School of Engineering
Director of the W.M. Keck Nanoimaging Lab,
Laboratory for Fluorescence Dynamics (LFD)
Advanced Imaging, Non-Invasive Detection and Molecular Dynamics: Our lab merges label-free deep-tissue optics with advanced mathematical analysis of single-molecule dynamics. We apply the “phasor approach” for Fluorescence Lifetime Imaging Microscopy (FLIM) to characterize autofluorescence from metabolic co-factors and map cellular metabolism without destructive dyes. We also combine 2-photon excitation, Second Harmonic Generation (SHG), and Third Harmonic Generation (THG) microscopy for non-invasive tissue imaging while using multi-color bioluminescence analysis to visually untangle overlapping chemical signals in real time. Beyond capturing these static structures, use Raster Image Correlation Spectroscopy (RICS), Fluorescence Correlation Spectroscopy (FCS), and Number and Molecular Brightness (N&B) analysis to track and count individual proteins within living cells, fundamentally advancing our ability to measure precise molecular concentrations and protein aggregation at the nanoscale.
Engineering new imaging systems: We have engineering advanced optical hardware and open-access imaging packages that overcome the fundamental physics constraints of light-sensitive microscopy. In particular we have built a low cost-effective hyperspectral imaging systems that enables multi-color bioluminescent outputs without toxic external excitation light. This specialized hardware setup bypasses traditional multi-camera calibration hurdles by channeling weak emissions straight into a unified mathematical algorithm, allowing us to cleanly separate up to six distinct biological probes simultaneously and track live tissue operations in real time.
Automated Image Analysis Software: We developed Mitometer, an automated segmentation and tracking algorithm that eliminates human bias by utilizing precise background-removal and feature-linking logic to monitor fast-moving mitochondria in live-cell time-lapse images. Our lab also developed FIBIS (FLIM Intensity-Based Image Segmentation), a data-driven algorithm tailored to segment fluorescence lifetime data, which was used to profile the energetics of cancer cells and tracking cellular nanoparticles. Most recently, we pioneered ESPRESSO (Environmental Sensor Phenotyping RElayed by Subcellular Structures and Organelles), a groundbreaking spatiotemporal omics framework that blends advanced microscopy with bioimage data analysis pipelines to map single-cell structural and functional organelle transformations across space and time. Through these algorithms we have successfully bridged the gap between optical engineering and software development, providing the scientific community with highly scalable tools for automated cellular discovery.
Lopez KL, Palomba F, Mock M, Srinivasan N, Pelegri-O’Day E, Digman MA. A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development. Methods Appl Fluoresc. 2026 Apr 30;14(2) PubMed ID: 41962589.
Scipioni L, Torrado B, Tedeschi G, Halbers L, Torrey Z, Fuller E, Fersini F, Gohlke C, Luptak A, Prescher J, Digman M. An Open, Integrated Platform for Multiplexed Bioluminescence Microscopy. Chemical & Biomedical Imaging. 2026 April 15. Available from: https://doi.org/10.1021/cbmi.5c00225 DOI: 10.1021/cbmi.5c00225
Fazel M, Hoseini R, Saurabh A, Xu LWQ, Scipioni L, Tedeschi G, Gratton E, Digman MA, Pressé S. Resolving fluorescently labeled species using highly multiplexed spectral FLIM. Sci Rep. 2026 Apr 11;16(1) PubMed Central ID: PMC13230939.
Halbers LP, Brennan CK, Scipioni L, Tedeschi G, Torrey ZR, Parag-Sharma K, Labra B, Gohlke C, Amelio AL, Digman MA, Prescher JA. Expanded applications of bioluminescence microscopy with phasor analysis. Cell Rep Methods. 2026 Apr 20;6(4):101344. PubMed Central ID: PMC13106971.
Fuller EB, Cole KH, Halbers LP, Chan CET, Ng KK, Scipioni L, Gohlke C, Digman MA, Steward O, Prescher JA, Lupták A. Bioluminescent Probes for Multiplexed RNA Imaging. J Am Chem Soc. 2026 Mar 25;148(11):11521-11530. PubMed Central ID: PMC13022865.
