Phasor Fluorescence Lifetime Imaging Microscopy Research Articles

Phasor identifier: A cloud-based analysis of phasor-FLIM data on Python notebooks

This paper introduces an innovative approach utilizing Google Colaboratory for the versatile analysis of phasor fluorescence lifetime imaging microscopy (FLIM) data collected from various samples (e.g., cuvette, cells, tissues) and in various input file formats. In fact, phasor-FLIM widespread adoption has been hampered by complex instrumentation and data analysis requirements. We mean to make advanced FLIM analysis more accessible to researchers through a cloud-based solution that 1) harnesses robust computational resources, 2) eliminates hardware limitations, and 3) supports both CPU and GPU processing. We envision a paradigm shift in FLIM data accessibility and potential, aligning with the evolving field of artificial intelligence-driven FLIM analysis. This approach simplifies FLIM data handling and opens doors for diverse applications, from studying cellular metabolism to investigating drug encapsulation, benefiting researchers across multiple domains. The comparative analysis of freely distributed FLIM tools highlights the unique advantages of this approach in terms of adaptability, scalability, and open-source nature.

FLUTE: A Python GUI for interactive phasor analysis of FLIM data.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The fit-free phasor approach to FLIM data is increasingly being used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available in Python, thus limiting the widespread use of phasor analysis in biomedical research. Here, we present Fluorescence Lifetime Ultimate Explorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of the analysis of FLIM data acquired in the time domain, such as calibrating the FLIM data, performing interactive exploration of the phasor plot, displaying phasor plots and FLIM images with different lifetime contrasts simultaneously, and calculating the distance from known molecular species. After applying desired filters and thresholds, the final edited datasets can be exported for further user-specific analysis. FLUTE has been tested using several FLIM datasets including autofluorescence of zebrafish embryos and in vitro cells. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets, and accelerates FLIM analysis for non-specialized labs.

Abstract 1639: Human immune system mouse model of ER+ metastatic breast cancer

Abstract In a recent clinical trial in heavily pretreated estrogen receptor alpha-positive (ER+) metastatic breast cancer (MBC), ~23% of patients reached the primary endpoint of clinical benefit of >24 weeks on Fulvestrant plus the anti-androgen Enzalutamide. ESR1 mutant biopsies had more T regulatory cells (t test p<0.021) and macrophages (p<0.003) than ESR1 wild type (WT), which had more TP53 mutations and higher levels of cytotoxic T effector cells (p<0.01). In addition, PD-L1 increased in post- vs. pre-treatment biopsies (paired t test p<0.002). This increase in PD-L1 post-treatment warrants testing if the addition of a checkpoint inhibitor to the Fulvestrant-Enzalutamide combination will enhance therapy efficacy in ER+ MBC resistant to standard endocrine therapy. However, the lack of immune competent ER+ breast cancer pre-clinical models is a major barrier to these studies. Human immune system (HIS) mouse models were developed as a platform in which to test immune checkpoint inhibitors in other human cancers, and one HIS model was recently shown to support the growth and immune infiltration of ER+ patient-derived xenograft (PDX) primary tumors. We tested whether HIS mice are a robust model in which to define ER+ tumor-immune microenvironment interactions at metastatic sites. We implanted NCG mice (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) humanized by engraftment of human umbilical cord blood-derived CD34+ (huCD34+) stem cells with the estrogen-independent HCI-013EI (013EI) PDX, derived from an ER+, invasive lobular MBC which harbors the Y537S ESR1 mutation. Mice were monitored for tumor latency and growth rate, and primary tumors and organs were collected for immunohistochemistry, single-cell RNA sequencing (scRNAseq), and phasor-fluorescence lifetime imaging microscopy (FLIM). The ER mutated PDX 013EI had a longer tumor latency in HIS (median 77 days) vs. age-matched immune deficient control mice (27.5 days, log-rank p=0.043). Once formed, 013EI tumors in HIS mice grew slower than those in controls (mixed-effects analysis, p=0.035). HIS mice had visible lung, liver, and/or gastrointestinal tract metastases despite small primary tumors. Lung metastases from HIS mice retain ER expression and are infiltrated by human CD4+, CD8+, and CD68+ immune cells. Initial scRNAseq analysis also suggests infiltration by diverse immune cell populations in primary tumors from HIS mice. Initial phasor-FLIM for NADH autofluorescence suggests that 013EI tumors are highly glycolytic in HIS mice. In conclusion, our results show that huCD34+ HIS mice effectively support growth, metastasis, and immune infiltration of an ESR1 mutant MBC PDX. Ongoing studies will further define the tumor-immune microenvironment in this and ESR1 WT MBC PDX, focusing on immune cell infiltration, PD-L1 expression, steroid receptor status, metabolic signaling and changes in these parameters following novel combinations of endocrine, immunotherapy and metabolic agents. Citation Format: Ayodeji Olukoya, Shaymaa Bahnassy, Nicole S. Spoelstra, Lu Jin, Suman Ranjit, Anthony Elias, Jennifer K. Richer, Rebecca B. Riggins. Human immune system mouse model of ER+ metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1639.

