The future's bright: Imaging cell biology in the 21st century

Abstract

In November 2008, Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize for Chemistry, to recognize their truly groundbreaking work identifying green fluorescent protein (GFP) and showing that it can be used as a tool to study a wide range of cellular processes. Chalfie and Tsien's special seminar at the ASCB 48th Annual Meeting in San Francisco the following month was an opportunity for the community to hear first-hand the excitement and inspiration behind learning how to manipulate this humble jellyfish protein. Very soon after its discovery, GFP was catapulted literally into the limelight, and is now in use in cell biological laboratories worldwide. It started a microscopy revolution.We are marking the 1-year anniversary of this well-deserved recognition with a special issue of Trends in Cell Biology devoted to state-of-the-art imaging approaches. Through a group of 12 topical reviews by experts in the field, we provide a snapshot of some of the most exciting work being done in cell biology using GFP, its relatives and derivatives, and other innovative tools and techniques.A central question for cell biologists is intracellular communication. How do cascades of signals travel from the cell periphery to the interior of the cell? The ability to follow individual signaling molecules in space and time is vital to the understanding of the molecular mechanisms involved. A class of exciting new photoactivatable fluorescent proteins (PA-FPs) are described by one of us (J.L.-S.) and George Patterson. The PA-FPs can be used to ‘highlight’ subpopulations of a particular intracellular species, leading to the development of novel imaging technologies. PA-FPs are now being developed for use in live cell imaging and for tracking single particles. Diane Lidke and Bridget Wilson give an account of the development of Quantum Dots, another cutting-edge tool that allows intricate tracking of protein localization and dynamics in living cells. Visualization of membrane bound receptors corralled by the actin cytoskeleton has provided spatiotemporal information that is not available using traditional biochemical techniques. Tamas Balla describes the latest developments in our understanding of GPCR activation and second messenger signaling, with an appraisal of how Forster resonance energy transfer (FRET) and the related techniques fluorescent lifetime imaging microscopy (FLIM) and bioluminescence fluorescence resonance energy transfer (BRET) are used to probe second messenger activity and localization.Taking a very different approach, Carolyn Larabell and colleagues consider the contribution of soft X-ray tomography (SXT) to analyzing cellular architecture. Recent development in microscopes capable of integrating cryogenic light microscopy with SXT allow correlated light and X-ray imaging to be carried out on the same specimen. Individual molecules and compartments tagged with fluorescent probes can therefore be localised in a high-resolution 3D reconstruction of a cell, as demonstrated by the beautiful central cover image for this special issue.Internalization of membranes and cytoskeletal dynamics are also integral to cellular signaling and vital processes such as cytokinesis. Tom Kirchhausen reports on the latest live-cell imaging data that sheds new light on the interesting and complex field of clathrin dynamics. Imaging the highly dynamic process of membrane budding is leading to many exciting new discoveries about the nature of the various types of clathrin patches on the plasma membrane. Innovative methodologies in this field are leading to great leaps and bounds in our understanding of the mechanisms involved in plasma membrane dynamics. Another long-standing conundrum of membrane dynamics is in the field of abscission, the final step in cytokinesis following mitosis. Daniel Gerlich and Patrick Steigemann discuss the variety of imaging-based assays that have recently been employed to understand the nature and dynamics of the midbody. Given its small size, this structure has been difficult to visualize using traditional imaging methods and so the generation of new fluorescent probes and perturbation techniques has accelerated the research in this exciting area.Another field in which novel perturbation techniques are aiding the progression of research in tandem with the latest visualization methodologies is DNA repair. Double strand break (DSB) dynamics can now be induced and followed in living cells, giving detailed mechanistic insights into the spatiotemporal regulation of DNA repair. Evi Soutoglou and Zita Nagy consider the pros and cons of the panoply of methodologies now available. Imaging individual mRNAs is also now possible. Robert Singer and colleagues relate the advances in single molecule studies that are allowing the analysis of gene expression at the level of the individual cell.The ability to visualize cells in vivo has clear implications for monitoring disease progression. Patricia Keely and colleagues describe recent advances in the development of ‘optical biomarkers’ that allow tracking of individual cells metastasizing from a primary tumour. The potential to harness these novel imaging techniques to improve mechanistic understanding of processes such as metastasis is compelling. Precise three-dimensional imaging in vivo has now become an achievable goal for the community.The development of new techniques will of course present new challenges for researchers, and three opinion pieces in this collection address broader issues related to some of these potential problems. Erik Snapp provides a brief primer on the initial selection of a fluorescent protein from the ‘GFP toolbox’; Jason Swedlow and Kevin Eliceiri discuss open source solutions for how to store and analyze data; and Sanford Simon takes a more philosophical standpoint, and using total internal reflection fluorescence (TIRF) microscopy as his example, invites us to consider: how do you ensure that the questions you ask can really be answered with the tools you are using?What does the future of imaging hold for cell biology? The techniques described in this special issue point towards further technical revolutions on the cell imaging horizon. Overcoming the diffraction barrier of the light-microscope is now possible for several techniques, and the concomitant advances in computer algorithms means that generation and processing of large data sets is fast becoming achievable for even modest set-ups. The conflation of expertise in biology, computer science, physics and mathematics generates new subfields in areas such as systems biology and single molecule visualization, and encourages further interdisciplinary collaborations. Improvements in whole organism imaging, combined with visualization of individual cells or particles within that same organism, mean that the ability of the cell biologist to unlock the secrets of the cell is increasing exponentially. The advent of less damaging live cell imaging and in vivo imaging will lead to truly holistic overviews of signal transduction, motility and morphology, from single cells to functioning organisms. In the long term, imaging individual cells and tissues for diagnostic purposes is an exciting potential future application of these new techniques.We hope the sense of excitement that drove Shimomura, Chalfie and Tsien onwards in their quest to see more with the help of a small fluorescent protein is palpable in this excellent collection of reviews and perspectives. The thirst for understanding events on the molecular scale and the innovative approaches taken by the authors of all the work reviewed here is a testament to the inspiration and delight that can be gained by actually seeing what the cells we study are doing - see Figure 1Figure 1 for an example.Figure 1An NIH 3T3 cell displaying the initials of Trends in Cell Biology after photoconversion of the Dendra 2 protein. The originally green fluorescent protein Dendra 2 was converted to red using the 405 nm blue diode. The image was taken with the Sp5 Leica confocal microscope using the Region of Interest (ROI) function at the imaging facility of IGBMC in Strasbourg with the help of Marc Koch.View Large Image | Download PowerPoint SlideFinally, we’d like to thank all the contributors to this special issue for their extensive efforts, both the authors and referees, who worked to extremely tight deadlines to meet this auspicious anniversary. Congratulations also to Tsien, Chalfie and Shimomura on their first Nobel Birthday, and to all pioneers of exciting imaging techniques for the cell biology community.We hope you enjoy reading the articles in this special issue as much as we have.