Dissertations / Theses on the topic 'Two-photon excitation fluorescence of proteins'

Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions. The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso. The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding. We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur. In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence.

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed [1]. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

We have studied fundamental aspects of time-resolved two-photon excited fluorescence depolarisation. The thesis presents experimental as well as theoretical progress. We show that a multi-photon induced instrumental response function obtained from a suspension of gold nanoparticles is appropriate for the analysis of two-photon excited fluorescence decays obtained using time-correlated single photon counting detection. Theoretical expressions have been derived for the fluorescence anisotropy decay obtained upon two-photon excitation of various molecular systems in liquid solutions: a) an anisotropic rigid rotor that undergoes rotational diffusion in the presence of ultrafast unresolved restricted reorientations, e.g. librations. b) a molecular group covalently attached to a stationary macromolecule, and undergoing local reorientation in a uniaxial ordering potential. A new approach to the analysis of two-photon excited fluorescence depolarisation experiments was developed, which combines data obtained by using linearly and circularly polarised excitation light, in a global manner. In the analysis, knowledge about unresolved reorientations was obtained from one-photon excitation studies of the corresponding systems. By means of this procedure it has been possible to obtain quantitative information about the molecular two-photon absorption tensor for perylene and two of its derivatives. Thereby the symmetry of the final excited and intermediate vibronic states could be assigned. The analysis reveals that the two-photon transition studied with the 800 nm laser exhibits mixed character. An important finding from the experiments was that the two-photon absorption tensor appears to be solvent dependent. Furthermore, the thesis presents the first theoretical treatment of two-photon excited donor-donor energy migration in the presence of molecular reorientation and which applies the extended Förster theory. Explicit expressions for molecules that belong to the point groups D_2h, D₂ and C_2v are given. Preliminary experiments are finally also reported on a two-photon excited donor-donor energy migration system consisting of a bisanthryl-bisteroid.

Fluorescence microscopy allows for spatial and temporal resolution of systems which are inherently fluorescent or which can be selectively labeled with fluorescent molecules. Temporal resolution is crucial for imaging real time processes in living samples. A common problem in fluorescence microscopy of biological samples is autofluorescence, fluorescence inherent to the system, which interferes with detection of fluorescence of interest by decreasing the signal to noise ratio. Two current methods for improved imaging against autofluorescence are two-photon excitation and total internal reflection microscopy. Two-photon excitation occurs when two longer wavelength photons are absorbed quasi-simultaneously by a single fluorophore. For this to take place there must be a photon density on the order of 1030 photons/(cm2)(s), which is achieved through use of a femtosecond pulsed laser and a high magnification microscope objective. Two-photon excitation then only occurs at the focal spot, significantly reducing the focal volume and therefore background autofluorescence. The second method, total internal reflection, is based on evanescent wave excitation, which decreases exponentially in intensity away from the imaging surface. This allows for excitation of a thin (~200 nm) slice of a sample. Since only a narrow region of interest is excited, an optical slice can be imaged, decreasing excitation of out-of-focus autofluorescence, and increasing the signal to noise ratio. By coupling total internal reflection with two-photon excitation, an entire cell can be imaged while still maintaining the use of lower energy photons to irradiate the sample and achieve two-photon excitation along the length traveled by the evanescent wave. This system allows for more sensitive detection of fluorescence of interest from biological systems as a result of a significant decrease in excitation volume and therefore a decrease in autofluorescence signal. In the two-photon total internal reflection microscopy setup detailed in this work, an excitation area of 20 μm by 30 μm is achieved, and used to image FITC-stained actin filaments in BS-C-1 cells

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of 1-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular features of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality which overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. In this thesis, I describe the development of 2PLSM combined with STED microscopy for superresolution fluorescence imaging of neurons embedded in thick tissue. Furthermore, I describe the application of this method to studying the biophysics connecting synaptic structure and function in dendritic spines.

