Modern computational approaches based on quantum mechanical methods to characterize structures and optical spectra of biological chromophores in the gas phase, in solutions and proteins are discussed. Primary attention is paid to the chromophores from the family of the green fluorescent protein (GFP) widely used as a biomarker in living cells. Beyond GFP, photophysical properties of the monomeric teal fluorescent protein (mTFPI) and the kindling fluorescent protein asFP595 are simulated. We apply modern quantum chemical approaches for high level calculations of the structures of the chromophore binding pockets and to estimate spectral bands corresponding to the S₀-S₁ optical transitions. A special attention is paid to evaluate effects of point mutations in the vicinity of the chromophore group. Theoretical data provide important information on the chromophore properties aiming to interpret the results of experimental studies of fluorescent proteins.

We study 2PA spectra of red fluorescent proteins (FPs), including DsRed2, mRFP, tagRFP, and mFruits in a wide range of excitation wavelengths, 600 - 1200 nm. For evaluation of mature FP extinction coefficient and concentration we propose a pure optical method which is based on Strickler - Berg equation, relating fluorescence radiative lifetime with molecular extinction coefficient. 2PA spectra and maximum cross sections are very sensitive to either changes in the chromophore structure (mOrange vs mRFP) or mutations in chromophore surrounding (DsRed and mFruits). All red FPs show two pronounced 2PA transitions, the first peaking in the 1000 - 1100-nm region, and the second - near 700 - 760 nm. For each region we have found a mutant, which is 3 - 4 times two-photon brighter than the benchmark EGFP.

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.

Biosensors designed on the principle of fluorescent resonance energy transfer (FRET) have been widely applied to visualize signaling cascades in live cells with high spatiotemporal resolution. In this paper, we review the work in our lab related to the application of FRET biosensors in studying molecular events in live cells, and our work using computational analysis methods to explore complex biological information implicated in FRET images. Membrane-tethered Src biosensors were used to visualize the dynamics of Src activity in subcellular microdomains. We have developed a finite element (FE) method to analyze the movement of biosensors. Based on fluorescence recovery after photobleaching (FRAP) experiments, the estimation and subtraction of biosensor diffusion revealed a high Src activity at cell periphery upon growth factor stimulation. In addition to Src, a RhoA biosensor was used to study the subcellular feature of RhoA activity in migrating HeLa cells. We have developed an image registration method to automatically track and quantify the FRET signals within user-defined subcellular regions, and classify the dynamics of subcellular pixels according FRET signals. The results revealed that the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. Therefore, FRET biosensors integrated with computational analysis provide powerful tools to precisely decode the complex dynamics of signaling transduction regulated in subcellular locations of live cells.

Fluorescent proteins (FPs) are extremely useful tools for whole-cell, tissue, and animal labeling. For these purposes, FPs may be monomeric or oligomeric, but should meet the criteria of being tolerated at high expression levels in cells and having desirable photophysical properties. Our goal was to create a variant of DsRed-Express that maintains the brightness, fast-maturation, and photostability of this protein, while exhibiting decreased cytotoxicity. For this purpose, we mutated the surface of DsRed-Express to decrease aggregation and created DsRed-Express2. DsRed-Express2 retains the favorable photophysical properties of DsRed-Express while showing dramatically reduced cytotoxicity and higher expression in bacterial and mammalian systems. Further, it was shown that DsRed-Express2 outperforms other red FPs as a label for bacterial and mammalian cells.

In this paper, nebulized or intravenous cetuximab (also known as Erbitux) labeled with NIR dyes is administered in the lungs of the mouse and imaged using a time-domain fluorescence imaging system (Optix^(R)). Time resolved measurements provide lifetime of the fluorescent probes. In addition, through time-of-flight information contained in the data, one can also assess probe localization and concentration distribution quantitatively. Results shown include suppression of tissue autofluorescence by lifetime gating and recovery of targeted and non-targeted distributions of cetuximab labeled with the NIR fluorophores.

