Phasor Approach Research Articles

Label Free Linear and Non-Linear Excitation Nanoscopy

Despite tremendous progress in super-resolution microscopy techniques, the imaging of non-fluorescent species with sub-diffraction resolution is a challenging task. To overcome this limit the subtraction microscopy approach has been proposed recently [1,2]. The concept is based on the acquisition and the successive mathematical subtraction of two images acquired with Gaussian and doughnut shaped focal spots, respectively.Here, we will show a novel rescaling method that avoids artifacts, maintaining the contrast and resolution enhancement. Nevertheless, Infrared absorption/saturation microscopy is also designed to be a label free super-resolution approach[3]. The main idea of InfraRed Nanoscopy (IRN) is an absorption/saturation effect similar to the conventional pump-probe technique[4], where the pump beam modifies the carrier density inside the sample, followed by the intensity changes in the transmitted probe beam. The introduction of an additional doughnut shaped pump beam can transiently saturate the induced transition in the periphery of the focal spot and enabling super-resolution. Finally we will show a novel implementation based on single wavelength 2PE-STED nanoscopy[5] by means of random access scanning, and a phasor approach for the analysis of polarization SHG images, that, providing quantitative information, can be used to extract a fingerprint of tissues.The LANIR research leading to these results has received funding from FP7/20012-2015 under grant agreement n°280804.1.Harold, D., Piche, M., De Koninck, Y.(2013) Optics express 21,13:15912-15925.2.Wang, N., Miyazaki, J., He, J., Seto, K., Kobayashi, T.(2015). Optics Communications 343,28-33.3.Silien, C., Liu, N., Hendaoui, N., Tofail, SAM., Peremans, A.(2012). Optics Express 20,29694-29704.4.Dong CY, So PT, French T, Gratton E.(1995) Biophysical Journal. 69(6):2234-2242.5. Bianchini, P., Peres, C., Oneto, M., Galiani, S., Vicidomini, G., Diaspro, A. (2015). Cell and Tissue Research 360,143-150.

NADH Conformation Assessed using Spectral Phasor Analysis: Illustrating Concepts from Molecular Folding to Metabolic Monitoring

The wide applicability of spectroscopy makes fluorescence measurements on biomolecules an attractive modality for introducing biophysical topics into undergraduate physics laboratory courses. Here we show how the spectroscopic assessment of reduced nicotinamide adenine dinucleotide (NADH) conformation is useful for conveying concepts spanning multiple size scales. While advanced sources of optical contrast for NADH conformation include fluorescence excitation transfer efficiency, excited-state lifetime, and anisotropy decay rate, our recently published studies have shown how the quantification of autofluorescence spectrum shape using spectral phasor analysis is useful for monitoring NADH conformations during metabolic transitions. Here we present the pedagogical use of spectral phasor approaches to assess NADH conformation, with educational modules illustrating concepts ranging from molecular folding to protein binding, in addition to cellular metabolic monitoring. In the first module, we explore the NADH folding-unfolding transition in solution during methanol denaturation. The two-state nature of the transition and the degree of unfolding are evaluated using spectral phasors. A two-state solvent-denaturation model is used to estimate the free energy of transition. Next, we consider the protein binding of NADH to various dehydrogenases, showing that binding to different proteins can be discriminated optically, providing a spectroscopic means of examining ligand-binding behavior. The NADH binding constant to malate dehydrogenase is also measured, with the degree of ligand binding quantified using spectral phasors. Finally at the cellular level, the response of energy metabolism in Saccharomyces cerevisiae to various functional modifiers is monitored. We note that all modules were originally research projects involving undergraduate students, and so results here illustrate the relationship between student-driven, faculty-mentored research and the translation of those outcomes to courses centered around discovery-oriented learning.