Abstract 2337: Label-free single-cell drug response determined by fluorescence lifetime imaging microscopy (FLIM) and RNA sequencing

Abstract Colorectal cancer (CRC) is the second-leading cause of cancer-related death worldwide, and treatment failure in patients with metastatic CRC portends a poor prognosis. Early detection of chemoresistance would help reduce the usage of ineffective and possibly toxic therapies and facilitate alternative treatment strategies. We present a new technique combining phasor-fluorescence lifetime imaging microscopy (phasor-FLIM) and a machine learning algorithm that quantitatively identifies chemoresistant single cells, which may also be potentially applicable for evaluating heterogeneous circulating tumor cells (CTCs) obtained by liquid biopsy. We used parental HCT116 CRC cells sensitive to 5-fluorouracil (5-FU), a common chemotherapeutic agent used in CRC, to generate a cell line resistant to 5-FU; we confirmed resistance by a cell proliferation assay (MTS). We then analyzed phasor-FLIM patterns in sensitive and resistant cells before and after treatment with 5-FU of two endogenously fluorescent biomarkers, NADH and NADPH, which are key co-enzymes involved in energy metabolism. This was performed at 21% and 1% oxygen, which simulates both standard cell culture and tumor hypoxic conditions. Differences in phasor-FLIM signatures were found in resistant cells that suggested upregulation of the electron transport chain under normoxia (21% oxygen) and reprogrammed glucose metabolism under hypoxia (1% oxygen). Such changes were confirmed using NADH, lactate, and pyruvate production colorimetric tests, which are readouts of energy metabolism. To improve the quantification of changes in phasor-FLIM patterns, a new drug resistance indexing algorithm was developed to separate sensitive and resistant cells based on fluorescence lifetime multiparametric analysis of the intrinsic fluorescent biomarkers at the single-cell level. We then used single-cell total RNA sequencing (scRNA-Seq) to explore gene expression differences between sensitive and resistant HCT116 cells. Principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) clustering showed a clear separation of 5-FU-sensitive and -resistant cells. There was increased expression of proliferation markers in resistant cells and upregulation of genes involved in electron transport from NADPH to ferredoxin, consistent with our single-cell phasor-FLIM findings. We demonstrate proof of concept that the phasor-FLIM method combined with our new drug resistance indexing algorithm provides a marker-free technology for quantitatively assaying drug response and discriminating drug resistance at single-cell resolution. Citation Format: Ning Ma, Hui Ren, Naveen Ramalingam, David King, Banafshé Larijani, Stefanie S. Jeffrey. Label-free single-cell drug response determined by fluorescence lifetime imaging microscopy (FLIM) and RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2337.

Development and characterization of phasor-based analysis for FLIM to evaluate the metabolic and epigenetic impact of HER2 inhibition on squamous cell carcinoma cultures.

