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For over 20 years, genetically encoded Ca2+ indicators have illuminated dynamic Ca2+ signaling activity in living cells and, more recently, whole organisms. We are just now beginning to understand how they work. Various fluorescence colors of these indicators have been developed, including red. Red ones are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a red fluorescent protein that is circularly permuted and fused to a Ca2+-sensing domain. When Ca2+ binds, a conformational change in the sensing domain causes a change in fluorescence. Three factors can contribute to this fluorescence change: 1) a shift in the protonation equilibrium of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient or the two-photon cross section, depending on if it is excited with one or two photons. Here, we conduct a systematic study of the photophysical properties of a range of red Ca2+ indicators to determine which factors are the most important. In total, we analyzed nine indicators, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We find that these could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases, the magnitude of the change in fluorescence was larger with two-photon excitation compared to one-photon because of a change in the two-photon cross section, by up to a factor of two.

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in the mammalian brain and can be readily applied to neuroimaging.

Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP—the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green 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.

Two-photon (2P) microscopy is the preferred method for imaging fluorescent proteins and biosensors in living, thick tissues. Over the past decade, many groups have worked to improve single photon (1P) properties of fluorescent proteins, but little has been done to improve their 2P properties. This is important because 2P properties can be quite different from the 1P properties. Our goal was to create a system for screening libraries of randomly mutagenized fluorescent proteins for better 2P properties. We created an optical system for collecting 1- and 2-photon induced fluorescence images from entire petri dishes of E. coli colonies. Libraries of randomly mutagenized eGFP were screened for improved 2P/1P fluorescence ratios. Screening the first round of evolution showed a slightly larger ratio spread outside the range of parent eGFP. One hundred of these clones were subjected to a second round of screening. Colonies that showed reproducible 2P/1P ratios greater than the parent eGFP were selected to move forward. A selective pool of the best 18 clones (library A), and a more inclusive pool of the best 59 clones (library B), were used for a new round of gene shuffling and random mutagenesis. After only two rounds of evolution, libraries A and B contained at least 5 distinct mutant populations with significantly increased 2P/1P ratios. Sequence analysis of 96 clones from these libraries revealed that every clone harbored at least one mutation. Eleven unique mutants were used for a third round of evolution. Screening and sequence analysis of the third library, reveals thirteen mutations that either individually (V68M, S72G, V163A, T203I) or in combination (E6G, T65S, Q80R, N105S, D117G, N121S, Q184R, S202N, V219I) significantly shift the 2P/1P fluorescence ratios.

Fluorescent proteins (FPs) are widely used in two-photon microscopy as genetically-targeted probes. Understanding the mechanisms of their two-photon absorption (2PA) and photobleaching is therefore of prime importance for creation of brighter and more photostable mutants.We study the effect of the local electrostatic environment of the FP chromophore on its optical properties and photostability. To this end, we select the series of red FPs, including DsRed and a number of its Fruits derivatives, all possessing the same red anionic chromophore but different local surrounding.2PA spectroscopy provided us a direct experimental access to the absolute values of the local electric field on the chromophore site [1]. Using crystallography data available for several of the proteins, we perform here a series of QM/MM calculations of local electric potentials and fields on the chromophore site and determine the “hot” spot on the chromophore where the calculated fields almost quantitatively match the experimental data. This procedure also allows us to validate the protonation state of certain amino acids near the chromophore.We further analyze our experimental 2PA spectra of the Fruits [2] and show that the local electric field at the “hot” spot of the chromophore drives their 2PA absorptivity. The variations of the field result in different vibronic band intensities and eventually are responsible for the 2PA (vibronic) peak amplitude.We finally present our experimental data on photobleaching kinetics and demonstrate that there is a correlation between the rate of chromophore degradation and local electrostatic potential on the chromophore.Our results suggest the directions of mutagenesis that can simultaneously improve the 2PA brightness and photostability.

Two-Photon laser scanning microscopy, which makes use of genetically encoded fluorescent protein (FP) probes, is becoming a method of choice for studying biological systems from sub-cellular to the whole body level. However, reliable information on two-photon absorption (2PA) properties of FPs, specially for the more popular orange and red variants, is still very fragmentary. 2PA spectra, measured in absolute cross section values, will allow us to select the two-photon brightest FP variant with desired fluorescence properties and also to choose the optimum laser system and excitation wavelength. Here we study 2PA spectra of a large set of orange and red FPs, including DsRed2, mRFP, TagRFP, and mFruits series in a wide range of excitation wavelengths, 600 -1200 nm. We have found the the 2PA spectra and maximum cross sections are very sensitive to either changes in chromophore structure (mOrange vs mRFP) or to mutations in chromophore surrounding (DsRed and mFruits series). All red FPs show two pronounced 2PA transitions, the first peaking in the 1000 - 1100 nm region, and second - near 700 - 760 nm. We quantitatively describe the first transition within the framework of two-level model, and the second - within three-level model with strong resonance enhancement. Excitation in the longer wavelength region, accessible for Nd- and Yb-doped short-pulse lasers, has advantages of producing less two-photon autofluorescence and deeper penetration. For each region of wavelengths, we have found a mutant, which is 3-4 times two-photon brighter than the benchmark EGFP.

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 in series of mutants with the same chromophore structure, but different surrounding, is however not understood. Here we study the FP series with acylimine-containing red chromophore, in which the absorption maximum varies from 540 nm to 590 nm, and a series of green FPs with phenolate chromophore, where the absorption peak shifts from 450 to 500 nm. We use two-photon absorption spectroscopy to show that the different colors in each series can be explained by quadratic Stark effect due to variations of the strong local electric field within the beta barrel. The model allows us to experimentally access the chromophore parameters, such as vacuum transition frequency (ν0) and vacuum changes of permanent dipole moment (Δμ0) and polarizability (Δα0) upon excitation. Using this purely experimental and all-optical approach, we estimate, for the first time to our knowledge, the amplitudes of the internal electric field (namely its projection on Δμ0) in a protein. These values amount 10 to 100 MV/cm in the mFruits series. Although these fields appear to be very large, they fall well in the range previously estimated theoretically for different other proteins, and are still 1 −2 orders of magnitude smaller than the fields required to ionize the chromophore. Our model brings simplicity to a bewildering diversity of fluorescent protein properties, and it suggests a new way to sense electrical fields in biological systems. On the other hand, it opens up the way to create two-photon brighter FP probes by tuning internal electric field with smart mutagenesis around the chromophore.

Two-photon laser scanning microscopy (TPLSM) has several advantages over one-photon confocal microscopy, including deeper tissue penetration, higher signal-to-background ratio, and less photodamage in the out-of-focus volume. However, due to very high instantaneous light intensities in the focal volume, the probability of further, stepwise resonant photon(s) absorption increases dramatically, leading to very efficient bleaching of a probe. To deal with this challenge one has to understand the underlying mechanisms. Here we measured the power dependence of multiphoton bleaching rates of several red fluorescent proteins expressed in live E. coli cells under two-photon microscope conditions. To clarify the photophysical mechanisms, we also used much lower repetition rate (1 kHz) and different pulse durations in experiments in vitro with Ti:Sa amplifier excitation. Our experimental data supported by quantum mechanical calculations of the chromophore in protein environment are consistent with the mechanism of ultrafast (