Long-term Time-lapse Microscopy Research Articles

Daf-42 is an evolutionarily young gene essential for dauer development in Caenorhabditis elegans.

Under adverse environmental conditions, nematodes arrest into dauer, an alternative developmental stage for diapause. Dauer endures unfavorable environments and interacts with host animals to access favorable environments, thus playing a critical role in survival. Here, we report that in Caenorhabditis elegans, daf-42 is essential for development into the dauer stage, as the null mutant of daf-42 exhibited a "no viable dauer" phenotype in which no viable dauers were obtained in any dauer-inducing conditions. Long-term time lapse microscopy of synchronized larvae revealed that daf-42 is involved in developmental changes from the pre-dauer L2d stage to the dauer stage. daf-42 encodes large, disordered proteins of various sizes that are expressed in and secreted from the seam cells within a narrow time window shortly before the molt into dauer stage. Transcriptome analysis showed that the transcription of genes involved in larval physiology and dauer metabolism is highly affected by the daf-42 mutation. Contrary to the notion that essential genes that control the life and death of an organism may be well conserved across diverse species, daf-42 is an evolutionarily young gene conserved only in the Caenorhabditis genus. Our study shows that dauer formation is a vital process that is controlled not only by conserved genes but also by newly emerged genes, providing important insights into evolutionary mechanisms.

CellTracker: an automated toolbox for single-cell segmentation and tracking of time-lapse microscopy images.

Recent advances of long-term time-lapse microscopy have made it easy for researchers to quantify cell behavior and molecular dynamics at single-cell resolution. However, the lack of easy-to-use software tools optimized for customized research is still a major challenge for quantitatively understanding biological processes through microscopy images. Here, we present CellTracker, a highly integrated graphical user interface software, for automated cell segmentation and tracking of time-lapse microscopy images. It covers essential steps in image analysis including project management, image pre-processing, cell segmentation, cell tracking, manually correction and statistical analysis such as the quantification of cell size and fluorescence intensity, etc. Furthermore, CellTracker provides an annotation tool and supports model training from scratch, thus proposing a flexible and scalable solution for customized dataset analysis. CellTracker is an open-source software under the GPL-3.0 license. It is implemented in Python and provides an easy-to-use graphical user interface. The source code, instruction manual and demos can be found at https://github.com/WangLabTHU/CellTracker. Supplementary data are available at Bioinformatics online.

Alignment of Cell Lineage Trees Elucidates Genetic Programs for the Development and Evolution of Cell Types

SummaryA full understanding of the developmental process requires fine-scale characterization of cell divisions and cell types, which are naturally organized as the developmental cell lineage tree (CLT). Technological breakthroughs facilitated determination of more CLTs, but complete comprehension of the data remains difficult without quantitative comparison among CLTs. We hereby quantified phenotypic similarity between CLTs using a novel computational method that exhaustively searches for optimal correspondence between individual cells meanwhile retaining their topological relationships. The revealed CLT similarities allowed us to infer functional similarity at the transcriptome level, identify cell fate transformations, predict functional relationships between mutants, and find evolutionary correspondence between cell types of different species. By allowing quantitative comparison between CLTs, our work is expected to greatly enhance the interpretability of relevant data and help answer the myriad of questions surrounding the developmental process.

Sample-based modeling reveals bidirectional interplay between cell cycle progression and extrinsic apoptosis.

Apoptotic cell death can be initiated through the extrinsic and intrinsic signaling pathways. While cell cycle progression promotes the responsiveness to intrinsic apoptosis induced by genotoxic stress or spindle poisons, this has not yet been studied conclusively for extrinsic apoptosis. Here, we combined fluorescence-based time-lapse monitoring of cell cycle progression and cell death execution by long-term time-lapse microscopy with sampling-based mathematical modeling to study cell cycle dependency of TRAIL-induced extrinsic apoptosis in NCI-H460/geminin cells. In particular, we investigated the interaction of cell death timing and progression of cell cycle states. We not only found that TRAIL prolongs cycle progression, but in reverse also that cell cycle progression affects the kinetics of TRAIL-induced apoptosis: Cells exposed to TRAIL in G1 died significantly faster than cells stimulated in S/G2/M. The connection between cell cycle state and apoptosis progression was captured by developing a mathematical model, for which parameter estimation revealed that apoptosis progression decelerates in the second half of the cell cycle. Similar results were also obtained when studying HCT-116 cells. Our results therefore reject the null hypothesis of independence between cell cycle progression and extrinsic apoptosis and, supported by simulations and experiments of synchronized cell populations, suggest that unwanted escape from TRAIL-induced apoptosis can be reduced by enriching the fraction of cells in G1 phase. Besides novel insight into the interrelation of cell cycle progression and extrinsic apoptosis signaling kinetics, our findings are therefore also relevant for optimizing future TRAIL-based treatment strategies.