Song Y, Hao Y, Latacz M, Cykowiak M, Kirylczuk J, Quan X, Palomba F, Ni S, Liu L, Hu J, Shi B, Posey A, Li Q, Yuan H, Sun J, Pappu R, Digman MA, Huang K, Jiang H. Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer. bioRxiv. 2026 Feb 20; PubMed Central ID: PMC12934752.
Huang YK, Zhuang M, Digman MA. FLIM intensity-based image segmentation reveals upregulated energy metabolism and chemotherapy sensitivity in MCF-7 cells. J Cell Sci. 2025 Dec 1;138(23) PubMed Central ID: PMC12752511.
Scipioni L, Tedeschi G, Navarro MX, Jia YY, Zhu S, Halbers LP, Di Bona M, Atwood SX, Prescher JA, Gratton E, Digman MA. ESPRESSO: spatiotemporal omics based on organelle phenotyping. Nat Methods. 2025 Nov;22(11):2349-2361. PubMed ID: 41184551.
Woytash JA, Lefebvre AEYT, Zhang Z, Xu B, Harchenko SA, Le HT, McColloch AR, Shi X, Digman MA, Razorenova OV. CDCP1/mitochondrial Src axis increases electron transport chain function to promote metastasis in triple-negative breast cancer. Br J Cancer. 2025 Nov;133(9):1265-1277. PubMed Central ID: PMC12530134.
Scipioni L, Torrado B, Tedeschi G, Halbers LP, Torrey ZR, Fuller EB, Fersini F, Gohlke C, Luptak A, Prescher JA, Digman MA. An open, integrated platform for multiplexed bioluminescence microscopy. bioRxiv. 2025 Aug 22; PubMed Central ID: PMC12393305.
Fazel M, Hoseini R, Saurabh A, Xu LWQ, Scipioni L, Tedeschi G, Gratton E, Digman MA, Pressé S. Highly multiplexed spectral FLIM via physics informed data analysis. bioRxiv. 2025 Aug 12; PubMed Central ID: PMC12363814.
Tedeschi G, Palomba F, Scipioni L, Digman MA. Multimodal Phasor Approach to Study Breast Cancer Cell Invasion in a 3D Spheroid Model. Chem Biomed Imaging. 2025 Jul 28;3(7):433-442. PubMed Central ID: PMC12308588.
Gallagher T, Leemans S, Dvornikov AS, Perinbam K, Fong J, Kim C, Kapcia J, Kagawa M, Grosvirt-Dramen A, Hochbaum AI, Digman MA, Gratton E, Siryaporn A, Whiteson K. Fluorescence Lifetime Imaging Detects Long-Lifetime Signal Associated with Reduced Pyocyanin at the Surface of Pseudomonas aeruginosa Biofilms and in Cross-Feeding Conditions. ACS Infect Dis. 2025 Mar 14;11(3):543-549. PubMed Central ID: PMC11915379.
Di Franco E, Tedeschi G, Scipioni L, Gratton E, Digman M, Castello M, Diaspro A, Vicidomini G, Bianchini P, Lanzanò L. Exploiting the detector distance information in image scanning microscopy by phasor-based SPLIT-ISM. Biomed Opt Express. 2025 Mar 1;16(3):1270-1283. PubMed Central ID: PMC11919353.
Sanchez-Velasquez J, Solano A, Digman MA, Gratton E, Cardarelli F, Hinde E. Pair correlation microscopy of intracellular molecular transport. Nat Protoc. 2025 Jun;20(6):1651-1677. PubMed ID: 39915621.
Georgakoudi I, Skala MC, Quinn KP, Stringari C, Sorrells JE, Heikal AA, Li LZ, Xu HN, You S, Walsh AJ, Datta R, Samimi K, Gillette AA, Eliceiri KW, Balu M, Boppart SA, Digman MA, Dunning KR, Evans CL, Garcia AA, Houston JP, Hwang W, Lindley MM, Li X, Liu Z, Marcu L, Murugkar S, Nichols MG, Niesner R, Parekh SH, Rajaram N, Ranjit S, Shen K, Shi L, Torrado B, Vallmitjana A, Wang-Evers M, Zemp R. Consensus guidelines for cellular label-free optical metabolic imaging: ensuring accuracy and reproducibility in metabolic profiling. J Biomed Opt. 2025 Feb;30(Suppl 2):S23901. PubMed Central ID: PMC12604823.
