Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.     

Super-resolution 3D microscopy of live whole cells using structured illumination

Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resolution of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per volume for >50 time points with 120-nm lateral and 360-nm axial resolution. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.     

Partial nephrectomy margin imaging using structured illumination microscopy

Partial nephrectomy (PN) is the recommended procedure over radical nephrectomy (RN) for patients with renal masses less than 4 cm in diameter (Stage T1a). Patients with less than 4 cm renal masses can also be treated with PN, but have a higher risk for positive surgical margins (PSM). PSM, when present, are indicative of poor clinical outcomes. The current gold‐standard histopathology method is not well‐suited for the identification of PSM intraoperatively due to processing time and destructive nature. Here, video‐rate structured illumination microscopy (VR‐SIM) was investigated as a potential tool for PSM detection during PN. A clinical image atlas assembled from ex vivo renal biopsies provided diagnostically useful images of benign and malignant kidney, similar to permanent histopathology. VR‐SIM was then used to image entire parenchymal margins of tumor resection covering up to >1800× more margin surface area than standard histology. Aided by the image atlas, the study pathologist correctly classified all parenchymal margins as negative for PSM with VR‐SIM, compared to standard postoperative pathology. The ability to evaluate large surgical margins in a short time frame with VR‐SIM may allow it to be used intraoperatively as a “safety net” for PSM detection, allowing more patients to undergo PN over RN.      

Optical sectioning microscopy with planar or structured illumination

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.     

3D structured illumination microscopy of mammalian embryos and spermatozoa

Background

Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass.

Results

Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa.

Conclusions

Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa.

     

Detection of single quantum dots in model organisms with sheet illumination microscopy

Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 μm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.     

Protein families in multicellular organisms.

The complete sequence of the nematode worm Caenorhabditis elegans contains the genetic machinery that is required to undertake the core biological processes of single cells. However, the genome also encodes proteins that are associated with multicellularity, as well as others that are lineage-specific expansions of phylogenetically widespread families and yet more that are absent in non-nematodes. Ongoing analysis is beginning to illuminate the similarities and differences among human proteins and proteins that are encoded by the genomes of the multicellular worm and the unicellular yeast, and will be essential in determining the reliability of transferring experimental data among phylogenetically distant species.     

High-speed volumetric imaging in vivo based on structured illumination microscopy with interleaved reconstruction

Wide-field fluorescence microscopy (WFFM) is widely adopted in biomedical studies, due to its high imaging speed over large field-of-views. However, WFFM is susceptible to out-of-focus background. To overcome this problem, structured illumination microscopy (SIM) was proposed as a wide-field, optical-sectioning technique, which needs multiple raw images for image reconstruction and thus has a lower imaging speed. Here we propose SIM with interleaved reconstruction, to make SIM of lossless speed. We apply this method in volumetric imaging of neural network dynamics in brains of zebrafish larva in vivo.     

Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy

Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured illumination microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct an SR image. Using an alternative approach, we demonstrate that the additional spatial information encoded in the knowledge of the position of the illumination pattern can be efficiently decoded using a generalized version of separation of photon by lifetime tuning (SPLIT) that does not require lifetime measurements. In the resulting SPLIT-SIM, the SR image is obtained by isolating a fraction of the intensity corresponding to the center of the diffraction-limited point spread function. This extends the use of the SPLIT approach from stimulated emission depletion microscopy to SIM. The SPLIT-SIM algorithm is based only on phasor analysis and does not require deconvolution. We show that SPLIT-SIM can be used to generate SR images of chromatin organizational motifs with tunable resolution and can be a valuable tool for the imaging of functional sites in the nucleus.     

Evolution of mitotic cell-lineages in multicellular organisms

Adaptive evolution in multicellular organisms is generally assumed to occur through natural selection acting differentially among the phenotypes programmed by sexually-generated zygotic genotypes. Under this view, only genetic changes in the gamete-zygote-germline-gamete cycle are considered relevant to the evolutionary process. Yet asexuality - production of progeny through proliferation of mitotic cell-lineages - is found in over one half of all eukaryotic phyla, and is likely to contribute to adaptive changes, as suggested by recent evidence from both animals and plants. Adaptive changes in mitotic lineages can be reconciled with contemporary evolutionary thought by fully abandoning the weismannian concept of individuality.     

Synthetic biology in multicellular organisms: Opportunities in nematodes

Synthetic biology has mainly focused on introducing new or altered functionality in single cell systems: primarily bacteria, yeast, or mammalian cells. Here, we describe the extension of synthetic biology to nematodes, in particular the well-studied model organism Caenorhabditis elegans, as a convenient platform for developing applications in a multicellular setting. We review transgenesis techniques for nematodes, as well as the application of synthetic biology principles to construct nematode gene switches and genetic devices to control motility. Finally, we discuss potential applications of engineered nematodes.     

Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy

Summary The potential of tuber disc culture for chromosome doubling was investigated in somaclonal populations of four dihaploid genotypes and one tetraploid cultivar of potato (Solanum tuberosum). Laser scanning confocal microscopy (LSCM) was used for rapid determination of the ploidy level based on the number of chloroplasts in stomatal guard cells of leaves. Factorial analysis of chloroplast number in 58 clones and two leaf types showed that somaclones were clearly divided in two groups. Clones with 5–7 chloroplasts per cell as observed in tuber derived diploid controls were classified as 2X (not doubled), while those with 9–14 chloroplasts resembled the tuber derived tetraploid controls and were considered 4X (doubled). A high frequency of spontaneous chromosome doubling, 42% – 50%, was detected in 3 dihaploid genotypes, whereas no doubling was observed in one of the dihaploids as well as the tetraploid cultivar Yukon Gold. Effects of leaf type on chloroplast number was also significant. The middle leaf showed significantly higher chloroplast number than the younger leaves.     

Codon usage and tRNA content in unicellular and multicellular organisms

Choices of synonymous codons in unicellular organisms are here reviewed, and differences in synonymous codon usages between Escherichia coli and the yeast Saccharomyces cerevisiae are attributed to differences in the actual populations of isoaccepting tRNAs. There exists a strong positive correlation between codon usage and tRNA content in both organisms, and the extent of this correlation relates to the protein production levels of individual genes. Codon-choice patterns are believed to have been well conserved during the course of evolution. Examination of silent substitutions and tRNA populations in Enterobacteriaceae revealed that the evolutionary constraint imposed by tRNA content on codon usage decelerated rather than accelerated the silent-substitution rate, at least insofar as pairs of taxonomically related organisms were examined. Codon-choice patterns of multicellular organisms are briefly reviewed, and diversity in G+C percentage at the third position of codons in vertebrate genes--as well as a possible causative factor in the production of this diversity--is discussed.