.Significance: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy.Aim: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established.Approach: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825’s impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition.Results: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration.Conclusions: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

The phasor FLIM method reveals a link between a change in energy metabolism and mHtt protein spread in healthy Mammalian cells when co-cultured with Huntington diseased cells

Huntington Disease (HD) is a late-onset autosomal neurodegenerative disease characterized by the aggregations of mutant Huntingtin proteins (mHTT). A glutamine stretch (PolyQ) at the N-terminal of the Huntingtin protein is generated by the abnormal expansion of CAG trinucleotide repeats in exon 1 of the HTT gene. While the resulting polyQ aggregates are the predominate feature of HD, the intercellular spread of the expanded protein and the effect upon this transfer inside healthy cells have not yet fully understood. Here, we have employed the phasor Fluorescence Lifetime Imaging Microscopy (FLIM) method to measure NADH fluorescence lifetime change after the internalization of the PolyQ protein. Based on our analysis, we have found a significant decrease in the fraction of bound NADH in both cytoplasmic and nucleus regions when cells are co-cultured or when healthy cells uptake the supernatant containing polyQ proteins and aggregates. Overall, our FLIM study combined with confocal fluorescence imaging visualizes the absorption of the mutant Htt protein aggregates which results in a distinct NADH fluorescence lifetime between control cells and acceptor cells. These studies show, for the first time, the influence of how neighboring cells expressing the expanded Htt protein can regulate energy metabolism in healthy cells.

Caffeine and Cisplatin Effectively Targets the Metabolism of a Triple-Negative Breast Cancer Cell Line Assessed via Phasor-FLIM.

Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM). We found that solely using caffeine to treat MDA-MB-231 shifts their metabolism towards respiratory-chain phosphorylation with a lower ratio of free to bound NADH, and a similar trend is seen in MCF7. However, MDA-MB-231 cells shifted to a higher ratio of free to bound NADH when cisplatin was added. The combination of cisplatin and caffeine together reduced the survival rate for MDA-MD231 and shifted their energy metabolism to a higher fraction of bound NADH indicative of oxidative phosphorylation. The FLIM and viability results of MCF10A cells demonstrate that the treatments targeted cancer cells over the normal breast tissue. The identification of energy metabolism alteration could open up strategies of improving chemotherapy for malignant breast cancer.

Carbon Dots for Intracellular pH Sensing with Fluorescence Lifetime Imaging Microscopy.

The monitoring of intracellular pH is of great importance for understanding intracellular trafficking and functions. It has various limitations for biosensing based on the fluorescence intensity or spectra study. In this research, pH-sensitive carbon dots (CDs) were employed for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time. FLIM is a highly sensitive method that is used to detect a microenvironment and it can overcome the limitations of biosensing methods based on fluorescence intensity. The different groups on the CDs surfaces changing with pH environments led to different fluorescence lifetime values. The CDs aqueous solution had a gradual change from 1.6 ns to 3.7 ns in the fluorescence lifetime with a pH range of 2.6–8.6. Similar fluorescence lifetime changes were found in pH buffer-treated living cells. The detection of lysosomes, cytoplasm, and nuclei in living cells was achieved by measuring the fluorescence lifetime of CDs. In particular, a phasor FLIM analysis was used to improve the pH imaging. Moreover, the effects of the coenzymes, amino acids, and proteins on the fluorescence lifetime of CDs were examined in order to mimic the complex microenvironment inside the cells.

Discriminating different grades of cervical intraepithelial neoplasia based on label-free phasor fluorescence lifetime imaging microscopy.

This study proposed label-free fluorescence lifetime imaging and phasor analysis methods to discriminate different grades of cervical intraepithelial neoplasia (CIN). The human cervical tissue lesions associated with cellular metabolic abnormalities were detected by the status changes of important coenzymes in cells and tissues, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging microscopy (FLIM) was used to study human cervical tissues, human cervical epithelial cells, and standard samples. Phasor analysis was applied to reveal the interrelation between the metabolic changes and cancer development, which can distinguish among different stages of cervical lesions from low risk to high risk. This approach also possessed high sensitivity, especially for healthy sites of CIN3 tissues, and indicated the dominance of the glycolytic pathway over oxidative phosphorylation in high-grade cervical lesions. This highly adaptive, sensitive, and rapid diagnostic tool exhibits a great potential for cervical precancer diagnosis.

