Multiphoton microscopy: a personal historical review, with some future predictions.

The historical development of multiphoton microscopy is described, starting with a review of two-photon absorption, and including two- and three-photon fluorescence microscopies, and second- and third-harmonic generation microscopies. The effects of pulse length on signal strength and breakdown are considered. Different contrast mechanisms, including use of nanoparticles, are discussed. Two new promising techniques that can be applied to multiphoton microscopy are described.

Switchable Fluorophores for Single-Molecule Localization Microscopy.

The past decade has witnessed an explosion in the use of super-resolution fluorescence microscopy methods in biology and other fields. Single-molecule localization microscopy (SMLM) is one of the most widespread of these methods and owes its success in large part to the ability to control the on-off state of fluorophores through various chemical, photochemical, or binding-unbinding mechanisms. We provide here a comprehensive overview of switchable fluorophores in SMLM including a detailed review of all major classes of SMLM fluorophores, and we also address strategies for labeling specimens, considerations for multichannel and live-cell imaging, potential pitfalls, and areas for future development.

Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

Light and life in Baltimore--and beyond.

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores.

Better imaging through chemistry.

The 2014 Nobel Prize in Chemistry has been awarded jointly to William E. Moerner, Stefan W. Hell, and Eric Betzig "for the development of super-resolved fluorescence microscopy." I discuss the contributions made by this year's awardees and how advances in understanding the behavior of fluorophores and research in light microscopy converged to allow the improved visualization of biological structures.

Fluorescence microscopy-a historical and technical perspective.

For a little more than a century, fluorescence microscopy has been an essential source of major discoveries in cell biology. Recent developments improved both visualization and quantification by fluorescence microscopy imaging and established a methodology of fluorescence microscopy. By outlining basic principles and their historical development, I seek to provide insight into and understanding of the ever-growing tools of fluorescence microscopy. Thereby, this synopsis may help the interested researcher to choose a fluorescence microscopic method capable of addressing a specific scientific question.

FRET microscopy in 2010: the legacy of Theodor Förster on the 100th anniversary of his birth.

Theodor Förster would have been 100 years old this year, and he would have been astounded to see the impact of his scientific achievement, which is still evolving. Combining his quantitative approach of (Förster) resonance energy transfer (FRET) with state-of-the-art digital imaging techniques allows scientists to breach the resolution limits of light (ca. 200 nm) in light microscopy. The ability to deduce molecular or particle distances within a range of 1-10 nm in real time and to prove or disprove interactions between two or more components is of vital interest to researchers in many branches of science. While Förster's groundbreaking theory was published in the 1940s, the availability of suitable fluorophores, instruments, and analytical tools spawned numerous experiments in the last 20 years, as demonstrated by the exponential increase in publications. These cover basic investigation of cellular processes and the ability to investigate them when they go awry in pathological states, the dynamics involved in genetics, and following events in environmental sciences and methods in drug screening. This review covers the essentials of Theodor Förster's theory, describes the elements for successful implementation of FRET microscopy, the challenges and how to overcome them, and a leading-edge example of how Förster's scientific impact is still evolving in many directions. While this review cannot possibly do justice to the burgeoning field of FRET microscopy, a few interesting applications such as threecolor FRET, which greatly expands the opportunities for investigating interactions of cellular components compared with the traditional two-color method, are described, and an extensive list of references is provided for the interested reader to access.

Introduction to fluorescence microscopy.

This chapter is an overview of basic principles of fluorescence microscopy, including a brief history on the invention of this type of microscopy. The chapter highlights important points related to properties of fluorochromes, resolution in fluorescence microscopy, phase contrast and fluorescence, fluorescence filters, construction of a fluorescence microscope, and tips on the correct use of this equipment.

Two-photon excitation fluorescence microscopy.

Two-photon fluorescence microscopy is one of the most important recent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast images and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine.