Beginners Guide to Fluorescence Lifetime Imaging in Life Sciences

Virtually everywhere

Virtually everywhere

Ali Gheisari (CMCB Light Microscopy Facility, TU Dresden) , Mariano Gonzalez Pisfil (Core Facility Bioimaging, Biomedical Center, LMU München) , Stefanie Weidtkamp-Peters (Center for Advanced Imaging, HHU Düsseldorf) , Steffen Dietzel (Core Facility Bioimaging, Biomedical Center, LMU München) , Thomas Zobel (Imaging Network, WWU Münster)

The Beginners Guide to Fluorescence Lifetime Imaging (FLIM) in Life Sciences takes place on November 23rd to 27th 2020 via Zoom. The online FLIM symposium is co-organized by CMCB Light Microscopy Facility of Technische Universität Dresden, Imaging Network of WWU Münster, Center of Advanced Imaging of HHU Düsseldorf University and the Core Facility Bioimaging at the Biomedical Center of the Ludwig-Maximilians-Universität München.

The talks are live and online while the hands-on sessions (WS1 - WS9) are combinations of interactive sessions at the microscope and respective recorded videos. There, the experts will showcase further developments in the field, and explain how FLIM contributes to take their research to the next level.

Our aim is to address the applications of FLIM microscopy in life science and discuss the tips and tricks in FLIM data analysis and data acquisition.


Invited speakers

Ammasi Periasamy - University of Virginia, the United States

Angelika Rück - Universität ULM, Germany

Alessandro Esposito - University of Cambridge, United Kingdom

Carsten Grashof - Westfälische Wilhelms-Universität Münster, Germany

Gerhard Holst – PCO AG, Germany

Rupsa Datta - Morgridge Institute of Research, the United States

Sumeet Rohilla - Charité-Universitätsmedizin Berlin, Germany

Werner Zuschratter - Otto-von-Guericke-Universität Magdeburg, Germany

Yvone Stahl - Heinrich-Heine-Universität Düsseldorf, Germany



Ali Gheisari - CMCB light microscopy facility of Technische Universität Dresden

Thomas Zobel - Imaging Network of WWU Münster,

Stefanie Weidtkamp-Peters - Center of advanced imaging of HHU Düsseldorf University and

Steffen Dietzel - Core Facility Bioimaging at the Biomedical Center, LMU München

Mariano Gonzalez Pisfil - Core Facility Bioimaging at the Biomedical Center, LMU München


Header image: Sumandibular Gland section excited with 488 nm laser courtesy of Mariano Gonzalez Pisfil

Survey - Beginners Guide to Fluorescence Lifetime Imaging (FLIM) in Life Sciences - November 23rd to 27th 2020
    • 14:00 14:10
      Opening 10m
      Speaker: Dr Ali Gheisari (Technische Universität Dresden)
    • 14:10 14:50
      Introduction to Fluorescence Lifetime Imaging Microscopy (FLIM) 40m
      Speaker: Dr Stefanie Weidtkamp-Peters (WWU Düsseldorf)
    • 14:50 15:30
      Application of Förster Resonance Energy Transfer (FRET) in plant biology 40m

      Yvonne Stahl holds a diploma degree in Biology from the University of Cologne (Germany). She finished her PhD under the supervision of Prof. Peter C. Morris at Heriot-Watt University, Edinburgh (UK) in 2003. Thereafter, she worked as a Research Fellow and then as a group leader at the Institute for Developmental Genetics, Heinrich Heine University, Düsseldorf (Germany). She habilitated in developmental genetics in 2016. Her research group focusses on root development and architecture in plants using fluorescence spectroscopic methods. She utilizes interdisciplinary approaches consisting of genetic and fluorescence spectroscopic methods to demonstrate that dynamic interactions and complexes of information molecules are important for root stem cell maintenance.

      Speaker: Dr Yvone Stahl (Heinrich-Heine-Universität Düsseldorf, Germany)
    • 15:30 16:00
      Virtual Coffee Break 30m
    • 16:00 16:40
      Piconewton-sensitive biosensors to investigate molecular forces in cells 40m

      Carsten Grashoff studied Applied Science at the University of Freiberg in Germany and performed his diploma studies at the Robert-Koch Institute in Berlin. He worked as a PhD student at the Max Planck Institute of Biochemistry in Munich and obtained his PhD degree in 2007, before moving for his postdoctoral work to the University of Virginia (USA). In 2010, he was awarded an Emmy-Noether fellowship and started an independent research group at the Max Planck Institute of Biochemistry; in 2014 he obtained the Paul Gerson Unna Fellowship from the Max Planck Foundation. In 2018, he moved to the University of Münster, where he was appointed Professor for Quantitative Cell Biology. Carsten Grashoff received numerous awards including the Early Career Award of the German National Academy of Science (2014) and the Binder Innovation Award (2018) by the German Society of Cell Biology.