Spatial Dynamics of SIRT1 Dictate Metabolic Transitions in the Cell Nucleus

Sirtuins are a highly conserved family of NAD+-dependent deacylases that function as metabolic sensors by detecting the cellular energy state to modulate biochemical pathways to allow the cell to adapt to the environment. SIRT1, one of the seven mammalian sirtuins, has been widely studied. Previous reports have implicated that SIRT1 activity can be regulated by fluctuations in intracellular NAD+ concentrations. However the direct impact of SIRT1 on the regulation of the metabolism of its own cofactor, NAD+, remains unclear. Here, we demonstrate the nuclear organization of NAD+/NADH states are determined by SIRT1. Phasor approach to two photon autofluorescence lifetime microscopy in live cells shows that the redox state is compartmentalized inside of the nucleus, and that the subnuclear distribution as well as the redox ratio itself depends on SIRT1. Moreover, this effect seems specific for SIRT1, as the other nuclear sirtuin SIRT6, doesn't play a major role. Fluorescence fluctuation spectroscopy studies, RICS and phasor FCS in single living cells, reveal that NAD+ metabolism in the nucleus impacts subnuclear dynamics of active SIRT1 enzyme. These results provide direct evidence of an intimate crosstalk between NAD+ states and SIRT1 activity in the nucleus of live cells, and open the path to study connections between nuclear organization and metabolism.Grants: NIH- DA036408, AG041504, P41-GM103540 and P50-GM076516

Non-Invasive Fluorescence Lifetime Imaging(FLIM) of Stem Cell Signature Metabolic States

Development of the early post-implantation gastrulating human embryo has largely been inaccessible to comprehensive molecular study for obvious moral and ethical reasons. During gastrulation, a flat sheet of pluripotent cells called the epiblast undergoes ordered differentiation, giving rise to three primary germ layers that form fetal tissues. These events, triggered in part by specific growth factors (GFs), involve changes in cell shape, cell migration, chromatin architecture, cell cycle, etc., all processes requiring metabolic resources. Human embryonic stem cells (hESCs), thought to be equivalent to human epiblast cells, could provide an important new window into development and allow us to better understand how this stage is controlled at the molecular level and risks to early human development. Here we explore the metabolic changes that occur in Human embryonic Stem Cells (hESCs) exposed to growth factors such as WNT3 and BMP4. We take advantage of the Non-invasive phasor approach to fluorescence lifetime imaging microscopy (FLIM). This method can map the fluorescence lifetime of intrinsic autofluorescencent biochemical species in cells and living tissues. Our goal is to define how individual vertebrate pluripotent epiblast cells respond metabolically to key GFs and establish a baseline for subsequent studies on hESCs. To image the response of hESCs during exposure with growth factors, hESC are cultured with different media and are imagined using 2-photon excitation to excite NADH at 740nm to detect autofluorescence in the microenvironment. Such methods could be used to understand heterogeneity in hESC cultures, how that metabolic heterogeneity is affected by culture conditions, how it affects cell fate decisions in hESCs and if it reflects conditions in developing embryos. Such information could provide new information on human embryonic development and inform us about the risks to embryos during this time period.

Organization of Inner Cellular Components as Reported by a Viscosity-Sensitive Fluorescent Bodipy Probe Suitable for Phasor Approach to Flim

Abstract According to the recent developments in imaging strategies and in tailoring fluorescent molecule as probe for monitoring biological systems, we coupled a Bodipy-based molecular rotor ( BoMe ) with FLIM phasor approach to evaluate the viscosity in different intracellular domains. BoMe rapidly permeates cells, stains cytoplasmic as well as nuclear domains, and its optical properties make it perfectly suited for widely diffused confocal microscopy imaging setups. The capability of BoMe to report on intracellular viscosity was put to the test by using a cellular model of a morbid genetic pathology (Hutchinson–Gilford progeria syndrome, HGPS). Our results show that the nucleoplasm of HGPS cells display reduced viscosity as compared to normal cells. Since BoMe displays significant affinity towards DNA, as demonstrated by an in vitro essay, we hypothesize that genetic features of HGPS, namely the misassembly of lamin A protein within the nuclear lamina, modulates chromatin compaction. This hypothesis nicely agrees with literature data.