Ramos R, Pham KT, Prince RC, Leiser-Miller LB, Prasad MS, Wang X, Nordberg RC, Bielajew BJ, Hu JC, Yamaga K, Oh JW, Peng T, Datta R, Astrowskaja A, Almet AA, Burns JT, Liu Y, Guerrero-Juarez CF, Tran BQ, Chu YL, Nguyen AM, Hsi TC, Lim NT, Schoeniger S, Liu R, Pai YL, Vadivel CK, Ingleby S, McKechnie AE, van Breukelen F, Hoehn KL, Rasweiler JJ 4th, Kohara M, Loughry WJ, Weldy SH, Cosper R, Yang CC, Lin SJ, Cooper KL, Santana SE, Bradley JE, Kiebish MA, Digman M, James DE, Merrill AE, Nie Q, Schilling TF, Astrowski AA, Potma EO, García-Castro MI, Athanasiou KA, Behringer RR, Plikus MV. Superstable lipid vacuoles endow cartilage with its shape and biomechanics. Science. 2025 Jan 10;387(6730):eads9960. PubMed Central ID: PMC12279017.
Tedeschi G, Navarro M, Scipioni L, Sondhi T, Prescher J, Digman M. Monitoring Macrophage Polarization with Gene Expression Reporters and Bioluminescence Phasor Analysis. Chemical & Biomedical Imaging. 2024 November 25; 2(11):765-774. Available from: https://doi.org/10.1021/cbmi.4c00049 DOI: 10.1021/cbmi.4c00049
Rojas-Andrade MD, Perinbam K, Nguyen QT, Kim JS, Palomba F, Whiteson K, Digman MA, Siryaporn A, Hochbaum AI. Rapid Antibiotic Susceptibility Determination by Fluorescence Lifetime Tracking of Bacterial Metabolism. ACS Infect Dis. 2024 Dec 13;10(12):4057-4065. PubMed ID: 39572010.
Hedde PN, Zhu S, Barylko B, Chiu CL, Nelson LT, Digman MA, Albanesi JP, James NG, Jameson DM. Effect of Pathogenic Mutations on the Formation of High-Order Dynamin 2 Assemblies in Living Cells. Biochemistry. 2024 Nov 5;63(21):2750-2758. PubMed ID: 39390788.
Tedeschi G, Navarro MX, Scipioni L, Sondhi TK, Prescher JA, Digman MA. Monitoring Macrophage Polarization with Gene Expression Reporters and Bioluminescence Phasor Analysis. Chem Biomed Imaging. 2024 Nov 25;2(11):765-774. PubMed Central ID: PMC11600157.
Torrey ZR, Halbers LP, Scipioni L, Tedeschi G, Digman MA, Prescher JA. A versatile bioluminescent probe with tunable color. RSC Chem Biol. 2024 Oct 30;5(11):1097-1103. PubMed Central ID: PMC11414822.
Butenko S, Nagalla RR, Guerrero-Juarez CF, Palomba F, David LM, Nguyen RQ, Gay D, Almet AA, Digman MA, Nie Q, Scumpia PO, Plikus MV, Liu WF. Hydrogel crosslinking modulates macrophages, fibroblasts, and their communication, during wound healing. Nat Commun. 2024 Aug 9;15(1):6820. PubMed Central ID: PMC11315930.
Tedeschi G, Navarro MX, Scipioni L, Sondhi TK, Prescher JA, Digman MA. Monitoring macrophage polarization with gene expression reporters and bioluminescence phasor analysis. bioRxiv. 2024 Jun 12; PubMed Central ID: PMC11195121.
Tedeschi G, Palomba F, Scipioni L, Digman MA. Multimodal Phasor Approach to study breast cancer cells invasion in 3D spheroid model. bioRxiv. 2024 Jun 12; PubMed Central ID: PMC11195137.