      Speaker: Prof. Carsten Grashoff (Westfälische Wilhelms-Universität Münster, Germany)
    • 16:40 17:20
      Live cell imaging of biochemical networks and cellular decisions 40m

      The integration of contemporary genetic and optical microscopy tools is permitting researchers to control and observe biochemical networks in single living cells and organisms. I will provide a brief introduction and examples on how we can use FRET, FLIM and optogenetics to study how cell biochemistry encodes for cellular decisions. I’ll discuss some of the challenges and opportunities that biochemical imaging presents to improve our understanding of the mechanisms underpinning disease.

      Speaker: Dr Alessandro Esposito (University of Cambridge, United Kingdom)
    • 17:20 18:00
      Spectral FLIM (Multiplexing) 40m

      Sumeet Rohilla studied physics (M.Sc.) and optics (M.Tech) at the Indian Institute of Technology Delhi, India.
      He worked as a Marie Curie Research Fellow (Ph.D.) at the Charité – Universitätsmedizin Berlin and PicoQuant GmbH, where the primary focus of his research work was to develop spectrally-resolved fluorescence lifetime imaging (spectral-FLIM) systems and corresponding data analysis algorithms. 
      In his talk, he will provide a brief introduction to the data acquisition & investigation routines especially tailored towards spectral-FLIM techniques for tissue imaging.

      Speaker: Dr Sumeet Rohilla (Charité-Universitätsmedizin Berlin)
    • 14:00 14:40
      Wide-field FLIM by Single Photon Counting with LINCam 40m

      Werner Zuschratter studied Biology at TU-Darmstadt and managed the Special Laboratory for Electron- and Laserscanning Microscopy at Leibniz Institute for Neurobiology (LIN) for more than 25 years before he became coordinator and head of the Combinatorial NeuroImaging Core Facility at LIN (CNI). Starting his career in the field of electron microscopy his research activities focus on structural-functional relationships on the ultrastructural, i.e. nm-range level and the analysis of dynamics of biochemical, molecular-biological and cell biological processes in neurons and the immune system. As unique technology in our lab an ultra-sensitive, time-resolving wide-field camera has been developed for FLIM and spectroscopy. The quantum detector based on Time-Correlated-Single-Photon-Counting (TCSPC) is mainly used to examine protein complexes and molecular interactions via FRET, long term cell dynamics under low light conditions and metabolic changes by auto-fluorescence imaging of NADH. The patented and award-winning technology is market now by the spin-off Photonscore under the name LINCam.

      Yury Prokazov studied physics in Moscow and got his doctoral degree in engineering at OvGU Magdeburg. From 2005 to 2018 he was co-worker in the group of WZ at LIN, where he invented the wide-field quantum detector together with Evgeny Turbin. YP’s main focus is software development for the determination and analysis of the space coordinates together the with precise timing information from the quanta data stream. Since 2017 he is managing director of Photonscore, a spin-off from LIN that further develops and markets the technology of the LINCam single-photon-counting camera for various applications.

      André Weber studied physics at OvGU Magdeburg and joined the group of Werner Zuschratter at LIN in 2010. He started his scientific career with studying glycolytic oscillations in yeast cells. By imaging the autofluorescence of the coenzyme NADH at single cell level, he could show that the dynamics of glycolytic oscillations in yeast are cell density dependent. While sparse cell populations show individual desynchronized NADH oscillations, cells synchronize their metabolic activity in dense cell populations and oscillate in concert.
      Using time-correlated single photon counting with the LINCam he identified several fluorescence lifetimes of NADH corresponding to the free or bound state of the coenzyme. Moreover, in cell and tissue cultures of neuronal and immune cells he demonstrated that the amount and the lifetime contributions of NADH correlate with the cellular and network activity. Currently, AW runs the FLIM laboratory of CNI at LIN and uses the LINCam for new applications, e.g. Light-Sheet FLIM, Metal induced energy transfer (MIET), time resolved Raman spectroscopy and single molecule detection of spectrally overlapping dyes.