Fluorescent nucleic acid base analogues (FBAs) are an important class of molecule used to study the structure and dynamics of DNA and RNA. These base analogues are molecules with structures that resemble one of the natural bases but which, unlike the natural bases, have high fluorescence quantum yields. 2-Aminopurine (2AP) has long been the most widely used fluorescent base analogue and is one of the few base analogues commercially available. One problem with 2AP is that it undergoes significant quenching when incorporated into DNA: the quantum yield decreases 100 fold from that of the free base, thus becoming too low for use in, for example, single molecule studies. A secondary problem is that the 305 nm absorption peak requires excitation in the UV. A variety of new fluorescent base analogues are being produced, with a view to remedying the deficiencies of 2AP and expanding the current range of use. The first part of this thesis explores the one-photon photophysical properties of several of these novel FBAs. The first of these novel FBAs is the 6-aza-uridine family. These compounds, analogues of uridine, have large Stokes shifts and their absorption and emission spectra are red-shifted in comparison to 2AP; their quantum yields as free bases have been shown to exceed that of 2AP and their environmental sensitivity has been demonstrated. Time-resolved measurements reported in this thesis indicate the presence of multiple emitting species. A density functional theory (DFT) study has been carried out to rationalise these emitting species as rotational isomers. Similar fluorescence lifetime measurements were made on a second class of FBAs, the quadracyclic adenine analogues, qANs; these results also indicated the presence of multiple emitting species. Experimental results show that these FBAs undergo excited-state proton transfer. The final FBA studied in this thesis is pentacyclic adenine, pA. This FBA showed some of the most promising characteristics of all the analogues investigated, such as a high quantum yield in both polar and non-polar solvents. A time-resolved investigation into pA-containing oligonucleotides indicated that in an oligonucleotide pA adopts multiple stacked conformations and its behaviour is highly sequence dependent. Several of these aforementioned fluorescent base analogues have absorption spectra in a region that makes them accessible to two-photon (2P) excitation with a Ti:Sapphire laser. In biological systems, multiphoton excitation has several advantages over one-photon excitation. By avoiding the use of ultraviolet light there is reduced phototoxicity. Out of focus photobleaching and autofluorescence are also minimised which leads to a higher signal-to-background ratio and allows deeper tissue penetration to be achieved. Fluorescent base analogues tend to have small Stokes shifts; this is another problem that can be overcome by using two-photon excitation. To be of potential use in multiphoton microscopy, a FBA must have a high two-photon absorption cross-section and a high two-photon brightness. Previously, the highest two- photon brightness measured for a fluorescent base analogue was less than 2 GM. Amongst the base analogues investigated here, are several that have higher two-photon brightness than ever reported for FBAs; these include pA which is shown to have the highest 2P brightness of a FBA in an oligonucleotide, 1.3 GM, and a member of the 6-azauridine family which as a free base has a 2P brightness of 18 GM. Detection of individual molecules represents the ultimate level of sensitivity and enables details about a molecular system that would otherwise be concealed using conventional ensemble techniques to be revealed. With the improved 2P brightness of the molecules measured in this thesis, it has become feasible to detect single FBA molecules using 2P excitation. To maximise the chance of detection, ultrafast, shaped laser pulses have been used as the excitation source. For the first time, the signal has been high enough and the molecule of interest sufficiently photostable such that 2P fluorescence correlation spectroscopy of a fluorescent base analogue in an oligonucleotide could be measured. In summary, this thesis reports the fluorescence lifetimes and two-photon cross-sections of a series of novel fluorescent base analogues, as well as fluorescence correlation spectroscopy measurements of the most promising candidates.

A new and general procedure is described for a detailed analysis of time-resolved fluorescence depolarisation data in the presence of electronic energy migration. An isotropic ensemble of bifluorophoric molecules (D1-R-D2) has been studied to demonstrate its utility. Intramolecular donor-donor energy migration occurs between the two donor groups (D), which are covalently connected to a rigid linker group (R). These groups undergo restricted reorientational motions with respect to the R group. The analysis of depolarisation data basically involves the search for best-fit parameters which describe the local reorienting motions, the interfluorophore D1-D2 distance, as well as the mutual orientations of the donors. For this, the analysis is partly performed in the Fourier domain and the best-fit parameters are determined by using an approach based on a Genetic Algorithm. The energy migration process has been described by using Monte Carlo simulations and an extended Förster theory. It is found that this theory provides the least time-consuming computational method. Since one-photon and two-photon excited fluorescence experiments can be applied for energy migration studies, a general and unified theoretical formulation is given. To exemplify the developed quantitative approach the depolarisation of the fluorescence in the presence of electronic energy migration within a bis-(9-anthrylmethylphosphonate) bisteroid molecule has been studied by time-resolved two-photon excited fluorescence depolarisation experiments. To solely obtain information about local reorientations of the 9-anthrylmethyl group, also the mono-(9-anthrylmethylphosphonate) bisteroid was studied, which enabled modelling of the ordering potential of the donor. Values of the two-photon absorption tensor components were obtained. To describe the discrepancy between the measured values of the initial anisotropy and fundamental anisotropy predicted by theory the distribution of absorption tensor caused by fast processes have been introduced. An angular parameter of absorption tensor was determined. Reasonable values of the distance between the 9-anthrylmethyl groups, as well as for their mutual orientation were obtained.

