StayGold photostability under different illumination modes.

StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated.

StayGold variants for molecular fusion and membrane-targeting applications.

Although StayGold is a bright and highly photostable fluorescent protein, its propensity for obligate dimer formation may hinder applications in molecular fusion and membrane targeting. To attain monovalent as well as bright and photostable labeling, we engineered tandem dimers of StayGold to promote dispersibility. On the basis of the crystal structure of this fluorescent protein, we disrupted the dimerization to generate a monomeric variant that offers improved photostability and brightness compared to StayGold.

A highly photostable and bright green fluorescent protein.

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen.

Single-cell bioluminescence imaging of deep tissue in freely moving animals.

Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals.

Visualization of an endogenous retinoic acid gradient across embryonic development.

In vertebrate development, the body plan is determined by primordial morphogen gradients that suffuse the embryo. Retinoic acid (RA) is an important morphogen involved in patterning the anterior-posterior axis of structures, including the hindbrain and paraxial mesoderm. RA diffuses over long distances, and its activity is spatially restricted by synthesizing and degrading enzymes.

Diffusion of large molecules into assembling nuclei revealed using an optical highlighting technique.

The nuclear envelope (NE) defines the nuclear compartment, and nuclear pore complexes (NPCs) on the NE form aqueous passages through which small water-soluble molecules can passively diffuse. It is well known that proteins smaller than 50 kDa can diffuse though NPCs, whereas proteins larger than 60 kDa rarely enter by passive diffusion. Little, however, is known about how this size cutoff develops as the NE reassembles and the nucleus expands.

Development of microscopic systems for high-speed dual-excitation ratiometric Ca2+ imaging.

For quantitative measurements of Ca(2+) concentration ([Ca(2+)]), ratiometric dyes are preferable, because the use of such dyes allows for correction of uneven loading or partitioning of dye within the cell as well as variations in cell thickness. Although dual-excitation ratiometric dyes for measuring [Ca(2+)], such as Fura-2, Fura-Red, and ratiometric-pericam, are widely used for a variety of applications, it has been difficult to use them for monitoring very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cells. To overcome this problem, we have developed three new dual-excitation ratiometry systems.

Engineering FRET constructs using CFP and YFP.

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Here we discuss how to engineer constructs for FRET between the cyan- and yellow-emitting variants of green fluorescent protein (GFP) from Aequorea victoria (CFP and YFP, respectively). Throughout this chapter, we stress the fact that FRET is highly sensitive to the relative orientation and distance between the donor and the acceptor.

Identification of mitochondrial DNA polymorphisms that alter mitochondrial matrix pH and intracellular calcium dynamics.

Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus.

Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer.

Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.

Fast dual-excitation ratiometry with light-emitting diodes and high-speed liquid crystal shutters.

Dual-excitation ratiometric dyes permit quantitative measurements of Ca2+ concentrations ([Ca2+]s), by minimizing the effects of several artifacts that are unrelated to changes in [Ca2+]. These dyes are excited at two different wavelengths, and the resultant fluorescence intensities are measured sequentially. Therefore, it is difficult to follow fast [Ca2+] dynamics or [Ca2+] changes in highly motile cell samples.

Slow Ca2+ dynamics in pharyngeal muscles in Caenorhabditis elegans during fast pumping.

The pharyngeal muscles of Caenorhabditis elegans are composed of the corpus, isthmus and terminal bulb from anterior to posterior. These components are excited in a coordinated fashion to facilitate proper feeding through pumping and peristalsis. We analysed the spatiotemporal pattern of intracellular calcium dynamics in the pharyngeal muscles during feeding.

Optical recording of oscillatory neural activities in the molluscan brain.

The procerebrum (PC) of the terrestrial slug shows a coherent oscillatory activity. Information is encoded in the PC by neurons with synchronized oscillatory activity, and the oscillatory activity will propagate to other brain regions as the PC transmits information. Using the optical recording of membrane potentials and the correlation analysis, we showed that the metacerebrum/mesocerebrum (MC) region also shows an oscillatory activity coherent with that of the PC.

Simultaneous dual-excitation ratiometry using orthogonal linear polarized lights.

Dual-excitation ratiometric dyes permit quantitative Ca2+ measurements by minimizing the effects of several artifacts that are unrelated to changes in the concentration of free Ca2+ ([Ca2+]). These dyes are excited alternately at two different wavelengths, and the pair of intensity measurements must be collected sequentially. Therefore, it is difficult to follow very fast Ca2+ dynamics or Ca2+ changes in highly motile cell samples.

Confocal imaging of subcellular Ca2+ concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein.

Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca(2+) indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin.