The use of photoactive compounds in microscopy has a long history. Caged compounds have been used for almost forty years, not only to elicit chemical reactions in cells, but also to mark specific cells or regions within cells by photoactivation of fluorescence. During the last seven years, the advent of photoactivatable GFP (PA-GFP) and its successors has opened up a myriad of new applications. All of this work has, of course, been greatly facilitated in live cells through the possibility of genetic labeling that is given by the fluorescent proteins. However, even as more photo-activatable and photo-switchable proteins are discovered, they are still limited in terms of wavelength ranges and photophysical properties. Thus, there has been a resurgence of interest in small organic photoactive molecules for cell biology experiments. In this short introductory overview, we will present the basic concepts of photoactivation and discuss many of the strengths and limitations of various approaches. We will also provide a general description of the kinds of applications for which these probes can be used.

When living cells are far below the surface, they pose particularly complex problems for researchers trying to view dynamic life activities in the laboratory. Many fluorescence imaging systems rely on short-wavelength ultraviolet (UV) or blue light, which is then absorbed by the specimen and emitted as visible light. But living tissue scatters so much short-wavelength light that some of the emitted fluorescence from the region of interest does not reach the detector. The deeper the area of interest, the more severe this problem becomes. Indeed, for every specimen, there is a point at which so much scatter occurs that traditional fluorescence imaging techniques are no longer effective. Raising the intensity of the excitation light in order to get more light out of the system can itself be lethal for living systems, as it causes increased photobleaching and phototoxicity. Adding to the problem is the fact that in deep imaging, regions of the specimen above and below the focal plane that are not of interest are exposed to light, causing unwanted fluorescence. Finally, excessive scattering of the excitation light when imaging deep below the specimen's surface results in an image with poor signal-to-noise ratio. These images tend to look soft and dull instead of crisp and full of contrast. Assuming that the scientist's research protocol will not allow the use of thinner-cut sections, deep imaging is still possible via multiphoton microscopy.

The nucleolus represents a highly dynamic nuclear domain arising from an equilibrium between the level of ribosomal RNA synthesis and the efficiency of ribosomal RNA processing [1, 2]. Although the nucleolus is primarily associated with ribosome biogenesis, several lines of evidence now demonstrate that it has additional functions, such as regulation of mitosis, cell-cycle progression and proliferation, many forms of stress response, and biogenesis of multiple ribonucleoprotein particles. Ribosome biogenesis is regulated throughout interphase and ceases during mitosis (Figure 1). Thus, there is a direct relationship between cell growth and nucleolar activities. Nucleoli are well known to be dramatically modified in cancer cells. Additionally, a large number of key proteins from both DNA- and RNA-containing viruses are localized in the nucleolus, including the human immunodeficiency virus (HIV)-1 Rev and Tat proteins. Targeting of viral proteins to the nucleolus not only facilitates virus replication, but may also be required for pathogenic processes. The nucleolus can also be considered a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm through its disruption.

A unique class of impurity-based quasi-liquid films has been widely observed at free surfaces, grain boundaries (GBs), and hetero-phase interfaces in ceramic and metallic materials (Figure 1). These nanometer-thick interfacial films can be alternatively understood to be: (a) quasi-liquid layers that adopt an “equilibrium” thickness in response to a balance of attractive and repulsive interfacial forces (in a high-temperature colloidal theory) or (b) multilayer adsorbates with thickness and average composition set by bulk dopant activities [1–2]. In several model binary systems, such quasi-liquid, interfacial films are found to be thermodynamically stable well below the bulk solidus lines, provoking analogies to the simpler interfacial phenomena of premelting in unary systems [3] and prewetting in binary de-mixed liquids [4]. These interfacial films exhibit structures and compositions that are neither observed nor stable as bulk phases, as well as transport, mechanical, and physical properties that are markedly different from bulk phases.

It is well known that there is a profitable cooperation between archaeology and the scientific disciplines of chemistry, geology, biology, and physics with the aim of producing better interpretations of archaeological materials. This field of science is known as archaeometry. Two main goals of archaeometry are to analyze and characterize historic objects to preserve them and to investigate the knowledge and skills required to fabricate them. This latter information is essential in the evaluation of cultural and technological aspects of past societies and to further understand the transference of technological knowledge through time periods and geographical contexts.

The Hague, Netherlands, 1690. Christiaan Huygens and Sir Isaac Newton, at odds with their competing wave/particle theories of light, today reached a compromise with their unveiling of a new multibeam SEM/FIB instrument featuring an SEM column designed by Huygens that uses electromagnetic lenses to focus the beam of electrons and a FIB column employing electrostatic lenses to focus the beam of ions devised by Newton. Onlookers were dazzled by its extensive capabilities and range of applications.

Beam Deceleration is a relatively simple method to reduce electron beam energy and improve imaging parameters such as resolution and contrast. The scanning electron microscope (SEM) uses a sharply focused electron beam to probe the specimen surface. The energy of the electrons forming such a probe is determined by the electrical potential of the electron source, referred to as accelerating voltage or high voltage (HV). No matter how many times the electrons are accelerated or decelerated inside the column, they leave the column with an energy corresponding to the high voltage. The high voltage is usually controllable within a range of 200 V to 30 kV for most commercially available SEMs, allowing the operator to select the electron beam energy suitable for the application. Imaging with very low electron beam energy has great importance, which is illustrated by SEM instrumentation development over the last few decades [1–2]. Low voltage microscopy is a topic discussed at most microscopy-related conferences these days, but generally, it is approached with an immersion lens and field emission gun (FEG) SEM system because of the better beam current densities. However, beam deceleration is also a means to bring low kV improvement to SEMs with thermionic electron sources.

Capabilities for in-situ studies of materials at elevated temperatures and under gaseous environments have received increasing attention in recent years [1]. With the advent of electron microscopes that provide routine imaging at the atomic level (e.g. aberration-corrected TEM and STEM instruments), it is of particular interest to be able to record images at high temperatures while retaining the inherent resolution of the microscope; that is, the resolution is not limited by drift in the heating holder or other instabilities associated with its operation. A number of commercial and experimental heating devices have been used over the years; some holders are designed with miniature furnaces that heat entire grids [2], while a more recent development used a tiny spiral filament coated with a carbon film as the heater element [3]. These devices, while very useful for some applications (particularly in “environmental microscopes” that employ differential pumping to allow gases at some elevated pressure to be injected around the specimen), are invariably not as stable as might be desired for sub-Ångström imaging experiments. They are also limited by the speed at which the sample can be heated to temperature for stable operation. In collaboration with Protochips Inc. (Raleigh, NC), our laboratory is developing a novel new technology for in-situ heating experiments that overcomes a number of performance problems associated with standard heating stage technologies [4].

The analysis was finally completed, the data graphed, and I had to admit to myself, it was looking like an auspicious moment. Only a few weeks earlier, Charlie Lyman and I had been discussing a project that I had been working on for some time, one in which I was admittedly being deliberately vague concerning the details. However, I had promised to keep him updated when it succeeded. Given the date (April 1st) and the data in hand, I couldn't resist sharing with him (and a few colleagues) the first results from that experiment—and my thinly veiled attempt at a quasi-April Fools Day joke. After we exchanged a number of emails that day, Charlie concluded his last message with the line that many of us have heard from both him and Ron Anderson: “A little more text and you can have an article in the July issue of MT.” Have I gotten you curious? Well then read on!