Kristin L. Hazelwood

Improving the photostability of bright monomeric orange and red fluorescent proteins

All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

A bright and photostable photoconvertible fluorescent protein

Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the ∼10-nm localization precision of its parent.

Far-red fluorescent tags for protein imaging in living tissues

A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.

Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors

Fluorescence resonance energy transfer (FRET) with fluorescent proteins is a powerful method for detection of protein-protein interactions, enzyme activities and small molecules in the intracellular milieu. Aided by a new violet-excitable yellow-fluorescing variant of Aequorea victoria GFP, we developed dual FRET–based caspase-3 biosensors. Owing to their distinct excitation profiles, each FRET biosensor can be ratiometrically imaged in the presence of the other.

Photoconversion in orange and red fluorescent proteins.

We found that photoconversion is fairly common among orange and red fluorescent proteins, as in a screen of 12 proteins, 8 exhibited photoconversion. Specifically, three red fluorescent proteins could be switched to a green state, and two orange variants could be photoconverted to a far-red state. The orange proteins are ideal for dual-probe highlighter applications, and they exhibited the most red-shifted excitation of all fluorescent proteins described to date.

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

Components of the minichromosome maintenance complex (Mcm2-7) remain indefinitely bound to chromatin during G1 phase and replication arrest.

Entering the Portal: Understanding the Digital Image Recorded Through a Microscope

The primary considerations in imaging living cells in the microscope with a digital camera are detector sensitivity (signal-to-noise), the required speed of image acquisition, and specimen viability. The relatively high light intensities and long exposure times that are typically employed in recording images of fixed cells and tissues (where photobleaching is the major consideration) must be strictly avoided when working with living cells. In virtually all cases, live-cell microscopy represents a compromise between achieving the best possible image quality and preserving the health of the cells. Rather than unnecessarily oversampling time points and exposing the cells to excessive levels of illumination, the spatial and temporal resolutions set by the experiment should be limited to match the goals of the investigation. This chapter describes the fundamentals of digital image acquisition, spatial resolution, contrast, brightness, bit depth, dynamic range, and CCD architecture, as well as performance measures, image display and storage, and imaging modes in optical microscopy.

Searching the fluorescent protein color palette for new FRET pairs

One of the most promising imaging techniques for monitoring dynamic protein interactions in living cells with optical microscopy, universally referred to as FRET, employs the non-radiative transfer of energy between two closely adjacent spectrally active molecules, often fluorescent proteins. The use of FRET in cell biology has expanded to such a degree that hundreds of papers are now published each year using biosensors to monitor a wide spectrum of intracellular processes. Most of these sensors sandwich an environmentally active peptide between cyan and yellow fluorescent protein (CFP and YFP) derivatives to assay variables such as pH, calcium ion concentration, enzyme activity, or membrane potential. The availability of these sensitive indicators is growing rapidly, but many are hampered by a low dynamic range that often is only marginally detectable over the system noise. Furthermore, extended periods of excitation at wavelengths below 500 nm have the potential to induce phototoxic effects that can mask or alter the biological events under observation. Recent success in expanding the fluorescent protein color palette offers the opportunity to explore new FRET partners that may be suitable for use in advanced biosensors.

Evaluating and improving the photostability of fluorescent proteins

Fluorescent proteins are the most common and versatile class of genetically encoded optical probes. While structure-guided rational design and directed evolution approaches have largely overcome early problems such as oligomerization, poor folding at physiological temperatures, and availability of wavelengths suitable for multi-color imaging, nearly all fluorescent proteins have yet to be fully optimized. We have developed novel methods for evaluating the current generation of fluorescent proteins and improving their remaining suboptimal properties. Little is yet known about the mechanisms responsible for photobleaching of fluorescent proteins, and inadequate photostability is a chief complaint among end users. In order to compare the performance of fluorescent proteins across the visual spectrum, we have standardized a method used to measure photostability in live cells under both widefield and confocal laser illumination. This method has allowed us to evaluate a large number of commonly used fluorescent proteins, and has uncovered surprisingly complex and unpredictable behaviors in many of these proteins. We have also developed novel methods for selecting explicitly for high photostability during the directed evolution process, leading to the development of highly improved monomeric orange and red fluorescent proteins. These proteins, most notably our photostable derivative of TagRFP, have remarkably high photostability and have proven useful as fusion tags for long-term imaging. Our methods should be applicable to any of the large number of fluorescent proteins still in need of improved photostability.

Confocal microscopy on the Internet.

In a few short years, the Internet (in terms of the World Wide Web) has become a powerful informational resource for the original scientific literature pertaining to biological investigations using the laser scanning confocal microscope. However, there still remains an obvious void in the development of educational Web sites targeted at beginning students and novices in the field. Furthermore, many of the commercial aftermarket manufacturers (for example, those offering live-cell imaging chambers) have Web sites that are not adequately represented in published compilations, and are therefore somewhat difficult to locate. In order to address this issue, several educational sites dedicated to optical microscopy and digital imaging that are being constructed and hosted at The Florida State University are currently turning their attention to the increasing application of confocal microscopy in the biological and materials sciences. The primary focus of this effort is to create new sections on the existing sites that address the important educational issues in confocal microscopy, as well as creating indices of links to both the confocal scientific literature and the Web sites of manufacturers who supply useful accessories.