L’objectif de cette thèse est l’élaboration de sondes organiques pour la microscopie optique non-linéaire par fluorescence excitée à deux photons (F2P) et génération de seconde harmonique (GSH). Dans une première partie, cette thèse décrit la synthèse de sondes pour l’imagerie de potentiels de membrane par GSH, comportant un ou plusieurs motifs sucres ainsi que leurs caractérisations spectroscopiques. Les premiers essais en imagerie biologique ont permis de démontrer une bonne affinité des sondes sucres pour la membrane cellulaire et un signal de GSH sur cellule neuronale a pu être observé sur une période de temps allant jusqu'à près de trois heures. La seconde approche a consisté à synthétiser et étudier des chromophores possédant des propriétés de fluorescence à l’état solide pour des applications dans la synthèse de nanoparticules fluorescentes pour l’imagerie biologique. 18 des 21 composés synthétisés ont pu être cristallisés et leur structure résolue par diffraction des rayons X et les propriétés spectroscopique en solution et à l’état solide ont été réalisées. Cette étude a permis de montrer que l’arrangement des molécules les unes par rapport aux autres avait une grande influence sur la fluorescence à l’état solide et donc que les substituants avaient une grande importance. Enfin, cette partie se termine sur les premiers essais effectués pour synthétiser des nanoparticules fluorescentes
The objective of this thesis is the design of new organic probes for nonlinear optical microscopy by two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). In the first part, we describe the synthesis of probes for voltage sensitive imaging by SHG, bearing one or more sugar units and their spectroscopic characterization. The first biological imaging tests have shown good affinity of the probes to the cell membrane and the SHG signal of neuronal cell was observed over a period of nearly three hours. The second part comprises the synthesis and the study of chromophores with solid state fluorescence properties for use in fluorescent nanoparticles for biological imaging. 18 of the 21 compounds synthesized have been crystallized, their crystal structures determined by X-ray diffraction and their spectroscopic properties studied in solution and in the solid state. These studies showed that the arrangement of molecules relative to each had a great influence on the solid state fluorescence and therefore that the substitution was very important. The chapter ends with the first tests of fluorescent nanoparticles synthesis

Etude par fluorescence des durees de vie radiatives et des probabilites de transition vers l'etat de resonance (ms mp)**(1)p::(1) du zinc et du magnesium, montrant de fortes perturbations des series **(1)d::(2) et **(1)s::(0). Mise en evidence par des calculs theoriques des melanges tres important avec la configuration (mp**(2))

V oblasti nových nízkomolekulárních organických materiálů patří deriváty difenyldiketopyrrolopyrrolu (DPP), používané dříve jako barviva a pigmenty, k objektům vysokého zájmu pro jejich potencionální aplikace v moderních technologiích. Studium jejich optických vlastností ve vztahu k jejich chemické struktuře umožní využití jejich vysokého potenciálu ve vývoji pokročilých inteligentních materiálů. Přehled chemických a fyzikálních vlastností DPP derivátů a zhodnocení současného stavu řešené problematiky jsou uvedeny v teoretické části této práce. Tři hlavní procesy studované v této práci jsou: klasická absorpce a emise, dvoufotonová absorpce (TPA) a zesílená spontánní emise (ASE). Výsledky budou diskutovány a shrnuty ve dvou částech: první zahrnuje první dvě výše zmíněné oblasti a druhá problematiku zesílené spontánní emise.

Ce travail concerne la spectroscopie de la molecule et du cristal de 4-(n, n diethylamino)-beta-nitrostyrene (deanst). Dans un premiere partie, les proprietes de la molecule en solution et en phase vapeur sont etudiees. Un calcul de chimie quantique permet de quantifier le transfert de charge intramoleculaire dont la resonance est observee experimentalement. Ce calcul permet egalement d'obtenir des distributions de charges associees aux dipoles moleculaires. Le spectre d'absorption a deux photons du deanst en solution dans le tetrahydrofurane est mesure par la technique d'ellipsometrie kerr. Ce spectre est bien decrit par une theorie simplifiee prenant en compte l'elargissement inhomogene et les etats vibrationnels de la transition a transfert de charge. Dans une seconde partie, les interactions intermoleculaires sont calculees dans le cadre de la theorie de frenkel-davydov. Le modele du dipole etendu est generalise a l'aide des distributions de charges calculees precedemment. Ce modele ameliore la description de la molecule lorsque la distance entre proches voisins est du meme ordre de grandeur que les dimensions moleculaires. Les differentes hypothese du modele utilises sont discutees. Des spectres de reflectivite enregistres selon deux plans cristallographiques demontrent les effets du couplage intermoleculaire : un eclatement de davydov de 5400 cm##1 est mesure. Le spectre d'excitation de fluorescence pompee a deux photons permet d'obtenir les informations sur les etats excitoniques pairs. Dans la derniere partie, l'influence de la focalisation du faisceau et de l'epaisseur de l'echantillon sur l'absorption non lineaire est etudiee. Une longueur effective d'interaction non lineaire est introduite. Un modele simplifie permet d'obtenir une expression de cette longueur effective sur l'axe optique et pour une faible non linearite. Des resultats experimentaux mesures sur des cristaux de 2-amino-5-nitro-pyridinium-chlorure illustrent ce calcul qui est ensuite generalise, par la technique numerique de propagation de faisceaux (bpm) a de plus fortes non linearites. Enfin, des effets d'absorption d'ordre plus eleve ainsi que de refraction non lineaire sont discutes.

