George H. Patterson

Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy

We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.

Advances in fluorescent protein technology.

Current fluorescent protein (FP) development strategies are focused on fine-tuning the photophysical properties of blue to yellow variants derived from the Aequorea victoria jellyfish green fluorescent protein (GFP) and on the development of monomeric FPs from other organisms that emit in the yellow-orange to far-red regions of the visible light spectrum. Progress toward these goals has been substantial, and near-infrared emitting FPs may loom over the horizon. The latest efforts in jellyfish variants have resulted in new and improved monomeric BFP, CFP, GFP and YFP variants, and the relentless search for a bright, monomeric and fast-maturing red FP has yielded a host of excellent candidates, although none is yet optimal for all applications. Meanwhile, photoactivatable FPs are emerging as a powerful class of probes for intracellular dynamics and, unexpectedly, as useful tools for the development of superresolution microscopy applications.

Photobleaching in two-photon excitation microscopy.

The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.

Quantitative imaging of the green fluorescent protein (GFP).

Publisher Summary This chapter discusses the properties of green fluorescent protein (GFP) that are important for quantitative imaging. Spectral and physical properties of GFP affect the accuracy and usefulness of any quantitative measurement. Many of these properties, such as extinction coefficient, quantum yield, photobleaching rate, and pH dependence, can be measured with purified GFP in vitro. However, other important properties, especially the time course of chromophore formation and protein degradation in vivo , cannot be easily determined. In general, one chooses the brightest, most photostable GFP available, which may make complicated corrections for background and photobleaching unnecessary in less demanding applications. The chapter also discusses the properties of the fluorescence microscope that are important in quantitative imaging, such as microscope components (objective lenses, fluorescence filters, etc.), signal-to-noise ratio, detection linearity, and fluorophore saturation. Because of the large number of GFP mutants and the variety of potential biological applications, a comprehensive description of all possible quantitative imaging situations is not possible. Thus, most descriptions of methods for quantitative imaging will be limited to the use of fluorescein-like GFP mutants with laser scanning confocal microscopy.

Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation.

The ability to analyze independently the expression of multiple reporter gene constructs within single cells is a potentially powerful application of flow cytometry. In this paper, we explore the simultaneous detection of two variants of the reporter molecule, green fluorescent protein (GFP) that both fluoresce when excited with 488-nm light. One of these, enhanced GFP (EGFP) (excitation max. 490 nm; > 90% efficiency at 488 nm), has been widely used for studies that involve flow cytometric detection of reporter gene expression. As a partner for EGFP, we employed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm; approximately 35% efficiency at 488 nm). Using 488-nm excitation, EYFP fluorescence could be readily detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGFP. Importantly, we describe an optical filter configuration that permits the fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configuration employed a 510/20-nm bandpass filter for EGFP detection, a 550/30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dichroic mirror to separate the two signals. With these filters, expression of either reporter protein could be detected, alone or in combination, within a mixed population of cells over a broad range of signal intensities.

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue.

Significance Superresolution microscopy has made much progress in improving resolution and imaging speed over the past several years, but the ability to image below the diffraction limit in thick scattering specimens has not kept pace. In many interesting samples, such as Caenorhabditis elegans, Drosophila melanogaster, mouse, or human tissues, resolution is limited primarily by scattering rather than diffraction. In this paper, we show that the combination of multiphoton excitation with multifocal structured illumination microscopy gives high quality resolution-doubled images even in thick opaque samples, which until now have resisted superresolution techniques. Since the majority of model organisms and human tissues are opaque to some degree, this advance brings superresolution imaging to a substantial fraction of biological problems. Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 µm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.

Quantitative imaging of TATA-binding protein in living yeast cells

We describe the quantitative monitoring of TATA‐binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) · TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP · TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP · TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real‐time quantitation in living cells and subcellular localization information. Time‐lapse confocal imaging of GFP · TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0·7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP · TBP, we suggest that intracellular levels of TBP are near rate‐limiting for growth and viability.

Two-photon-like microscopy with orders-of-magnitude lower illumination intensity via two-step fluorescence

We describe two-step fluorescence microscopy, a new approach to non-linear imaging based on positive reversible photoswitchable fluorescent probes. The protein Padron approximates ideal two-step fluorescent behaviour: it equilibrates to an inactive state, converts to an active state under blue light, and blue light also excites this active state to fluoresce. Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose. Here, we use Padrons quadratic non-linearity to demonstrate the principle of two-step microscopy, similar in principle to two-photon microscopy but with orders-of-magnitude better cross-section. As with two-photon, quadratic non-linearity from two-step fluorescence improves resolution and reduces unwanted out-of-focus excitation, and is compatible with structured illumination microscopy. We also show two-step and two-photon imaging can be combined to give quartic non-linearity, further improving imaging in challenging samples. With further improvements, two-step fluorophores could replace conventional fluorophores for many imaging applications.

A New Temporal Dimension for Multisignal Sedimentation Velocity as a Tool to Analyze Multicomponent Interactions

Multisignal sedimentation velocity (MSSV) analysis is a powerful method for determining the stoichiometry of reversibly formed multi-protein complexes. For example, it has been used to unravel the number and stoichiometry of co-existing complexes, for example, in receptor-ligand interactions and in systems of adaptor proteins. By globally analyzing absorbance data acquired simultaneously at different wavelengths, differences in the component spectra can be exploited to obtain information on the composition of all hydrodynamically resolved species. For fluorescence data that are acquired only at a single excitation wavelength, it was recently shown that the characteristic quantum yield of photoswitching of reversibly photoswitchable fluorescent proteins leads to a temporal fluorescence signal change on the time-scale of sedimentation, which can substitute for spectral discrimination of components in an analysis analogous to MSSV. In the present work, we have embarked on combining the two concepts by considering the temporal extinction coefficient changes associated with reversible photoswitching in the context of spectral decomposition of components. To this end, we report pilot experiments, for the first time acquiring and analyzing multi-wavelength absorbance data with temporal modulation of extinction amplitudes. Theoretically, the combination of spectral and temporal signal domain is expected to greatly enhance the discrimination of sedimenting components, thereby facilitating the study of multi-protein complexes with a higher number of components in solution.

Synthesis of Caged Q-Rhodamine Dye

The Caged Q-rhodamine derivative, along with the synthesis of the caging group had been prepared using two synthetic approaches. UV-Vis profile showed an absorbance at 365nm which can be used to uncage the dye.