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.

Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.patterned excitation | moiré | subdiffraction

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it lightefficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.extended resolution | frequency mixing | multicolor | patterned excitation F luorescence microscopy allows noninvasive 3D imaging of the interior of living specimens with molecular specificity, and is therefore an invaluable resource to the biological sciences. Unfortunately, the resolving power of fluorescence microscopy is fundamentally limited by the diffraction of light.Lately, many techniques have been introduced to extend the resolution beyond the classic diffraction limit (1-8). Although the improvement of spatial resolution is impressive, the practical impact of these new techniques will largely depend on whether they can keep the two key advantages of fluorescence microscopy, namely, the capability of imaging living cells in three dimensions and multicolor labeling. Live-cell imaging over many time points has been demonstrated for some resolution-extending techniques (9-15) but, to our knowledge, not for multicolor 3D imaging of whole cells.Stimulated emission depletion (STED) microscopy increases the spatial resolution by suppressing the fluorescence emission on the rim of a focused laser spot, theoretically enabling unlimited resolution (16). STED microscopy has been demonstrated for live imaging at high frame rates (12, 15), but its point-scanning nature makes it unsuitable for fast volumetric imaging over large fields of view. Furthermore, to achieve high spatial resolution, very high power densities (38-540 MW/cm 2 ) (12) are necessary for the STED laser beam, which may cause increased photobleaching and phototoxicity, thus limiting the application of live-cell STED microscopy.In localization-based microscopy, only sparse subsets of photoswitchable fluorophores in a sample are imaged at a time, which allows the isolation and precise localizati...

Summary Paragraph While microorganisms are often studied as populations, the behavior of single, individual cells can have profound consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population rapidly dies. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed 1. In fact, mycobacteria create variability every time a cell divides, producing daughter cells with different sizes and growth rates 2, 3. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a FACS-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases the amount of heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologs in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (“the divisome”). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and certain drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure.

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.

Live‐cell fluorescence light microscopy has emerged as an important tool in the study of cellular biology. The development of fluorescent markers in parallel with super‐resolution imaging systems has pushed light microscopy into the realm of molecular visualization at the nanometer scale. Resolutions previously only attained with electron microscopes are now within the grasp of light microscopes. However, until recently, live‐cell imaging approaches have eluded super‐resolution microscopy, hampering it from reaching its full potential for revealing the dynamic interactions in biology occurring at the single molecule level. Here we examine recent advances in the super‐resolution imaging of living cells by reviewing recent breakthroughs in single molecule localization microscopy methods such as PALM and STORM to achieve this important goal. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 322–331, 2011.

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress.

A mutation in a microtubule-severing enzyme, katanin, causes flagella to become short due to a reduced cytoplasmic precursor pool. These results suggest that competition between flagella and cytoplasmic microtubules for a limited tubulin pool is facilitated by katanin, which is confirmed by stochastic models.