Self-Assisted Optothermal Trapping of Gold Nanorods Under Two-Photon Excitation

We report self-assisted optothermal trapping and patterning of gold nanorods (GNRs) on glass surfaces with a femtosecond laser. We show that GNRs are not only the trapping targets, but also can induce convective flows that drive more particles toward the trap. The trapping phenomenon is the net result of thermophoresis and convective flow caused by localized heating, which is due to the conversion of absorbed photons into heat at GNR's longitudinal surface plasmon resonance wavelength. We investigated the optothermal trapping of GNRs at the glass surface which can be obtained with laser power as low as 0.5 mW at 840 nm. The attraction of particles toward the central hot spot can be larger than the typical field of view, e.g. attraction toward the trap was observed from a range of 210 µm × 210 µm. By moving the laser focus away from the glass surface, at certain distances, ring patterns of GNRs on the glass surface can be obtained visualizing the regions of flow. These patterns could be controlled by the laser power and the numerical aperture of the microscope objective. Moreover, we examined the spectral emission of GNRs under different trapping conditions using the spectral phasor approach to reveal the temperature and association status of GNRs. Our study will help understanding manipulation of flows in solution and in biological systems which could arise in future applications of GNRs induced heating and flows. Work done in part with funds from NIH P41-GM103540 and NIH P50-GM076516.

Controllability of linear passive network behaviors

Classical RLC realization procedures (e.g. Bott–Duffin) result in networks with uncontrollable driving-point behaviors. With this motivation, we use the behavioral framework of Jan Willems to provide a rigorous analysis of RLC networks and passive behaviors. We show that the driving-point behavior of a general RLC network is stabilizable, and controllable if the network contains only two types of elements. In contrast, we show that the full behavior of the RLC network need not be stabilizable, but is marginally stabilizable. These results allow us to formalize the phasor approach to RLC networks using the notion of sinusoidal trajectories, and to address an assumption of conventional phasor analysis. Finally, we show that any passive behavior with a hybrid representation is stabilizable. This paper relies substantially on the fundamental work of our late friend and colleague Jan Willems to whom the paper is dedicated.

Phasor Representation of Monomer-Excimer Kinetics: General Results and Application to Pyrene.

Phasor plots of the fluorescence intensity decay (plots of the Fourier sine transform versus the Fourier cosine transform, for one or several angular frequencies) are being increasingly used in studies of homogeneous and heterogeneous systems. In this work, the phasor approach is applied to monomer-excimer kinetics. The results obtained allow a clear visualization of the information contained in the decays. The monomer phasor falls inside the universal circle, whereas the excimer phasor lies outside it, but within the double-exponential outer boundary curve. The monomer and excimer phasors, along with those corresponding to the two exponential components of the decays, fall on a common straight line and obey the generalized lever rule. The clockwise trajectories described by both phasors upon monomer concentration increase are identified. The phasor approach allows discussing in a single graphic not only the effect of concentration but also that of rate constants, including the evolution from irreversible kinetics to fast excited-state equilibrium upon a temperature increase. The obtained results are applied to the fluorescence decays of pyrene monomer and excimer in methylcyclohexane at room temperature. A straightforward method of monomer-excimer lifetime data analysis based on linear plots is also introduced.

Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches

In this note, we present a discussion of the advantages and scope of model-free analysis methods applied to the popular solvatochromic probe LAURDAN, which is widely used as an environmental probe to study dynamics and structure in membranes. In particular, we compare and contrast the generalized polarization approach with the spectral phasor approach. To illustrate our points we utilize several model membrane systems containing pure lipid phases and, in some cases, cholesterol or surfactants. We demonstrate that the spectral phasor method offers definitive advantages in the case of complex systems.

Modulated CMOS camera for fluorescence lifetime microscopy.

Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition.

Imaging Fibrosis and Separating Collagens using Second Harmonic Generation and Phasor Approach to Fluorescence Lifetime Imaging.

