Ms. Dan L. Pham Profile

Dan L. Pham

at Morgridge Institute for Research

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Publications (6)

Proceedings Article | 13 March 2024 Presentation

Autofluorescence lifetime flow cytometry with time-correlated single-photon counting

Kayvan Samimi, Ojaswi Pasachhe, Emmanuel Contreras Guzman, Amani Gillette, Dan Pham, Kathy Wiech, Jeremiah Riendeau, Melissa Skala

Proceedings Volume PC12853, PC1285307 (2024) https://doi.org/10.1117/12.3003561

KEYWORDS: Time correlated single photon counting, Autofluorescence, Flow cytometry, Fluorescence lifetime imaging, Equipment, Ultraviolet radiation, Temporal resolution, Sodium, Single photon detectors, Semiconductor lasers

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SPIE Journal Paper | 29 February 2024 Open Access

Perspectives on label-free microscopy of heterogeneous and dynamic biological systems

Dan Pham, Amani Gillette, Jeremiah Riendeau, Kasia Wiech, Emmanuel Contreras Guzman, Rupsa Datta, Melissa Skala

JBO, Vol. 29, Issue S2, S22702, (February 2024) https://doi.org/10.1117/12.10.1117/1.JBO.29.S2.S22702

KEYWORDS: Microscopy, Tissues, Diseases and disorders, Imaging systems, Image analysis, Raman spectroscopy, Cancer, Tumors, Image segmentation, Image resolution

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SPIE Journal Paper | 9 October 2021 Open Access

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

Dan Pham, Christina Miller, Molly Myers, Dominick Myers, Laura Hansen, Michael Nichols

JBO, Vol. 26, Issue 10, 106501, (October 2021) https://doi.org/10.1117/12.10.1117/1.JBO.26.10.106501

KEYWORDS: Luminescence, Fluorescence lifetime imaging, Mode conditioning cables, Vehicle control, Cancer, Tumors, Tumor growth modeling, Statistical analysis, Skin cancer, Tissue optics

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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.

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Proceedings Article | 5 March 2021 Presentation

Multiphoton autofluorescence imaging of immune cells in cancer

Melissa Skala, Tiffany Heaster, Kelsey Tweed, Alexa Heaton, Dan Pham, Katherine Mueller, Dave Beebe, Paul Sondel, Krishanu Saha

Proceedings Volume 11648, 1164807 (2021) https://doi.org/10.1117/12.2579252

KEYWORDS: Tumors, Cancer, Auto-fluorescence imaging, Mode conditioning cables, 3D modeling, Systems modeling, Optical imaging, Nondestructive evaluation, Multiphoton microscopy, Mouse models

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Proceedings Article | 5 March 2021 Presentation

Time-domain single photon-excited autofluorescence lifetime is sensitive to activation state of T cells

Kayvan Samimi, Emmanuel Contreras Guzman, Steven Trier, Dan Pham, Tongcheng Qian, Melissa Skala

Proceedings Volume 11655, 116550L (2021) https://doi.org/10.1117/12.2577765

KEYWORDS: Fluorescence lifetime imaging, Imaging systems, Flow cytometry, Time correlated photon counting, System integration, Single photon, Resistance, Proteins, Nondestructive evaluation, Microscopy

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Proceedings Article | 5 March 2021 Presentation

Label-free metabolic imaging of tumor-induced T cell exhaustion

Dan Pham, Katherine Mueller, Krishanu Saha, Melissa Skala

Proceedings Volume 11655, 1165508 (2021) https://doi.org/10.1117/12.2578763

KEYWORDS: Tumors, Proteins, Mode conditioning cables, Luminescence, Fluorescence lifetime imaging, Statistical analysis, Radiotherapy, Microscopy, In vivo imaging, Flow cytometry

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