碩士
國立臺灣大學
醫學工程學研究所
99
In this thesis, we use the two-photon based fluorescence correlation spectroscopy system to measure the dynamic parameter of green fluorescence protein in PBS, and in cytosol. The concentration of GFP was increased with time during the period of the time, 6-27 hours after transfection. According the correlation we should know our system limitation of the concentration GFP. Our purpose is to set up a platform which is low bio-damage and for long-term observation for the study of the cell biology such as cytoskeleton and transportation in cell which are related to the metastasis of cancer cell or cell movement. In general, the studies of cytotskeleton or membrane transfusion rate are use Fluorescence recovery after photobleaching (FRAP) as a tool to study. The laser of FRAP was one photon excitation. Compare to the one photon excitation two photon excitation had less photobleaching, less cell damage and longer penetration depth. Not only for the immobile phase such as membrane, but also the protein in cytosol can be measured the parameter in our system – Two photon excitation fluorescence correlation spectroscopy (TPE-FCS). As for these reasons that two photon excitation was the suitable tool to develop a platform for long period of time in observation of cell biology. As a given excitation volume, the diffusion coefficient of molecules due to the Brownian motion into or out of the excitation volume is determined. In our system, the limit concentration of measurement is between sub-nanomolar to sub-micromolar. This is associated to the transfection. After tansfection, the HEK 293 cell started express the green fluorescence protein. However, less than 6 hour after transfection, the cells were at an unstable situation and there are less GFP in the cell. On the other hands, more than 30 hours that too much GFP were expressed in cell even secrete out to the medium. Thus became more background noise to the FCS measurement.

Coherent control of laser induced processes is based on the quantum interference among multiple excitation pathways. Progress in the field has been fueled by advances in pulse shaping techniques, allowing modulation of phase and amplitude across the bandwidth of ultra short pulses. This dissertation makes use of coherent control technique for the optimization of two-photon fluorescence (TPF) and its applications in selective excitation for biomedical imaging. Different physical processes, e.g. TPF, second harmonic generation (SHG) and their ratios (e.g. TPF/SHG) were optimized by using feedback control pulse shaping technique with an evolutionary algorithm. Various nonlinear effects, e.g. filamentation, intensity clamping and white light generation were studied using two-photon fluorescence and Z-scan technique with different dyes and biomarkers. Simultaneous measurements of different nonlinear effects were performed. Novel methods were proposed and implemented to obtain two-photon excitation characteristics in intensity-resolved manner. Understanding of these nonlinear effects can give new solution to the issues of spatial resolution and molecular contrast for cellular and tissue imaging.

Indiana University-Purdue University Indianapolis (IUPUI)
The sympathetic nervous system strongly modulates the contractile and electrical function of the heart. The anatomical underpinnings that enable a spatially and temporally coordinated dissemination of sympathetic signals within the cardiac tissue are only incompletely characterized. In this work we took the first step of unraveling the in situ 3D microarchitecture of the cardiac sympathetic nervous system. Using a combination of two-photon excitation fluorescence microscopy and computer-assisted image analyses, we reconstructed the sympathetic network in a portion of the left ventricular epicardium from adult transgenic mice expressing a fluorescent reporter protein in all peripheral sympathetic neurons. The reconstruction revealed several organizational principles of the local sympathetic tree that synergize to enable a coordinated and efficient signal transfer to the target tissue. First, synaptic boutons are aligned with high density along much of axon-cell contacts. Second, axon segments are oriented parallel to the main, i.e., longitudinal, axes of their apposed cardiomyocytes, optimizing the frequency of transmitter release sites per axon/per cardiomyocyte. Third, the local network was partitioned into branched and/or looped sub-trees which extended both radially and tangentially through the image volume. Fourth, sub-trees arrange to not much overlap, giving rise to multiple annexed innervation domains of variable complexity and configuration. The sympathetic network in the epicardial border zone of a chronic myocardial infarction was observed to undergo substantive remodeling, which included almost complete loss of fibers at depths >10 µm from the surface, spatially heterogeneous gain of axons, irregularly shaped synaptic boutons, and formation of axonal plexuses composed of nested loops of variable length. In conclusion, we provide, to the best of our knowledge, the first in situ 3D reconstruction of the local cardiac sympathetic network in normal and injured mammalian myocardium. Mapping the sympathetic network connectivity will aid in elucidating its role in sympathetic signal transmisson and processing.

