Nucleolar dynamics are determined by the ordered assembly of the ribosome.

Ribosome biogenesis is coordinated within the nucleolus, a biomolecular condensate that exhibits dynamic material properties that are thought to be important for nucleolar function. However, the relationship between ribosome assembly and nucleolar dynamics is not clear. Here, we screened 364 genes involved in ribosome biogenesis and RNA metabolism for their impact on dynamics of the nucleolus, as measured by automated, high-throughput fluorescence recovery after photobleaching (FRAP) of the nucleolar scaffold protein NPM1. This screen revealed that gene knockdowns that caused accumulation of early rRNA intermediates were associated with nucleolar rigidification, while accumulation of late intermediates led to increased fluidity. These shifts in dynamics were accompanied by distinct changes in nucleolar morphology. We also found that genes involved in mRNA processing impact nucleolar dynamics, revealing connections between ribosome biogenesis and other RNA processing pathways. Together, this work defines mechanistic ties between ribosome assembly and the biophysical features of the nucleolus, while establishing a toolbox for understanding how molecular dynamics impact function across other biomolecular condensates.

Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility.

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

High-content imaging-based pooled CRISPR screens in mammalian cells.

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software µManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging.

Photobleaching limits extended imaging of fluorescent biological samples. We developed DNA-based 'FluoroCubes' that are similar in size to the green fluorescent protein, have single-point attachment to proteins, have a ~54-fold higher photobleaching lifetime and emit ~43-fold more photons than single organic dyes. We demonstrate that DNA FluoroCubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometer precision.

Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.

Nanometer-accuracy distance measurements between fluorophores at the single-molecule level.

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

Cellular aspect ratio and cell division mechanics underlie the patterning of cell progeny in diverse mammalian epithelia.

Cell division is essential to expand, shape, and replenish epithelia. In the adult small intestine, cells from a common progenitor intermix with other lineages, whereas cell progeny in many other epithelia form contiguous patches. The mechanisms that generate these distinct patterns of progeny are poorly understood. Using light sheet and confocal imaging of intestinal organoids, we show that lineages intersperse during cytokinesis, when elongated interphase cells insert between apically displaced daughters. Reducing the cellular aspect ratio to minimize the height difference between interphase and mitotic cells disrupts interspersion, producing contiguous patches. Cellular aspect ratio is similarly a key parameter for division-coupled interspersion in the early mouse embryo, suggesting that this physical mechanism for patterning progeny may pertain to many mammalian epithelia. Our results reveal that the process of cytokinesis in elongated mammalian epithelia allows lineages to intermix and that cellular aspect ratio is a critical modulator of the progeny pattern.

Visualizing Calcium Flux in Freely Moving Nematode Embryos.

The lack of physiological recordings from Caenorhabditis elegans embryos stands in stark contrast to the comprehensive anatomical and gene expression datasets already available. Using light-sheet fluorescence microscopy to address the challenges associated with functional imaging at this developmental stage, we recorded calcium dynamics in muscles and neurons and developed analysis strategies to relate activity and movement. In muscles, we found that the initiation of twitching was associated with a spreading calcium wave in a dorsal muscle bundle. Correlated activity in muscle bundles was linked with early twitching and eventual coordinated movement. To identify neuronal correlates of behavior, we monitored brainwide activity with subcellular resolution and identified a particularly active cell associated with muscle contractions. Finally, imaging neurons of a well-defined adult motor circuit, we found that reversals in the eggshell correlated with calcium transients in AVA interneurons.

Impact of New Camera Technologies on Discoveries in Cell Biology.

New technologies can make previously invisible phenomena visible. Nowhere is this more obvious than in the field of light microscopy. Beginning with the observation of "animalcules" by Antonie van Leeuwenhoek, when he figured out how to achieve high magnification by shaping lenses, microscopy has advanced to this day by a continued march of discoveries driven by technical innovations. Recent advances in single-molecule-based technologies have achieved unprecedented resolution, and were the basis of the Nobel prize in Chemistry in 2014. In this article, we focus on developments in camera technologies and associated image processing that have been a major driver of technical innovations in light microscopy. We describe five types of developments in camera technology: video-based analog contrast enhancement, charge-coupled devices (CCDs), intensified sensors, electron multiplying gain, and scientific complementary metal-oxide-semiconductor cameras, which, together, have had major impacts in light microscopy.

Quantitative evaluation of software packages for single-molecule localization microscopy.

The quality of super-resolution images obtained by single-molecule localization microscopy (SMLM) depends largely on the software used to detect and accurately localize point sources. In this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. Our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. These metrics reflect the various tradeoffs of SMLM software packages and help users to choose the software that fits their needs.