An automated microfluidic system for efficient capture of rare cells and rapid flow-free stimulation.

Cell fates are controlled by environmental stimuli that rapidly change the activity of intracellular signaling. Studying these processes requires rapid manipulations of micro-environmental conditions while continuously observing single cells over long periods of time. Current microfluidic devices are unable to simultaneously i) efficiently capture and concentrate rare cells, ii) conduct automated rapid media exchanges via diffusion without displacing non-adherent cells, and iii) allow sensitive high-throughput long-term time-lapse microscopy. Hematopoietic stem and progenitor cells pose a particular challenge for these types of experiments as they are impossible to obtain in very large numbers and are displaced by the fluid flow usually used to change culture media, thus preventing cell tracking. Here, we developed a programmable automated system composed of a novel microfluidic device for efficient capture of rare cells in independently addressable culture chambers, a custom incubation system, and user-friendly control software. The chip's culture chambers are optimized for efficient and sensitive fluorescence microscopy and their media can be individually and quickly changed by diffusion without non-adherent cell displacement. The chip allows efficient capture, stimulation, and sensitive high-frequency time-lapse observation of rare and sensitive murine and human primary hematopoietic stem cells. Our 3D-printed humidification and incubation system minimizes gas consumption, facilitates chip setup, and maintains stable humidity and gas composition during long-term cell culture. This approach now enables the required continuous long-term single-cell quantification of rare non-adherent cells with rapid environmental manipulations, e.g. of rapid signaling dynamics and the later stem cell fate choices they control.

A microfluidic cell-trapping device to study dynamic host-microbe interactions at the single-cell level.

Single-cell imaging of host-microbe interactions over time is impeded by cellular motility because the cells under scrutiny tend to migrate out of the imaging field. To overcome this technical challenge, we developed a microfluidic platform for imaging hundreds of individual motile phagocytic cells and bacteria within microfluidic traps that restrict their movement. The interaction of trapped host cells and bacteria is monitored by long-term time-lapse microscopy, allowing direct visualization of all stages of infection at the single-cell level. The medium flowing through the microfluidic device can be changed quickly and precisely, permitting the real-time imaging of cellular responses to antibiotics or other environmental stresses. Here, we demonstrate the potential applications of this approach by co-culturing the phagocytic amoeba Dictyostelium discoideum with the intracellular pathogen Mycobacterium marinum. However, the platform can be adapted easily for use with other host cells or microorganisms. This approach will provide new insights into host-pathogen interactions that cannot be studied using conventional population-based assays.

Long-term time-lapse microscopy of C. elegans post-embryonic development

We present a microscopy technique that enables long-term time-lapse microscopy at single-cell resolution in moving and feeding Caenorhabditis elegans larvae. Time-lapse microscopy of C. elegans post-embryonic development is challenging, as larvae are highly motile. Moreover, immobilization generally leads to rapid developmental arrest. Instead, we confine larval movement to microchambers that contain bacteria as food, and use fast image acquisition and image analysis to follow the dynamics of cells inside individual larvae, as they move within each microchamber. This allows us to perform fluorescence microscopy of 10–20 animals in parallel with 20 min time resolution. We demonstrate the power of our approach by analysing the dynamics of cell division, cell migration and gene expression over the full ∼48 h of development from larva to adult. Our approach now makes it possible to study the behaviour of individual cells inside the body of a feeding and growing animal.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Microfluidic Cell Culturing Platform Combining Long-term, High-resolution Imaging with Impedance Spectroscopy

We present an integrated microfluidic system, which combines long-term cell culturing with electrical impedance spectroscopy (EIS) on a single chip. Non-adherent cells, such as yeast (S. cerevisiae), can be grown under constant media perfusion in a 2D layer under clamping pads that enable long-term time-lapse microscopy within the same focal plane. The addition of EIS provides a complementary method for continuous in-situ analysis of single cells and has been used here to monitor the growth rate of cell colonies in real time.

