We compare two recently developed multiple-frame deconvolution approaches for the reconstruction of structured illumination microscopy (SIM) data: the pattern-illuminated Fourier ptychography algorithm (piFP) and the joint Richardson-Lucy deconvolution (jRL). The quality of the images reconstructed by these methods is compared in terms of the achieved resolution improvement, noise enhancement, and inherent artifacts. Furthermore, we study the issue of object-dependent resolution improvement by considering the modulation transfer functions derived from different types of objects. The performance of the considered methods is tested in experiments and benchmarked with a commercial SIM microscope. We find that the piFP method resolves periodic and isolated structures equally well, whereas the jRL method provides significantly higher resolution for isolated objects compared to periodic ones. Images reconstructed by the piFP and jRL algorithms are comparable to the images reconstructed using the generalized Wiener filter applied in most commercial SIM microscopes. An advantage of the discussed algorithms is that they allow the reconstruction of SIM images acquired under different types of illumination, such as multi-spot or random illumination.
Various types of non-uniform illumination can be used for resolution improvement in fluorescence microscopy. Here we study the differences between several types of incoherent illumination patterns, such as multi-spot, line and pseudo-random patterns. This requires an imaging setup and an image reconstruction algorithm that are flexible enough to incorporate any type of illumination pattern. We employ fluorescence microscope with structured illumination generated by a Digital Micro-mirror Device (DMD) and the pattern-illuminated Fourier Ptychography reconstruction algorithm (piFP) to this end. The piFP method is modified and improved by identifying the algorithm as steepest descent optimization of a least squares function. We find that illumination patterns with regular structure are superior to those with irregular structure in terms of resolution enhancement and noise level in the reconstructed images.
Photobleaching is a major factor limiting the observation time in fluorescence microscopy. We achieve photobleaching reduction in structured illumination microscopy (SIM) by locally adjusting the illumination intensities according to the sample. Adaptive SIM is enabled by a digital micro-mirror device (DMD), which provides a projection of the grayscale illumination patterns. We demonstrate a reduction in photobleaching by a factor of three in adaptive SIM compared to the non-adaptive SIM based on a spot grid scanning approach. Our proof-of-principle experiments show great potential for DMD-based microscopes to become a more useful tool in live-cell SIM imaging.
We present a versatile fluorescence microscope, built by complementing a conventional fluorescence microscope with a digital micro-mirror device (DMD) in the illumination path. Arbitrary patterns can be created on the DMD and projected onto the sample. This patterned illumination can be used to improve lateral and axial resolution over the resolution of a wide-field microscope, as well as to reduce the illumination dose. Different illumination patterns require different reconstruction strategies and result in an image quality similar to confocal or structured illumination microscopy. We focus on the optical design and characterization of a DMD-based microscope. Estimation of the optical quality of the microscope has been carried out by measuring the modulation transfer function from edge profiles. We have obtained optically sectioned images by applying multi-spot illumination patterns followed by digital pinholing. The sectioning capabilities of our DMD-based microscope were estimated from the dependence of the signal-to-background and signalto-noise ratios on the pitch of the projected multi-spot patterns and the size of the digital pinhole. In addition, we provide an outlook on the use of pseudo-random illumination patterns for achieving both sectioning and resolution enhancement.
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