In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur. This is a label free approach towards fast automatable detection of fibrosis in tissue samples. FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy. Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated.

Graphical analysis of transient absorption spectra using the phasor approach

Transient absorption spectroscopy is a powerful tool to study chemical dynamic processes. Several methods have been developed for transient absorption spectra data analysis, such as the singular value decomposition with exponential fitting method. Here, we use a transient absorption phasor approach to transient absorption spectra data graphical analysis which is developed from the phasor approach. This expands the research processes of the phasor approach and makes the visual analysis of different kinetic processes possible. The lifetime can be evaluated in phasor space without professional exponential fitting. The method is computationally efficient and straightforward to implement. The simulation result validates the effectiveness of the presented method to transient absorption spectra data analysis. The transient absorption phasor approach has the potential to simplify transient absorption spectra data analysis and paves the way for the analysis of large data sets.

Organization of inner cellular components as reported by a viscosity-sensitive fluorescent Bodipy probe suitable for phasor approach to FLIM

According to the recent developments in imaging strategies and in tailoring fluorescent molecule as probe for monitoring biological systems, we coupled a Bodipy-based molecular rotor (BoMe) with FLIM phasor approach to evaluate the viscosity in different intracellular domains. BoMe rapidly permeates cells, stains cytoplasmic as well as nuclear domains, and its optical properties make it perfectly suited for widely diffused confocal microscopy imaging setups. The capability of BoMe to report on intracellular viscosity was put to the test by using a cellular model of a morbid genetic pathology (Hutchinson–Gilford progeria syndrome, HGPS). Our results show that the nucleoplasm of HGPS cells display reduced viscosity as compared to normal cells. Since BoMe displays significant affinity towards DNA, as demonstrated by an in vitro essay, we hypothesize that genetic features of HGPS, namely the misassembly of lamin A protein within the nuclear lamina, modulates chromatin compaction. This hypothesis nicely agrees with literature data.

Line voltage phasor reconstruction on capacitive voltage transformers using dynamic phasor approach

Capacitive voltage transformers (CVTs) are widely used in line voltage measurement of EHV/UHV power systems. When a fault occurs, CVT measurements may contain severe transients during the beginning 3–6 fault cycles, which have usually been ignored instead of processed in many previous algorithms. However, this may cause reach problems and postpone response time in digital relay operation. In the present paper, dynamic phasor approach was employed to reconstruct line voltage phasors. Using the proposed algorithm, line voltage phasors can be obtained rapidly after one-cycle plus 2 samples, which are still accurate even if transients are involved in the CVT measurements. The proposed algorithm is simulated and evaluated by the MATLAB/SIMULINK built system. Simulation and evaluation results illustrate the effectiveness and performance of the proposed algorithm in various fault conditions and CVT types.

Characterizing Fibrosis in Mouse Kidney Using Fluorescence Lifetime and Second Harmonic Generation Imaging Microscopy in Unilateral Ureteral Obstruction Model

Renal tubulointerstitial fibrosis is considered to be the final common pathway for most forms of chronic kidney disease (CKD). Fibrosis has been traditionally characterized by histological studies such as Picro‐Sirius Red (PSR) staining, Masson Trichrome staining or collagen immunohistochemistry. Such methods are limited due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to unilateral ureteral obstruction (UUO), a well‐characterized model of fibrosis. At 21 days, both kidneys were collected. Serial sections of both kidneys were analyzed by PSR staining or FLIM with SHG. Quantification of a whole kidney section from the PSR staining showed 34.32±0.99% area of fibrosis in the injured kidney compared to 5.55±1.07% in the control kidney. Using the Phasor approach to FLIM, comparisons between the two kidneys show that the auto fluorescence lifetime signature give rise to two well separate phasor clusters. Quantification of ten different fields of view for each kidney from SHG suggests the presence of more collagen I in the diseased kidneys (17.44±4.21% compared to 2.59±1.98% control kidney). In conclusion, the combined FLIM and SHG images let us establish a criterion for quantitative determination of fibrosis directly from the microscope images.

NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells.

α-Synuclein (aS) aggregation has been amply investigated for its involvement in Parkinson's disease because its amyloid fibrils are the main constituent of Lewy bodies, one of the hallmarks of the disease. aS aggregation was studied here in vitro and in cellular models to correlate aggregation products with toxicity mechanisms. Independent results published elsewhere suggested that aS overexpression and/or aggregation may impair cellular metabolism and cause mitochondrial damage. In this context, we report the characterization of changes in NADH fluorescence properties in vitro and in human embryonic kidney 293 cells upon aS aggregation. The application of the phasor approach to study NADH fluorescence lifetime and emission allowed us to identify changes that correlate with aS aggregation. In particular, the fraction of bound NADH, characterized by longer lifetimes in comparison to free NADH, is increased, and the maximum of the NADH emission is shifted toward shorter wavelengths in the presence of aggregating aS both in vitro and in cells. These data suggest that NADH binds to aggregated aS. NMR experiments in vitro substantiate such binding, which occurs during aggregation. NADH fluorescence is thus useful to detect aS aggregation and by extension the associated oxidative stress.

Characterizing Fibrosis in Mouse Kidney using Auto Fluorescence Flim and SHG in UUO Model

2410-Pos Board B547 Characterizing Fibrosis in Mouse Kidney using Auto Fluorescence Flim and SHG in UUO Model Moshe Levi 1 , Evgenia Dobrinskikh 1 , Suman Ranjit 2 , John Montford 1 , Alexander Dvornikov 3 , Allison Lehman 1 , Seth Furgeson 1 , Raphael Nemenoff 1 , Enrico Gratton 2 . Medicine, U of Colorado, Aurora, CO, USA, 2 Biomedical Engineering, U of California, Irvine, CA, USA, 3 Biomedical Engineering, U of California, Aurora, CA, USA. Chronic kidney disease (CKD) arises from a diverse list of renal injuries. Renal fibrosis is considered to be the final common pathway for most forms of CKD and involves glomerular sclerosis and/or interstitial fibrosis. Because of that there is great interest in identifying renal fibrosis in the early stages of CKD to prevent progression. Unilateral ureteral obstruction (UUO) is a well- described model of CKD and renal fibrosis. Kidney injury and fibrosis usually are assessed by histology. Histological methods include Picro-Sirius Red stain- ing, Masson’s Trichrome staining or immunohistochemistry for collagen iso- forms. Quantitation of fibrosis using histologic techniques may be difficult due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to UUO of right kidney. At 21 days, both kidneys were collected, fixed, and paraffin-embedded. The uninjured left kidney was used as a control. Serial sections of both kidneys were analyzed by Picro-Sirius Red staining or FLIM with SHG. Quantification of a whole kidney section from the Picro- Sirius Red staining showed 34.3250.99% area of fibrosis in the left kidney compare to 5.5551.07% in the control kidney. Using the Phasor approach to FLIM, comparisons between the two kidneys show that the auto fluorescence lifetime signature give rise to two well separate phasor clusters. Quantification of ten different fields of view for each kidney from SHG suggests the presence of more collagen I in the diseased kidneys (17.4454.21% compared to 2.5951.98% control kidney). Finally the combined FLIM and SHG images let us create a criterion to separate fibrosis directly from the microscope images.