Re-fusion of Small Mononucleated Hodgkin Cells Leads to Multinucleated Reed-Sternberg Cells

The multinucleated Reed-Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL) and their presence is essential for diagnosis. However, the development of these giant tumor cells is controversially discussed. It was postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single cell tracking of HL cell lines by long-term time-lapse microscopy showed that cell fusion is the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by size increase gives rise to giant RS cells. Importantly, we nearly exclusively observed fusion of cells originating from the same ancestor, termed re-fusion. In the majority of cases, re-fusion of daughter cells was preceded by an incomplete cytokinesis, visualized by a microtubule bond between the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further specifying small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells showed a shared propensity for re-fusion, which provides evidence of early RS cell fate commitment. Thus, RS cell generation is neither due to cell fusion of unrelated Hodgkin cells nor to endomitosis, but is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding indicates the existence of a novel mechanism for the generation of multinuclear RS cells, which might have implications beyond HL, as RS-like cells are frequently observed in several other lymphoproliferative diseases.

Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed–Sternberg cells

Multinucleated Reed-Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL), and their presence is essential for diagnosis. How these giant tumor cells develop is controversial, however. It has been postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single-cell tracking of HL cell lines by long-term time-lapse microscopy has identified cell fusion as the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by a size increase gives rise to giant RS cells. Of note, fusion of cells originating from the same ancestor, termed re-fusion, is seen nearly exclusively. In the majority of cases, re-fusion of daughter cells is preceded by incomplete cytokinesis, as demonstrated by microtubule bonds among the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further supporting small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells show a shared propensity for re-fusion, providing evidence of early RS cell fate commitment. Thus, RS cell generation is related neither to cell fusion of unrelated Hodgkin cells nor to endomitosis, but rather is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding supports the existence of a unique mechanism for the generation of multinuclear RS cells that may have implications beyond HL, given that RS-like cells are frequently observed in several other lymphoproliferative diseases as well.

An automatic method for robust and fast cell detection in bright field images from high-throughput microscopy

BackgroundIn recent years, high-throughput microscopy has emerged as a powerful tool to analyze cellular dynamics in an unprecedentedly high resolved manner. The amount of data that is generated, for example in long-term time-lapse microscopy experiments, requires automated methods for processing and analysis. Available software frameworks are well suited for high-throughput processing of fluorescence images, but they often do not perform well on bright field image data that varies considerably between laboratories, setups, and even single experiments.ResultsIn this contribution, we present a fully automated image processing pipeline that is able to robustly segment and analyze cells with ellipsoid morphology from bright field microscopy in a high-throughput, yet time efficient manner. The pipeline comprises two steps: (i) Image acquisition is adjusted to obtain optimal bright field image quality for automatic processing. (ii) A concatenation of fast performing image processing algorithms robustly identifies single cells in each image. We applied the method to a time-lapse movie consisting of ∼315,000 images of differentiating hematopoietic stem cells over 6 days. We evaluated the accuracy of our method by comparing the number of identified cells with manual counts. Our method is able to segment images with varying cell density and different cell types without parameter adjustment and clearly outperforms a standard approach. By computing population doubling times, we were able to identify three growth phases in the stem cell population throughout the whole movie, and validated our result with cell cycle times from single cell tracking.ConclusionsOur method allows fully automated processing and analysis of high-throughput bright field microscopy data. The robustness of cell detection and fast computation time will support the analysis of high-content screening experiments, on-line analysis of time-lapse experiments as well as development of methods to automatically track single-cell genealogies.

A microfluidic system for long-term time-lapse microscopy studies of mycobacteria