      Speakers: Mr André Weber (Photonscore GmbH) , Dr Werner Zuschratter (Otto-von-Guericke-Universität Magdeburg, Germany) , Dr Yury Prokazov (Photonscore GmbH)
    • 14:40 15:20
      Frequency-domain Fluorescence Lifetime Imaging Microscopy 40m

      Gerhard Holst, Head of Research & Science Department, PCO AG, Kelheim, Germany
      Gerhard Holst graduated at the Technical University Aachen, Germany, with a Diploma in Electrical Engineering in 1991 (Information Technology) and went on to complete his Doctorate at the University of Dortmund in collaboration with the Max-Planck-Institute for Systems Physiology in Dortmund, Germany from 1991 - 1994. Gerhard furthered his research as member of the Microsensor Research Group at the Max-Planck-Institute for Marine Microbiology in Bremen, Germany from 1994 – 2001. Since 2001 Gerhard is head of the research & science department at PCO AG, where he is responsible for new technologies and all research projects.

      Speaker: Dr Gerhard Holst (PCO AG)
    • 15:20 15:50
      Virtual Coffee Break 30m
    • 15:50 16:30
      Investigation of Drug Cellular Uptake in Prostate Cancer Cells: Two-photon FLIRR Microscopy 40m

      Dr. Periasamy received his Ph.D. at the Indian Institute of Technology, Madras and postdoctoral training at the University of Washington Seattle. Currently, Dr. Periasamy is a prof. of Biology and Biomedical Engineering, the director and founder of the internationally known WM Keck Center for Cellular Imaging (KCCI), University of Virginia, Charlottesville. Dr. Periasamy is one of the pioneers in the development of fluorescence lifetime imaging microscopy (FLIM). A key area of his research is focused on the design and development of optical methodology including advanced light microscopy techniques to investigate/monitor exogenous and endogenous molecular interactions in live cells, tissues, and animals. Dr. Periasamy has edited three books, series book editor on cellular and clinical imaging (11 books), Chairperson of an annual International conference on Multiphoton Microscopy in the Biomedical Sciences through SPIE (since 2001) and runs a hands on training annual workshop on FLIM & FRET Microscopy at the University of Virginia, Charlottesville during March (since 2002).

      Speaker: Prof. Ammasi Periasamy (University of Virginia, the United States)
    • 16:30 17:10
      Analysis of FLIM data 40m

      Rupsa Datta is an Assistant Scientist in the laboratory of Dr. Melissa Skala, a Professor of Biomedical Engineering at the University of Wisconsin – Madison and lead investigator at the Morgridge Institute for Research, Madison. Rupsa graduated from University of California, Irvine with Ph.D in Biomedical Engineering. Trained in fluorescence lifetime imaging microscopy, her research entails employing optical imaging techniques to study cancer, specifically, to decipher the influence of stroma in development and progression of pancreatic cancer and thereby device new approaches to cancer therapy.

      Speaker: Dr Rupsa Datta (Morgridge Institute of Research, the United States)
    • 17:10 17:50
      Simultaneous Metabolic FLIM and Oxygen PLIM (Phosphorescence LIfetime Imaging) in Biomedical Research 40m

      Angelika Rueck studied chemistry at the University of Ulm and did her PhD in physical
      chemistry. For more than 20 years she led the research on photodynamic therapy and
      advanced microscopy at the Institute for advanced Photonics and Optics (ILM) in Ulm. Since
      7 years she held the position of the director of the core facility and bioimaging research group
      confocal and multiphoton microscopy at the University Ulm and is responsible for
      microscopic applications and developments in the field of biomedical research and molecular
      medicine. Dr. Rueck´s scientific efforts are focused on new methods for time resolved
      luminescence spectroscopy and microscopy as FLIM, SLIM and PLIM in living cells and
      organisms. Imaging of cell metabolism, bioenergetic alterations and oxygen levels during
      tumor development, neurodegenerative and other diseases are investigated.

