Shane Tillo

Two-photon absorption properties of fluorescent proteins

Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.

Absolute Two-Photon Absorption Spectra and Two-Photon Brightness of Orange and Red Fluorescent Proteins

Fluorescent proteins with long emission wavelengths are particularly attractive for deep tissue two-photon microscopy. Surprisingly, little is known about their two-photon absorption (2PA) properties. We present absolute 2PA spectra of a number of orange and red fluorescent proteins, including DsRed2, mRFP, TagRFP, and several mFruit proteins, in a wide range of excitation wavelengths (640-1400 nm). To evaluate 2PA cross section (sigma(2)), we use a new method relying only on the optical properties of the intact mature chromophore. In the tuning range of a mode-locked Ti:sapphire laser, 700-1000 nm, TagRFP possesses the highest two-photon cross section, sigma(2) = 315 GM, and brightness, sigma(2)phi = 130 GM, where phi is the fluorescence quantum yield. At longer wavelengths, 1000-1100 nm, tdTomato has the largest values, sigma(2) = 216 GM and sigma(2)phi = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3-4 times improvement in two-photon brightness.

Color Hues in Red Fluorescent Proteins Are Due to Internal Quadratic Stark Effect

Intrinsically fluorescent proteins (FPs) exhibit broad variations of absorption and emission colors and are available for different imaging applications. The physical cause of the absorption wavelength change from 540 to 590 nm in the Fruits series of red FPs has been puzzling because the mutations that cause the shifts do not disturb the pi-conjugation pathway of the chromophore. Here, we use two-photon absorption measurements to show that the different colors can be explained by quadratic Stark effect due to variations of the strong electric field within the beta barrel. This model brings simplicity to a bewildering diversity of fluorescent protein properties, and it suggests a new way to sense electrical fields in biological systems.

A new approach to dual-color two-photon microscopy with fluorescent proteins

BackgroundTwo-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs) is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA) efficiency.ResultsHere we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein.ConclusionOur method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation

A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores-rhodamine B, a red fluorescent protein, and CdSe quantum dots-while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.

Live Imaging of Endogenous PSD-95 Using ENABLED: A Conditional Strategy to Fluorescently Label Endogenous Proteins

Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.

Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates

Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons.

How to Enhance the Two-Photon Brightness of Fluorescent Proteins?

Fluorescent proteins (FPs) are widely used in 2-photon absorption (2PA) microscopy as genetically-targeted probes. We provide the guidelines for increasing their peak 2PA cross section by tuning (via mutations) local electric field inside protein.

Herzberg-Teller Vibronic Contribution to Mesomeric Dipole Moment Determines Two-Photon Absorptivity of Fluorescent Proteins

Fluorescent proteins (FPs) are widely used in two-photon microscopy as genetically-targeted bio-probes. The physical basis of large variability of their two-photon absorption (2PA) brightness is however not understood.We have recently demonstrated that the mFruits series of FPs, having the same red anionic chromophore, show the 2PA band in the region of S0 - S1 electronic transition, corresponding to 900 - 1200 nm of laser wavelength. In this 2PA band, the vibronic 0-1 transition is stronger than the 0-0 transition, in contrast to one-photon absorption spectrum where the 0-0 transition dominates. It is also intriguing that the strength of the dominant vibronic 2PA transition strongly depends on the surrounding of the chromophore.Here we perform a comprehensive analysis of the 2PA spectral profiles of Fruits FPs. We show that a crucial factor which drives their optical properties is the local electric field at the chromophore site (varying from one mutant to another). Variation of the field promotes the shift of equilibrium between the two resonating forms of the chromophore π-conjugation structure, which, in turn, results in systematic changes of mesomeric dipole moment (the difference between the dipole moments in the excited and ground states, Δμ) and of the single-to-double bond-length alternations (BLA).Because the two-photon tensor of the S0 - S1 transition is proportional to Δμ2, wesuggest an interesting physical effect implying strong Herzberg-Teller coupling of Δμ with the BLA coordinate. This effect can only be observed in 2PA spectrum.Our model quantitatively explains the vibronic enhancement in 2PA spectrum of Fruits FPs and also provides the upper limit estimation for the 2PA peak cross section of any FP possessing red anionic chromophore. It also can guide mutagenesis efforts toward improvement of two-photon brightness of FPs.

Internal quadratic stark effect results in color hue variations in fluorescent proteins with the same chromophore structure

Genetically-encoded fluorescent proteins are widely used for bio-imaging. We employ two-photon absorption spectroscopy to show that their different hues can be explained by quadratic Stark effect due to variations of electric field within the protein.