Fluorescence imaging is an important tool for tracking molecular-targeting probes in preclinical studies. It offers high sensitivity, but nonetheless low spatial resolution compared to other leading imaging methods such CT and MRI. We demonstrate our methodological development in small animal in vivo whole-body imaging using fluorescence tomography. We have implemented a noncontact fluid-free fluorescence diffuse optical tomography system that uses a raster-scanned continuous-wave diode laser as the light source and an intensified CCD camera as the photodetector. The specimen is positioned on a motorized rotation stage. Laser scanning, data acquisition, and stage rotation are controlled via LabVIEW applications. The forward problem in the heterogeneous medium is based on a normalized Born method, and the sensitivity function is determined using a Monte Carlo method. The inverse problem (image reconstruction) is performed using a regularized iterative algorithm, in which the cost function is defined as a weighted sum of the L-2 norms of the solution image, the residual error, and the image gradient. The relative weights are adjusted by two independent regularization parameters. Our initial tests of this imaging system were performed with an imaging phantom that consists of a translucent plastic cylinder filled with tissue-simulating liquid and two thin-wall glass tubes containing indocyanine green. The reconstruction is compared to the output of a finite element method-based software package NIRFAST and has produced promising results.

Cytokinesis is a consecutive process during cell division. For systems biological studies, it is important to precisely monitor and quantify proteins in different cell stages and mitosis processes. However, the absolute quantities in living cells are usually difficult to quantify. Fluorescent protein tagged protein is one of the techniques that are usually applied to monitor biological behaviors and phenomena. In this study, an insect cell line, DPnE, which can stably express both green fluorescent protein (EGFP) and red fluorescent protein (DsRed) was established. This dual fluorescent cell line was chosen as a model system to monitor the protein partition during cytokinesis. A spectrum analysis system was established and integrated in an inverted microscope. The two-dimensional distribution of the full fluorescent spectra of the two fluorescent proteins was obtained in a time-lapse series. Furthermore, we also developed an algorithm to analyze the quantities of both fluorescent proteins in the daughter cells and parent cells during the process of cytokinesis, respectively. With this innovative optical system and algorithm, the proteins partition during cytokinesis can be monitored and quantified precisely.

Fluorescent proteins have become extremely popular tools for in vivo imaging as well as the study of localization, motility and interaction of proteins in living cells. Bimolecular fluorescence complementation (BiFC) analysis based on fluorescent proteins enables direct and high throughput visualization of protein-protein interactions in living cells. Two red Bimolecular Fluorescent Complementation (BiFC) systems based on mRFP variants have been reported. However, some physical-chemical characteristics of mRFP limited their applications, such as low pH-stability, relative low brightness and low maturation rate. We have developed a new red BiFC system based on TagRFP, a novel monomeric red fluorescent protein with high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. In this study, bFos and bJun were used as the positive protein-protein interaction pair, a mutant of bFos (bFos) and bJun were used as the negative protein-protein interaction pair. bFos/ΔbFos was fused to N-terminal fragment of TagRFP, and bJun was fused to C-terminal fragment of TagRFP. The BiFC systems based on TagRFP was confirmed in living mammalian cells. Furthermore, the BiFC based on TagRFP allow analyzing multi-protein interactions when combined with other BiFC systems. Thus, the BiFC based on TagRFP is very useful for investigating the complicated and significant molecular mechanisms of multi-protein complex in living cells.

The Stagger Extension Process (StEP), a recombination of DNA technique, has been used as a rapid molecular mutagenesis strategy. In this study, for obtaining the fluorescence proteins with new properties, six fluorescence proteins (EYFP, EGFP, ECFP, mCitrine, mCerulean and Venus) were used as the templates to recombine the mutation library by the Stagger Extension Process (StEP) technique. Through screening this mutation library, we have obtained some useful new FPs which are different fluorescent properties with ancestor. These protein will extend fluorescent proteins application.