      Wolfgang Becker
      is specialist in time-resolved optical detection techniques. He obtained his PhD 1979 in Berlin, Germany. Since 1993 he is the head of Becker & Hickl GmbH in Berlin. His field of interest is development and application of advanced Time-Correlated Single Photon Counting techniques. He started to develop multi-dimensional TCSPC techniques in 1989, and is the originator of the TCSPC FLIM technique that is now widely used in laser scanning microscopes. He is author of the bh TCSPC handbook, now out in the 8th edition, and of 'Advanced Single Photon Counting Techniques', Springer, 2005, and editor of 'Advanced Single Photon Counting Applications', Springer, 2015.
      He likes cats, skiing and beach volleyball.

      Speakers: Dr Angelika Rück (Universität ULM, Germany) , Dr Woflgang Becker (Becker & Hickl GmbH)
    • 17:50 18:00
      Closure of this session 10m

      THe organization team

    • 09:30 12:30
      WS1 - FRET application in plant biology 3h

      In many biological research projects, a high spatial and temporal resolution for the observation of in vivo protein interaction is needed, e.g. in order to follow changes of interactions and complex formation over time. In vivo Fluorescence or Förster resonance energy transfer (FRET) measurement allows detailed analysis of interacting molecules in their natural environment at a subcellular level. Especially, FRET-FLIM (Fluorescence lifetime imaging microscopy) measurements provide an extremely powerful and reliable tool meeting the demands for investigating in vivo protein interaction also quantitatively and with high precision. During the workshop, we will introduce in detail how to practically perform in vivo FRET measurements in living plant tissue and discuss potential pitfalls and points of consideration.

      Speakers: Dr Stefanie Weidtkamp-Peters (WWU Düsseldorf) , Dr Yvone Stahl (Heinrich-Heine-Universität Düsseldorf, Germany)
    • 12:30 13:30
      Virtual Lunch Break 1h
    • 13:30 15:30
      WS2 - FRET-based tension biosensors 2h
      Speaker: Prof. Carsten Grashoff (WWU Münster)
    • 15:30 15:40
      Virtual Coffee Break 10m
    • 15:40 18:40
      WS3 - FLIM application in environmental sensing 3h

      The fluorescence lifetime of many fluorophores is highly dependent on their environment, making fluorescence lifetime imaging (FLIM) a sensitive tool for analyzing subcellular distributions of certain ions or gradients in viscosity or hydrophobicity. In this workshop, we will exemplary demonstrate the usage of a pH-sensitive dye to monitor changes in lysosomal pH (Tannert et al, submitted). Lysosomes are small acidic degradation compartments of eukaryotic cells. Their physiological pH is usually in the range of 4.5-5, depending on their subcellular localization and maturation status. Different (patho)physiological conditions, like autoimmune diseases, neurodegenerative disorder, or age-related complications can induce alterations in lysosomal pH. In most of these cases the lysosomal pH is increased. Lysosomal neutralization can also be induced by chemical stimulation, for instance using ammonium chloride. We will demonstrate a lysosomal staining procedure of HeLa cells and subsequent FLIM measurement. To be able to follow lysosomal pH changes over time, the measurement time for individual FLIM measurements in a time series has to be short. Therefore, the rapidFLIMHiRes module is applied. During a time series measurement, we will increase lysosomal pH by addition of 10 mM ammonium chloride after a certain baseline and monitor the alterations by detecting the lifetime of a pH sensitive lysosomal marker dye. Subsequently, FLIM data analysis of this time series will be explained in detail. Thereby we will give various tips for data analysis of standard FLIM or rapidFLIM acquisition conditions using the SymPhoTime 64 software package.

      Speakers: Dr Astrid Tannert (Jena Biophotonics and Imaging Laboratory (JBIL).) , Dr Fabian Jolmes (PicoQuant GmbH)
    • 09:30 12:30
      WS 4 - Component seperation by means of lifetime measurement 3h

      The workshop’s aim is to introduce another potential use of lifetime: dye separation. Much like absorption and emission spectra, the fluorescence lifetime depends on the intrinsic characteristics of each fluorescent dye and its surroundings. If two fluorochromes have the same emission properties, separation by color may be impossible. But their lifetime can still be quite different. This difference can be exploited to unmix them after acquisition using a lifetime based analysis. The approach allows to distinguish more colors than by conventional spectral separation alone. In this session, we will focus on phasor based lifetime separation. The phasor plot is an excellent tool for color lifetime separation, with its ease of use and quick understanding. Thus enabling an accessible spectral and lifetime based protocol for multicolor imaging.
      Among others samples, the separation of Aberior Star 635P and Atto 647N mounted in CFM 3 will be shown on Leica SP8 Falcon.

