from a practical point of view, concentrating on advantages, limitations and future prospects of these techniques and their use in cell biology. Structured illumination can also be used to obtain super-resolution information if structures of higher frequency are projected onto the sample. This promising approach to super-resolution microscopy is also briefly discussed from a user's perspective.

Optical manipulation and detection of biological particulates are crucial procedures in biophotonics. Optical sectioning (OS) opens the avenue to three-dimensional (3D) microscopy, but nonoptical approaches, including sample adhesion and mechanical scanning, have always been required in this technique, rendering it impossible to image suspended cells. Here, we develop optical tweeze-sectioning microscopy by coupling structured illumination microscopy (SIM) with holographic optical tweezers (HOTs). By sculpting light in HOTs, we demonstrate that the position fluctuations of suspended yeast cells can be optically squeezed to tens of nanometers, which is sufficient for the implementation of OS with SIM. Sample scanning is achieved through optical delivery of the cells, instead of translation stages as in the conventional way. Our work presents an all-optical solution for OS, broadening its application to nonadherent, suspended cells. It further furnishes the original technique that enables both SIM-based 3D imaging and optical manipulation.

Current model-based deconvolution methods for three-dimensional structured illumination microscopy (3D-SIM) assume that the observed image can be modeled as the convolution of the fluorescence emission with the detection point spread function. However, such a model is not suitable when 3D data is acquired using axial scanning of the sample as in the case of commercial microscopes, because the structured illumination (SI) pattern is changing axially. Here, we implement and test an iterative deconvolution approach based on a 3D model that takes into account data-acquisition with axial scanning, by minimizing the mean squared error between the data and the model using a conjugate-gradient method. This method is applicable for 3D-SIM systems in which the SI pattern is separable into axial and lateral functions. Numerical results show improvement in the restoration when a model mismatch is avoided.

A new organ-based tube current modulation (NOB-TCM) method was designed with the intent to decrease tube current by 30% over a prescribed 90° radial arc across the anterior aspect of the radiosensitive organ, without increasing tube current in the remaining radial arc. We compared a reference scan and five other dose-reducing methods with regard to effects on dose, practicality, and image quality to determine the most effective method for the reduction of the radiation dose to the eyes during CT examinations of the head. We compared the radiation doses to the eyes and physical image quality in different regions of interest for TCM and shielding scans. Three types of TCM scans were performed: longitudinal TCM, angular TCM, and NOB-TCM. A bismuth sheet and lead goggles were each applied for the shielding scan. Relative to the reference scan, the dose to the eye was reduced to 25.88% with NOB-TCM, 44.53% with lead goggles, and 36.91% with a bismuth shield. Relative to the reference scan, the mean signal-to-noise ratio (SNR) was decreased to 8.02% with NOB-TCM, 28.36% with lead goggles, and 32.95% with the bismuth shield. The SNR of the anterior region of interest was decreased to 11.89% with NOB-TCM and 87.89% with the bismuth shield. The average figure of merit was increased by 11.7% with longitudinal TCM and 13.39% with NOB-TCM, compared with the reference scan. NOB-TCM is a superior solution for head CT, including the orbital area, due to the reduction in radiation exposure without significant loss in image quality.

The performance of a tunable three-dimensional (3D) structured illumination microscope (SIM) system and its ability to provide simultaneously super-resolution (SR) and optical-sectioning (OS) capabilities are investigated. Numerical results show that the performance of our 3D-SIM system is comparable with the one provided by a three-wave interference SIM, while requiring 40% fewer images for the reconstruction and providing frequency tunability in a cost-effective implementation. The performance of the system has been validated experimentally with images from test samples, which were also imaged with a commercial SIM based on incoherent-grid projection for comparison. Restored images from data acquired from an axially-thin fluorescent layer show a 1.6× improvement in OS capability compared to the commercial instrument while results from a fluorescent tilted USAF target show the OS and SR capabilities achieved by our system.

Optical sectioning structured illumination microscopy (OS‐SIM) is a fast, minimally invasive 3D imaging technique that has found widespread application in the biosciences. It is based on sample illumination with several illumination fringe patterns featuring distinct mutual phase shifts, from which an axially sectioned image is reconstructed. Its optical sectioning capability is commonly attributed to the attenuation of the fringe modulation of light collected from planes displaced from the focal plane. However, in addition to this effect, which is governed solely by the detection optics, optical sectioning can be further enhanced by confining the fringe modulation axially via partially coherent illumination (PCI). To establish guidelines for optimal illumination field shaping, both theoretically and experimentally are investigated, the optical sectioning strength of OS‐SIM upon variation of the two key parameters, modulation period and angular spectrum of the incident illumination. By using PCI with OS‐SIM, nearly fivefold and 1.4‐fold enhanced axial resolution are achieved for scattering (non‐fluorescent) and fluorescent samples, respectively. This work elucidates the optical sectioning mechanism of OS‐SIM and provides a perspective for further optimization.

Imaging with single-pixel detectors offers a valuable alternative to the conventional focal plane array strategy, especially for wavelengths where silicon-based sensor arrays exhibit lower efficiency. However, the absence of optical sectioning remains a challenge in single-pixel microscopy. In this paper, we introduce a single-pixel microscope with optical sectioning capabilities by integrating single-pixel imaging (SPI) techniques with structured illumination microscopy (SIM) methods. A spatial light modulator positioned at the microscope's input port encodes a series of structured light patterns, which the microscope focuses onto a specific plane of the 3D sample. Simultaneously, a highly sensitive bucket detector captures the light reflected by the object. Optical sectioning is achieved through a high-frequency grating positioned at the microscope's output port, which is conjugated with the spatial light modulator. Utilizing SPI reconstruction techniques and SIM algorithms, our computational microscope produces high-quality 2D images without blurred out-of-focus regions. We validate the performance of the single-pixel microscope (SPM) by measuring the axial response function and acquiring images of various 3D samples in reflected bright-field configuration. Furthermore, we demonstrate the suitability of the optical setup for single-pixel fluorescence microscopy with optical sectioning.

Rising numbers of computed tomography (CT) examinations worldwide have led to a focus on dose reduction in the latest developments in CT technology. Iterative reconstruction (IR) models bear the potential to effectively reduce dose while maintaining adequate image quality. To assess the impact of adaptive statistical iterative reconstruction (ASIR) technique on dose reduction and image quality in a dedicated whole body CT (WBCT) protocol for trauma patients. A total of 122 subjects with multiple trauma was prospectively included in our study. Subjects who had to undergo a WBCT following a severe trauma were randomly assigned to two different groups: Group A was examined with an ASIR protocol for the body series (n = 64), group B (n = 58) was examined using a standard filtered back projection (FBP) protocol. Image quality was assessed both quantitatively by calculating signal-to-noise ratio (SNR) and contrast-to-noise ratios (CNRs) and qualitatively by two observers who evaluated image quality using a 5-point scale system. Applied dose was analyzed as CTDIvol (mGy), total DLP (mGyxcm), and effective dose (mSv). Applied dose for the body series in group A was about 23% lower than in group B (P < 0.05). SNR and CNRs for different tissues were not significantly different. Subjective image quality ratings were excellent and showed no significant difference, with a high inter-reader agreement. ASIR contributes to a relevant dose reduction without any loss of image quality in a dedicated WBCT protocol for patients with multiple trauma.

Novel diffraction limit implied geometric lens imaging model based on a finite number of pixels for integral imaging display