Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

Lattice Light Sheet Microscopy: Imaging Molecules to Embryos at High Spatiotemporal Resolution

Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.

/pdf/super-resolution-fluorescence-microscopy-v84c1ojckz.pdf

Super-Resolution Fluorescence Microscopy

Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination

▪ Abstract Meiotic chromosomes have been studied for many years, in part because of the fundamental life processes they represent, but also because meiosis involves the formation of homolog pairs, ...

Meiotic chromosomes: integrating structure and function.

For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

/pdf/a-guide-to-super-resolution-fluorescence-microscopy-28ekkdaf4n.pdf

A guide to super-resolution fluorescence microscopy

During meiotic prophase, telomeres attach to the inner nuclear envelope and cluster to form the so-called meiotic bouquet. Although this has been observed in almost all organisms studied, its precise function remains elusive. The coincidence of telomere clustering and initiation of chromosome synapsis has led to the hypothesis that the bouquet facilitates homologous chromosome pairing and synapsis. However, recent mutant analysis suggests that the bouquet is not absolutely required for either homologous pairing or synapsis but that it makes both processes much faster and more efficient. The initiation of bouquet formation is independent of the initiation of recombination. However, the progression through recombination and synapsis may be required for exit from the bouquet stage. Little is known about the mechanism of telomere clustering but recent studies show that it is an active process.

/pdf/a-bouquet-of-chromosomes-2c8n3kac6k.pdf

A bouquet of chromosomes.

Publisher Summary This chapter discusses the genetic control of meiosis. Meiosis presents a seemingly paradoxical situation in which universality and uniqueness are harmoniously combined. All organisms, irrespective of their evolved complexity, meiotically reduce the chromosome number on beginning sexual reproduction. Genetic recombination and the associated cytological phenomena—chromosome pairing and formation of the synaptonemal complex (sc) and chiasmata—all occur in meiotically dividing cells. The majority of genes do not turn the meiotic process entirely off; they rather interfere with its distinct steps. These comparisons of meiotic mutants make it possible to distinguish between primary and secondary abnormalities caused by mutations in different genes. Other meiotic mutants can serve as points plotted on a genetic flowchart depicting the sequential switching on of meiotic genes. Chromosome behavior appears to be controlled at two levels. There seems to exist a group of genes controlling the behavior of all the chromosomes in the nucleus and another group that controls the behavior of single chromosomes.

Genetic control of meiosis.

REC8 is a master regulator of chromatin structure and function during meiosis. Here, we dissected the functions of absence of first division ( afd1 ), a maize rec8/ α -kleisin homolog, using a unique afd1 allelic series. The first observable defect in afd1 mutants is the inability to make a leptotene chromosome. AFD1 protein is required for elongation of axial elements but not for their initial recruitment, thus showing that AFD1 acts downstream of ASY1/HOP1. AFD1 is associated with the axial and later the lateral elements of the synaptonemal complex. Rescuing 50% of axial element elongation in the weakest afd1 allele restored bouquet formation demonstrating that extent of telomere clustering depends on axial element elongation. However, rescuing bouquet formation was not sufficient for either proper RAD51 distribution or homologous pairing. It provides the basis for a model in which AFD1/REC8 controls homologous pairing through its role in axial element elongation and the subsequent distribution of the recombination machinery independent of bouquet formation.

Alleles of afd1 dissect REC8 functions during meiotic prophase I.

Pairing, synapsis, and recombination are prerequisites for accurate chromosome segregation in meiosis. The phs1 gene in maize is required for pairing to occur between homologous chromosomes. In the phs1 mutant, homologous chromosome synapsis is completely replaced by synapsis between nonhomologous partners. The phs1 gene is also required for installation of the meiotic recombination machinery on chromosomes, as the mutant almost completely lacks chromosomal foci of the recombination protein RAD51. Thus, in the phs1 mutant, synapsis is uncoupled from recombination and pairing. The protein encoded by the phs1 gene likely acts in a multistep process to coordinate pairing, recombination, and synapsis.

https://science.sciencemag.org/content/sci/303/5654/89.full.pdf

Inna N. Golubovskaya

Papers

Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination

A bouquet of chromosomes.

Genetic control of meiosis.

Alleles of afd1 dissect REC8 functions during meiotic prophase I.

Coordination of Meiotic Recombination, Pairing, and Synapsis by PHS1