碩士
國立中央大學
照明與顯示科技研究所
101
This thesis is based on the two-photon excitation stimulated-emission depletion microscopy to discuss the effects of donut beam and fluorescence lifetime on the spatial resolution of this system and propose the methods to improve the resolution. In this research, both simulation and experimental measurements were applied to analyze the intensity distributions of donut beams generated in different ways and their distributions under tightly-focusing condition. On the other hand, only simulation was used to discuss the impact of fluorescence lifetime on the spatial resolution improvement. Finally, in order to obtain the experimental evidences of our results, the two-photon excitation stimulated-emission depletion microscopy was setup.

Indiana University-Purdue University Indianapolis (IUPUI)
Introduction: Multiphoton fluorescence excitation microscopy (MPM) is an invaluable tool for studying processes in tissue in live animals by enabling biologists to view tissues up to hundreds of microns in depth. Unfortunately, imaging depth in MPM is limited to less than a millimeter in tissue due to spherical aberration, light scattering, and light absorption. Spherical aberration is caused by refractive index mismatch between the objective immersion medium and sample. Refractive index heterogeneities within the sample cause light scattering. We investigate the effects of refractive index mismatch on imaging depth in MPM. Methods: The effects of spherical aberration on signal attenuation and resolution degradation with depth are characterized with minimal light absorption and scattering using sub-resolution microspheres mounted in test sample of agarose with varied refractive index. The effects of light scattering on signal attenuation and resolution degradation with depth are characterized using sub-resolution microspheres in kidney tissue samples mounted in optical clearing media to alter the refractive index heterogeneities within the tissue. Results: The studies demonstrate that signal levels and axial resolution both rapidly decline with depth into refractive index mismatched samples. Interestingly, studies of optical clearing with a water immersion objective show that reducing scattering increases reach even when it increases refractive index mismatch degrading axial resolution. Scattering, in the absence of spherical aberration, does not degrade axial resolution. The largest improvements in imaging depth are obtained when both scattering and refractive index mismatch are reduced. Conclusions: Spherical aberration, caused by refractive index mismatch between the immersion media and sample, and scattering, caused by refractive index heterogeneity within the sample, both cause signal to rapidly attenuate with depth in MPM. Scattering, however, seems to be the predominant cause of signal attenuation with depth in kidney tissue. Kenneth W. Dunn, Ph.D., Chair

Indiana University-Purdue University Indianapolis (IUPUI)
Advancements in human genomics have simultaneously enhanced our basic understanding of the human body and ability to combat debilitating diseases. Historically, research has shown that there have been many hindrances to realizing this medicinal revolution. One hindrance, with particular regard to the kidney, has been our inability to effectively and routinely delivery genes to various loci, without inducing significant injury. However, we have recently developed a method using hydrodynamic fluid delivery that has shown substantial promise in addressing aforesaid issues. We optimized our approach and designed a method that utilizes retrograde renal vein injections to facilitate widespread and persistent plasmid and adenoviral based transgene expression in rat kidneys. Exogenous gene expression extended throughout the cortex and medulla, lasting over 1 month within comparable expression profiles, in various renal cell types without considerably impacting normal organ function. As a proof of its utility we by attempted to prevent ischemic acute kidney injury (AKI), which is a leading cause of morbidity and mortality across among global populations, by altering the mitochondrial proteome. Specifically, our hydrodynamic delivery process facilitated an upregulated expression of mitochondrial enzymes that have been suggested to provide mediation from renal ischemic injury. Remarkably, this protein upregulation significantly enhanced mitochondrial membrane potential activity, comparable to that observed from ischemic preconditioning, and provided protection against moderate ischemia-reperfusion injury, based on serum creatinine and histology analyses. Strikingly, we also determined that hydrodynamic delivery of isotonic fluid alone, given as long as 24 hours after AKI is induced, is similarly capable of blunting the extent of injury. Altogether, these results indicate the development of novel and exciting platform for the future study and management of renal injury.