Development of a Single-cell Label-free Drug Testing Platform using Fluorescence Lifetime Imaging Microscopy (Flim) for Patients with Metastatic Cancer

In the United States, of cancers that affect both men and women, colorectal cancer (CRC) is the second leading cause of cancer-related deaths, resulting in over 50,000 deaths each year. Despite major advances in treatment (i.e., surgery, radiation therapy, chemotherapy, biologic/immune therapy) and resultant improved outcomes over time, it has been demonstrated that failure of treatment in patients with metastatic breast cancer portends a poor prognosis. As the number of potential FDA-approved cancer drugs increases, the likelihood of identifying effective combination strategies tailored to an individual patient's tumor increases. Therefore, to provide significant advances in cancer treatment and enable the most effective chemotherapy, development of more quantitative and objective means for assessing drug sensitivity of an individual patient's tumor that is safer, faster, sensitive and capable for single- or multi-drug testing is urgently needed for advancing personalized therapies. To develop such an approach, fluorescence spectroscopic technologies may serve as a non- invasive means of revealing cell health. We applied phasor-fluorescence lifetime imaging microscopy (phasor- FLIM) to measure drug response using human colorectal cancer cell line HCT116 and patient samples. FLIM can be served as a promising new approach for reading the metabolic states of cancer cells treated with anti-cancer therapeutics and identified specific alterations of metabolic states that are early indicators of irreversible cell demise and that such metabolic changes can be detected using a label-free imaging assay for single-cell drug testing of rare and/or heterogeneous cancer cells. Based on these findings, we apply the label-free imaging platform to measure drug response using patient samples.

Reversibility of Age-related Oxidized Free NADH Redox States in Alzheimer\u2019s Disease Neurons by Imposed External Cys/CySS Redox Shifts

Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD+) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer’s disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by 89% to levels corresponding to the young neurons. Using FLIM as a non-invasive approach, we were able to measure the reversibility of aging subcellular free NADH levels in live neurons. Our results suggest a potential reductive treatment to reverse the loss of free NADH in old and Alzheimer’s neurons.

Phasor-Fluorescence Lifetime Imaging Microscopy Analysis to Monitor Intercellular Drug Release from a pH-Sensitive Polymeric Nanocarrier.

The design of highly efficient drug carriers, and the development of appropriate techniques to monitor their mechanism of action and therapeutic effect, are both critical for improving chemotherapy. Herein, a polymeric nanoparticle, PAH-Cit/DOX (poly(allylamine)-citraconic anhydride/doxorubicin), was synthesized and used as a nanodrug system for the efficient delivery and pH-responsive release of doxorubicin (DOX) into cancer cells. The PAH-Cit/DOX nanoparticles were stable at physiological pH but effectively released DOX under weakly acidic conditions. The release efficiency was 90.6% after 60 h of dialysis in phosphate-buffered saline at pH 5.5. Confocal images showed the rapid movement of the drug from the cytoplasm to the nucleus, indicating the effective drug release MCF-7 cells. Notably, the combination of fluorescence lifetime imaging microscopy (FLIM) and phasor analysis (phasor-FLIM) provides an approach to monitor the dynamic change of DOX fluorescence lifetime in intercellular environments. Phasor-differentiated lifetime pixel intensity in FLIM images was quantified and used to evaluate the DOX release from nanocarriers, making it possible to detect the dynamics of intracellular release and transport of DOX.

Tracking Functional Tumor Cell Subpopulations of Malignant Glioma by Phasor Fluorescence Lifetime Imaging Microscopy of NADH.

Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer and there exists a need to non-invasively identify functional tumor subpopulations responsible for tumor recurrence. Reduced nicotinamide adenine dinucleotide (NADH) is a metabolic coenzyme essential in cellular respiration. Fluorescence lifetime imaging microscopy (FLIM) of NADH has been demonstrated to be a powerful label-free indicator for inferring metabolic states of living cells. Using FLIM, we identified a significant shift towards longer NADH fluorescence lifetimes, suggesting an increase in the fraction of protein-bound NADH, in the invasive stem-like tumor-initiating cell (STIC) subpopulation relative to the tumor mass-forming cell (TMC) subpopulation of malignant gliomas. By applying our previously studied model to transition glioma from a majority of STIC to a majority of TMC in serum-adherent culture conditions following serial passages, we compared changes in NADH states, cellular respirations (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth speed over passages. We identified a significant positive correlation between free-NADH fraction and cell growth, which was related to an increase of TMC fraction. In comparison, the increase of EGFR and cellular respirations preceded all these changes. In conclusion, FLIM of NADH provides a non-invasive method to monitor the dynamics of tumor heterogeneity before and after treatment.

