Deep Imaging: the next frontier in microscopy

Abstract

The microscope is the quintessential tool for discovery in cell biology. From its earliest incarnation as a tool to make the unseen visible, microscopes have been at the center of most revolutionizing developments in cell biology, histology and pathology. Major quantum leaps in imaging involved the dramatic improvements in resolution to see increasingly smaller structures, methods to visualize specific molecules inside of cells and tissues, and the ability to peer into living cells to study dynamics of molecules and cellular structures. The latest revolution in microscopy is Deep Imaging-the ability to look at very large numbers of samples by high-throughput microscopy at high spatial and temporal resolution. This approach is rooted in the development of fully automated high-resolution microscopes and the application of advanced computational image analysis and mining methods. Deep Imaging is enabling two novel, powerful approaches in cell biology: the ability to image thousands of samples with high optical precision allows every discernible morphological pattern to be used as a read-out in large-scale imaging-based screens, particularly in conjunction with RNAi-based screening technology; in addition, the capacity to capture large numbers of images, combined with advanced computational image analysis methods, has also opened the door to detect and analyze very rare cellular events. These two applications of Deep Imaging are revolutionizing cell biology.

Fig. 1

Imaging-based screening. The workflow of an imaging-based RNAi screens typically involves the plating of cells in multi-well plates, and their automated manipulation including addition of siRNA reagents before a specific biological assay is performed. Detection of signal may occur via immunocytochemical methods, by use of genetically encoded markers, or analysis of morphological features. Samples are imaged in a fully automated high-throughput fashion and dedicated image analysis algorithms used to detect and quantitate signals. Secondary data analysis identifies hits based on statistical analysis

Fig. 2

Observation of a rare cellular event by deep imaging. a Design of a cell-based system to capture the formation of chromosome translocations in living cells. (i) Double-strand breaks (DSBs) are induced at engineered ISceI restriction sites integrated on distinct chromosomes adjacent to the LacO and TetO array sequences, which can be visualized through the LacO/TetO operator/repressor system (red, green, respectively). (ii) The fluorescently marked chromosome ends are tracked in space and in time by timelapse deep imaging and (iii) the rare joining of broken chromosomes in the form of a translocation is captured by the persistent superimposition of the two fluorescently labeled chromosome ends as identified by automated image analysis. b workflow of a deep imaging experiment. Cells are plated in multi-well plates (96, 384 wells), thousands of cells imaged in a fully automated fashion over time and features of interest (LacO/TetO spots) automatically extracted using image analysis software. Imaging data are then mined for analysis. c visualization of a chromosome translocation by timelapse microscopy. Cells were transfected with the ISceI endonuclease, and 6 h later, timelapse microscopy was performed to follow DSBs marked by the LacO (green) and TetO (red) for up to 20 h. Broken chromosome ends are seen to congregate and ultimately fuse as they translocate. Maximal projected image sequences are shown. Scale bar 5 μm