High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation.

Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation. Using 3D time-lapse imaging of beating zebrafish hearts, we observed that the initiation of cardiac trabeculation can be divided into two processes. Before any myocardial cell bodies have entered the trabecular layer, cardiomyocytes extend protrusions that invade luminally along neighboring cell-cell junctions. These protrusions can interact within the trabecular layer to form new cell-cell contacts. Subsequently, cardiomyocytes constrict their abluminal surface, moving their cell bodies into the trabecular layer while elaborating more protrusions. We also examined the formation of these protrusions in trabeculation-deficient animals, including erbb2 mutants, tnnt2a morphants, which lack cardiac contractions and flow, and myh6 morphants, which lack atrial contraction and exhibit reduced flow. We found that, compared with cardiomyocytes in wild-type hearts, those in erbb2 mutants were less likely to form protrusions, those in tnnt2a morphants formed less stable protrusions, and those in myh6 morphants extended fewer protrusions per cell. Thus, through detailed 4D imaging of beating hearts, we have identified novel cellular behaviors underlying cardiac trabeculation.

Advanced methods of microscope control using µManager software.

µManager is an open-source, cross-platform desktop application, to control a wide variety of motorized microscopes, scientific cameras, stages, illuminators, and other microscope accessories. Since its inception in 2005, µManager has grown to support a wide range of microscopy hardware and is now used by thousands of researchers around the world. The application provides a mature graphical user interface and offers open programming interfaces to facilitate plugins and scripts. Here, we present a guide to using some of the recently added advanced µManager features, including hardware synchronization, simultaneous use of multiple cameras, projection of patterned light onto a specimen, live slide mapping, imaging with multi-well plates, particle localization and tracking, and high-speed imaging.

Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells.

Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. We further show that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, we show that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells.

Chromophore-assisted laser inactivation in neural development.

Chromophore-assisted laser inactivation (CALI) is a technique that uses photochemically-generated reactive oxygen species to acutely inactivate target proteins in living cells. Neural development includes highly dynamic cellular processes such as asymmetric cell division, migration, axon and dendrite outgrowth and synaptogenesis. Although many key molecules of neural development have been identified since the past decades, their spatiotemporal contributions to these cellular events are not well understood. CALI provides an appealing tool for elucidating the precise functions of these molecules during neural development. In this review, we summarize the principles of CALI, a recent microscopic setup to perform CALI experiments, and the application of CALI to the study of growth-cone motility and neuroblast asymmetric division.

Biological imaging software tools.

Few technologies are more widespread in modern biological laboratories than imaging. Recent advances in optical technologies and instrumentation are providing hitherto unimagined capabilities. Almost all these advances have required the development of software to enable the acquisition, management, analysis and visualization of the imaging data. We review each computational step that biologists encounter when dealing with digital images, the inherent challenges and the overall status of available software for bioimage informatics, focusing on open-source options.

Software tools, data structures, and interfaces for microscope imaging.

The arrival of electronic photodetectors in biological microscopy has led to a revolution in the application of imaging in cell and developmental biology. The extreme photosensitivity of electronic photodetectors has enabled the routine use of multidimensional data acquisition spanning space and time and spectral range in live cell and tissue imaging. These techniques have provided key insights into the molecular and structural dynamics of living biology. However, digital photodetectors offer another advantage-they provide a linear mapping between the photon flux coming from the sample and the electronic sample they produce. Thus, an image presented as a visual representation of the sample is also a quantitative measurement of photon flux. These quantitative measurements are the basis of subsequent processing and analysis to improve signal contrast, to compare changes in the concentration of signal, and to reveal changes in cell structure and dynamics. For this reason, many laboratories and companies have committed their resources to software development, resulting in the availability of a large number of image-processing and analysis packages. In this article, we review the software tools for image data analysis that are now available and give some examples of their use in imaging experiments to reveal new insights into biological mechanisms. In our final section, we highlight some of the new directions for image analysis that are significant unmet challenges and present our own ideas for future directions.

Polarized myosin produces unequal-size daughters during asymmetric cell division.

Asymmetric positioning of the mitotic spindle before cytokinesis can produce different-sized daughter cells that have distinct fates. Here, we found an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage that began with a centered spindle but generated different-sized daughters, the smaller (anterior) of which underwent apoptosis. During this division, more myosin II accumulated anteriorly, suggesting that asymmetric contractile forces might produce different-sized daughters. Indeed, partial inactivation of anterior myosin by chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and differentiation of the anterior daughter. Thus, the balance of myosin activity on the two sides of a dividing cell can govern the size and fate of the daughters.