Phenotypic heterogeneity in bacterial populations is thought to contribute to a number of important phenomena including sporulation and persistence. The latter has clinical implications in many diseases such as tuberculosis, where persistence of Mycobacterium tuberculosis within the human host is believed to be the root cause of latent tuberculosis and the ability of a minority population of cells to survive antibiotic exposure, despite being genetically identical to the bulk population that are killed. However, phenotypic variations caused by non-genetic mechanisms are difficult to study because of the transient nature of the persistent state and thereby the requirement to observe individual cells in real-time. Recently, microfluidics, combined with time-lapse microscopy, has become a powerful tool for studying population heterogeneity in bacteria. However, growth and replication of mycobacterial cells provide particular problems for the development of microfluidic systems due to their tendency to grow in three dimensions. We here describe a novel microfluidic device for the observation of growth and antibiotic killing in individual mycobacterial cells. We constructed a microfluidic device suitable for studying single cell behavior in mycobacteria. The growth of single cells of Mycobacterium smegmatis expressing green fluorescent protein was monitored using a confocal laser scanning microscope. Within the device M.smegmatis cells were tightly confined within a hydrogel matrix thus promoting planar growth. Cell growth and killing was observed in the device with dead cells highlighted by uptake of propidium iodide. Conclusions/Significance. We demonstrate that our device allows real-time analysis and long-term culture of single cells of mycobacteria, and is able to support the study of cell death during the application of antibiotics. The device will allow observation of individual cells' cell genealogy to be determined and direct observation of rare states, such as persistence.

Nongenetic Individuality in the Host–Phage Interaction

Isogenic bacteria can exhibit a range of phenotypes, even in homogeneous environmental conditions. Such nongenetic individuality has been observed in a wide range of biological processes, including differentiation and stress response. A striking example is the heterogeneous response of bacteria to antibiotics, whereby a small fraction of drug-sensitive bacteria can persist under extensive antibiotic treatments. We have previously shown that persistent bacteria enter a phenotypic state, identified by slow growth or dormancy, which protects them from the lethal action of antibiotics. Here, we studied the effect of persistence on the interaction between Escherichia coli and phage lambda. We used long-term time-lapse microscopy to follow the expression of green fluorescent protein (GFP) under the phage lytic promoter, as well as cellular fate, in single infected bacteria. Intriguingly, we found that, whereas persistent bacteria are protected from prophage induction, they are not protected from lytic infection. Quantitative analysis of gene expression reveals that the expression of lytic genes is suppressed in persistent bacteria. However, when persistent bacteria switch to normal growth, the infecting phage resumes the process of gene expression, ultimately causing cell lysis. Using mathematical models for these two host–phage interactions, we found that the bacteria's nongenetic individuality can significantly affect the population dynamics, and might be relevant for understanding the coevolution of bacterial hosts and phages.

Encapsulated Petri dish system for single-cell drug delivery and long-term time lapse microscopy

We have developed a system that allows focal drug application for cell culture microscopy. Single-cell drug delivery is achieved through the insertion of a patch-clamp-like micropipette in a microenvironment-controlled chamber mounted on a standard 35-mm Petri dish. The system has precise control of temperature, CO 2 concentration, and humidity, while preventing contamination during experiments. The use of standard Petri dishes allows long-term experiments by alternating in situ microscopy with incubator growth. Modern biological long-term experiments such as the characterization of drug effects on cell movement, axonal guidance, mitosis, apoptosis, differentiation, or volume regulation can be performed. The chamber is compatible with any inverted microscope without significant modifications.

Multidimensional long-term time-lapse microscopy of in vitro peripheral nerve regeneration.

In order to test the effectiveness of a new advanced time-lapse microscopy imaging and image processing and analysis system, and to do quantitative and qualitative temporal analyses of in vitro peripheral nerve regeneration, long-term time-lapse imaging of cultures of mouse dorsal root ganglia (DRGs) was performed. DRGs were placed in a Petri dish, covered with collagen gel, their attached peripheral nerves were cut in the middle, creating a gap, and the dish was filled with culture medium. Six preparations were kept on the time-lapse imaging system, which provides a suitable incubation environment and enables to capture images from multiple coordinates at x,y,z axes at desired time intervals for 13 days. In general, the time-lapse imaging system proved quite stable and efficient, although some improvements are certainly required. Two main components of peripheral nerve regeneration, outgrowth of axons and activities of resident cells, were examined. Axons started to grow during the first hour of incubation with a 16.5 microm/h rate and showed the slowest rates (0.7 microm/h) on days 8 and 9, after which they resumed higher speeds again. The first cell came out of the proximal end of the cut nerve on the second day and it was a Schwann cell (SC), which was the prominent cell type in the preparations throughout the experiment. SCs were higher in number (83.15% of all cells) but slower in migration (3.4 vs. 7.3 microm/h, P < 0.001) than other cells. Other observed characteristics of axonal outgrowth and cellular activity and interactions between axons and the cells are discussed.