The Colored Revolution of Bioimaging

With the recent development of fluorescent probes and new high-resolution microscopes, biological imaging has entered a new era and is presently having a profound impact on the way research is being conducted in the life sciences. Biologists have come to depend more and more on imaging. They can now visualize subcellular components and processes in vivo, both structurally and functionally. Observations can be made in two or three dimensions, at different wavelengths (spectroscopy), possibly with time-lapse imaging to investigate cellular dynamics. The observation of many biological processes relies on the ability to identify and locate specific proteins within their cellular environment. Cells are mostly transparent in their natural state and the immense number of molecules that constitute them are optically indistinguishable from one another. This makes the identification of a particular protein a very complex task—akin to finding a needle in a haystack. However, if a bright marker were attached to the protein of interest, it could very precisely indicate its position. Much effort has gone into finding suitable markers for this purpose, but it is only over the course of the past decade, with the advent of fluorescent proteins, that this concept has been revolutionized. These biological markers have the crucial properties necessary for dynamic observations of living cells: they are essentially harmless to the organism, and can be attached to other proteins without impacting their function. Fluorescence microscopy was invented almost a century ago, when microscopists were experimenting with ultraviolet light to achieve higher resolutions. In the very beginning, observations were limited to specimens that naturally fluoresce. Rapidly, fluorescent dyes for staining tissues and cells were investigated. But it was not until the 1940s that fluorescence microscopy became popular, when Coons and Kaplan introduced a technique to label antibodies with a fluorescent dye to study antibody-antigen interactions, profoundly changing the field of immunohistochemistry. The discovery that really brought fluorescence microscopy to the forefront came in 1994, when M. Chalfie et al. succeeded in expressing a naturally fluorescent protein, the now-famous green fluorescent protein (GFP), in living organisms. This was a landmark evolution in the field, fostering a whole new class of tagging methods. While genetic engineering is at the origin of this new methodology, a number of innovations from the fields of physics, optics, and mechanical and electrical engineering have been combined to provide the necessary instrumentation. Impressive enhancements in classical microscopy have been achieved, and new imaging systems are actively being developed. A key element for the evolution of microscopy in general was the shift to digital imaging in the 1990s, with the availability of affordable high-sensitivity acquisition devices and powerful computer hardware. The capabilities of today's systems often lead to enormous data sets that, in most cases, require postprocessing for their interpretation. Signal processing methods for biological research are only at their prelude; the needs are considerable and most probably not even clearly formulated yet. It is thus predictable that signal processing will be one of the main challenges of fluorescence microscopy in the forthcoming years. The goal of this article is to provide an overview of the main aspects of modern fluorescence microscopy. We first cover the principles of fluorescence and highlight the key discoveries in the history of fluorescence microscopy. In subsequent sections, we present the optics of fluorescence microscopes and examine various types of detectors. Finally, we discuss the signal and image processing challenges in fluorescence microscopy and highlight some of the present developments and future trends in the field.