Real-Time Analysis of Endogenous Nuclear NADH in Differentiating Cells using the Spectral Phasor Approach

369-Pos Board B149 Real-Time Analysis of Endogenous Nuclear NADH in Differentiating Cells using the Spectral Phasor Approach Belinda K. Wright 1,2 , Mark R. Jones 1 , Michelle A. Digman 2 , Enrico Gratton 2 . School of Science and Health, University of Western Sydney, Richmond, Australia, 2 Biomedical Engineering, Laboratory of Fluorescence Dynamics (LFD), University of California, Irvine, CA, USA. NADH is an endogenous autofluorescent regulatory metabolite detected in the nuclear regions of live cells which when analysed for the bound and free form can aid in determining a cell’s metabolic status and molecular activity. Detect- ing the spectral differences of free and bound NADH in live cells is currently limited due to the very small differences in emission. The Spectral Phasor tech- nique enables not only examination of small shifts in spectral emissions but also provides the spatial location of spectrally different components in live cells without any prior knowledge of the species. The phasor representation enables direct comparison of either optical sections (i.e. different focal planes) of one cell or multiple cells for global analysis. Here we describe the use of Spectral Phasors to spatially map NADH’s spectral emission in the nucleus of live cells under normal culture conditions and those stimulated into the early stages of differentiation. A comparison of undifferen- tiated cells and those stimulated to differentiate demonstrate differing spatial distributions of emission spectra associated with NADH. Undifferentiated cells displayed shorter emissions cantered in the nucleus, while longer wavelengths were localised around the perinuclear boarder. Cells stimulated into the early stages of differentiation displayed a redirection of the shorter emissions to regional clustering predominately in one area close to the nuclear/cytoplasmic boarder, while the longer wavelengths were localised throughout the remainder of the nucleus. Here we show the application of the Spectral Phasor technique to identify discrete wavelength shifts associated with endogenous NADH auto- fluorescence in the nucleus, and observed changes in their spatial distribution in live cells during the early stages of differentiation. Work in part supported by NIH P41-GM103540 and NIH P50-GM076516.

Mapping P53 Alterations in Metabolism upon DNA Damage using the Phasor/FLIM and Number & Molecular Brightness Analysis

p53 is a tumor suppressor protein that regulates target genes involved in DNA damage migation and repair. If cells become stressed due to DNA damage, p53 will induce genes that trigger cell cycle arrest and/or apoptosis. p53 has a dual role in promoting oxidative phosphorylation (oxsphos) and glycolysis upon cellular stress. However, the metabolic function of p53 has not been fully explored. p53 metabolic activities may play an important role in normal growth, development, and tumor suppression, it might also be misused to help promote, rather than hinder, tumor development. To further assess p53 activity, we investigated the effect of metabolic changes under the same conditions and tested if the concentration of p53 influences the balance between apoptosis and DNA repair. We used the fluorescent lifetime imaging microscopy (FLIM) phasor approach to detect changes in oxsphos and glycolysis. Overall our data show the formation of p53 protein near the nucleolus and in areas near the inner regions of the nucleus. We also show that upon DNA damage the concentration of p53 increases by a factor of two independent of initial expression levels. The FLIM/Phasor data show that low concentrations of p53 is enough to trigger glycolytic response in the nucleus of the cells upon DNA damage with Cisplatin and under high expression levels, a new lifetime phasor cluster is detected. This new lifetime correlates with dead phenotype cells and may be a new “apoptotic” lifetime signal. In addition, we are able to visualize the sub-nuclear spatial localization of p53 tetramers upon DNA damage with cisplatin and obtain spatial-temporal malls of cellular localization and dynamics. This work is supported in part by NIH grants P50 GM076516 and P41 GM103540.

Modelling and Detection of Turn-to-Turn Faults in Transformer Windings Using Finite Element Analysis and Instantaneous Exciting Currents Space Phasor Approach

In this paper, a novel method based on online monitoring of Instantaneous Exciting Current Space Phasor is presented in order to detect inter-turn faults on the transformer windings. This approach detects winding faults by comparison between presentation of the Instantaneous Exciting Current Space Phasor under healthy and faulty condition. In this work, the angular speed of Instantaneous Exciting Current Space Phasor has been introduced as one of the fault detection tools that has good sensitivity for detection of minor inter-turn faults. Firstly, a typical transformer is simulated based on Finite Element Analysis (FEA) to investigate the transformer behavior under different conditions. Then, the accuracy and performance of proposed diagnosis technique are studied by applying it to the simulated transformer.