      Speaker: Mariano Gonzalez Pisfil (LMU München)
    • 12:30 13:30
      Virtual Lunch Break 1h
    • 13:30 15:30
      WS 5 - Tau-STED - application of lifetime measurement in super-resolution microscopy 2h

      Fluorescence lifetime provides a new perspective for STED nanoscopy. This workshop will focus on Tau-STED, a new concept for STED Nanoscopy that exploits the fluorescence lifetime gradient induced by the STED beam in a novel way to identify and remove uncorrelated background, improve image quality and increase image resolution in an automated way. This is only possible thanks to the unique combination of STED, ultra-fast photon-counting detectors, and FLIM as provided by the FALCON approach (Alvarez et al, Nat Methods 2019). Tau-STED identifies the signal from the STED process to differentiate it from background using fluorescence lifetime. It identifies the fluorescence lifetime gradient generated by the STED process with the help of FLIM Phasor analysis to deliver the desired resolution at dramatically lower light dose, and to push the resolution beyond what conventional STED provides. The experiment raw data is always available for validation and quantification of results.

      We will work with a variety of single and multicolor fixed samples, focusing on structures typically observed with STED (nuclear pores, microtubules, mitochondria). The lower excitation and STED light dose protect the sample during imaging, and enable acquiring more frames for large volume imaging or time-lapse experiments, which we will demonstrate with living cells.

      Alvarez L, Widzgowski B, Ossato G, van den Broek B, Jalink K, Kuschel L, Roberti MJ, and Hecht F: “SP8 FALCON: a novel concept in fluorescence lifetime imaging enabling video-rate confocal FLIM”, Nat Methods (2019).

      Speakers: Dr Daniel Smeets (Leica Microsystems) , Mariano Gonzalez Pisfil (LMU München) , Dr Steffen Dietzel (LMU Munich)
    • 15:30 15:40
      Virtual Coffee Break 10m
    • 15:40 18:40
      WS 6 - Label-free FLIM - Studying the cellular metabolism by NADH autofluorescence FLIM 3h


      During this session, we explore the potentials of autofluorescence imaging to investigate the cellular metabolic state.
      Adenosine triphosphate (ATP), the energy molecule of the cells is the product of different energy production pathways - glycolysis and Krebs cycle and oxidative phosphorylation. Two co-factors plays important role in the energy production pathways: NAD(P)H (reduced nicotinamide adenosine dinucleotide (phosphate)[H]) and FAD (oxidized Flavin adenine dinucleotide). The autofluorescence of both NAD(P)H and FAD exhibit two distinctive fluorescence lifetimes for bound and free forms. The changes between the fractions of the short and long lifetime of these co-factors indicate changes in the metabolic activity and pathways in the specimen.

      Drosophila melanogaster

      Leica SP8 FALCON
      This fluorescence microscope enables two-photon NAD(P)H and FAD autofluorescence FLIM. The system is equipped with FOUR HyD detectors in te non-descanned (NDD) configuration and FALCON module for sensitive and fast lifetime imaging.

      Speakers: Dr Ali Gheisari (Technische Universität Dresden) , Dr Cornelia Wetzker (TU Dresden, Germany)
    • 09:30 11:00
      WS7 - Data analysis in depth I - SymPhoTime 64 (PicoQuant) 1h 30m
      Speakers: Dr Benedikt Krämer (PicoQuant GmbH) , Dr Maria Loidolt-Krüger (PicoQuant GmbH)
    • 11:00 12:30
      WS 8 - Data analysis in depth II - Phassor analysis with LAS X (Leica Microsystems) 1h 30m

      This workshop will show the analysis capabilites of the current Leica LAS X software release,focusing on the integrated Phasor Plot analysis.

      Speaker: Dr Giulia Ossato (Leica Microsystems)
    • 12:30 13:30
      Virtual Lunch Break 1h
    • 13:30 15:30
      WS9 - Open source FLIM data analysis 2h
      Speaker: Ali Gheisari
    • 15:30 15:40
      Virtual Coffee Break 10m
    • 15:40 18:40
      WS10 - Wide-field photon counting made easy - tutorial and online experiments with the LINCam 3h
      Speakers: Mr André Weber (Leibniz Institute for Neurobiology, Magdeburg, Germany) , Dr Yury Prokazov (Photonscore GmbH, , Magdeburg, Germany)