Phasor-FLIM as a Screening Tool for the Differential Diagnosis of Actinic Keratosis, Bowen's Disease, and Basal Cell Carcinoma.

The aim of this study was to distinguish basal cell carcinoma (BCC) from actinic keratosis (AK) and Bowen's disease (BD) by fluorescence lifetimes of hematoxylin and eosin (H&E) and phasor analysis. Pseudocolor images of average fluorescence lifetime (τm) exhibited more contrast than conventional bright field and/or fluorescence images of H&E-stained sections. The mean values (μ) of τm distribution (τmμ) in three layers of skin were first explored for comparison with the corresponding layers of AK, BD, and BCC. Moreover, analysis of the H&E fluorescence lifetimes in the phasor space was performed by observing clusters in specific regions of the phasor plot. Various structures in the skin were distinguished. Comparisons of phase distributions from the corresponding layers of skin resulted in quantitative separation and calculation of distinctive parameters including coordinate values, diagonal slopes, and phasor areas. The combination of fluorescence lifetime imaging microscopy (FLIM) and phasor approach (phasor-FLIM) provides a simple method for histopathology analysis and can significantly improve the accuracy of bright field H&E diagnosis. We therefore believe that phasor-FLIM is an aided tool with the potential to provide rapid confirmation of diagnostic criteria and classification of histological types of skin neoplasms.

Real-time Analysis of Metabolic Activity Within Lactobacillus acidophilus by Phasor Fluorescence Lifetime Imaging Microscopy of NADH

Nicotinamide adenine dinucleotide (NADH) is an endogenous fluorescent molecule commonly used as a metabolic biomarker. Fluorescence lifetime imaging microscopy (FLIM) is a method in which the fluorescence decay is measured at each pixel of an image. While the fluorescence spectrum of free and protein-bound NADH is very similar, free and protein-bound NADH display very different decay profiles. Therefore, FLIM can provide a way to distinguish free/bound NADH at the level of single bacteria within biological samples. The phasor technique is a graphical method to analyse the entire image and to produce a histogram of pixels with different decay profile. In this study, NADH fluorescence decay profiles within Lactobacillus acidophilus samples treated using different protocols indicated discernible variations. Clear distinctions between fluorescence decay profiles of NADH in samples of artificially heightened metabolic activity in comparison to those of samples lacking an accessible carbon source were obtained.

The Spatial Mapping of the Metabolic Cofactor NADH within Live Progenitor Stem Cells

NADH is a naturally occurring bi-product and regulatory metabolite associated with cellular respiration. The quantification using the difference lifetime of autofluorescence of free and bound NADH has the potential to enhance the understanding of a range of cellular processes including apoptosis, cancer pathology and enzyme kinetics. Fluorescence lifetime imaging microscopy (FLIM) enables not only examination of the spatial location of the cofactor within live cells but also of its state.Here we describe the use of phasor FLIM to spatially map the fluorescence lifetimes of NADH in both free and bound form within live undifferentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. The phasor representation enables the possibility for a direct comparison of either optical sections (i.e. different focal planes) of one cell or multiple cells to enable a global analysis.A comparison of myoblast cells induced to differentiate through serum starvation and undifferentiated cells show differing spatial distribution of the different forms of NADH. Cells due to undergo differentiation displayed a short lifetime representing free NADH situated around the cytoplasmic periphery and a longer lifetime attributed to the presence of bound NADH just outside of the nucleus. Differentiated cells displayed redirection of the distribution of free NADH located mainly within the nucleus while the bound form remained directly comparable to that of the other cells. Furthermore, there appears to be a spatial shift in the distribution of lifetimes at a pixel level within the phasor plot. We show that the states of differentiation of myocytes may be determined through the phasor FLIM analysis of the autofluorescent properties of NADH.