Fluorescence-lifetime imaging microscopy (FLIM) not only for biologists - practical aspects with hands-on experience - MB100P03

• Title: Mikroskopie dob života fluorescence nejen pro biology - praktické aspekty a cvičení na mikroskopech
• Guaranteed by: Biology Section (31-101)
• Faculty: Faculty of Science
• Actual: from 2025
• Semester: winter
• E-Credits: 2
• Examination process: winter s.:
• Hours per week, examination: winter s.:0/3, C [DS]
• Capacity: 11
• Min. number of students: unlimited
• 4EU+: no
• Virtual mobility / capacity: no
• State of the course: taught
• Language: English
• Explanation: Please register first at the webpage of the course.Selected participants will add this course to their list of subjects later.
• Additional information: http://web.natur.cuni.cz/IMCF/FNOB/index.html
• Note: enabled for web enrollment; the course is taught as cyclical
• Guarantor: Mgr. Aleš Benda, Ph.D.

Annotation

Fluorescence microscopy became an indispensable tool for biologists to uncover the secrets of live. The more we want to know, the more details we need. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a set of tools that allow to utilize extra information offered by fluorescence probes and sensors, which is otherwise difficult to access. Typical examples, which will be demonstrated during the course, are metabolic activity by NAD(P)H imaging, protein interactions by FLIM-FRET and lipid membrane micro-environment sensing by Laurdan imaging.
The course focuses on explaining the principles of nano-second time-resolved fluorescence detection, on demonstration of different hardware realizations, on trying various applications of FLIM in biological imaging and on testing of several ways of FLIM data analysis.
The aim is to uncover the richness of information hidden in multi-parametric fluorescence imaging and to inspire the participants to use the easily obtainable extra contrast in their imaging applications. An important part is to make the FLIM data analysis understandable.
The course will cover the most widespread FLIM realization – acquisition in time domain based on raster scanning combined with pulsed lasers and time-correlated single photon counting (TCSPC). Participants will hands-on three different systems, covering pulsed diode lasers, white-light laser source as well as two-photon excitation, SPAD, HyD and GaAsP NDD detectors and three different TCSPC systems and software.
A focus will be given on common pitfalls, instrumental artifacts and ways of avoiding them.
The demonstrated applications will include FLIM-FRET, environmental sensing, metabolic NAD(P)H imaging and FLIM data analysis.

Last update: Benda Aleš, Mgr., Ph.D. (23.09.2020)

Literature

The bible of FLIM is the handbook from Wolfgang Becker: https://www.becker-hickl.com/literature/documents/flim/the-bh-tcspc-handbook/

Technical aspects of the main applications are described here: https://www.tcspc.com/doku.php/applications#fluorescence_lifetime_imaging_microscopy_flim

A recent review on newest applications, including those demonstrated during the course, is J. of Biomedical Optics, 25(7), 071203 (2020). https://doi.org/10.1117/1.JBO.25.7.071203

Last update: Benda Aleš, Mgr., Ph.D. (08.06.2022)

Course assessment methods and requirements for successful completion, grading scheme

Active participation at the whole course plus short 3 min presentation of your own work or planned work..

Last update: Benda Aleš, Mgr., Ph.D. (07.10.2019)

Syllabus

http://web.natur.cuni.cz/IMCF/FNOB/programme.html

Last update: Benda Aleš, Mgr., Ph.D. (23.09.2020)

Learning outcomes

Knowledge and Understanding

1. Describes the physical principles of fluorescence lifetime, nano‑second time‑resolved detection, and the fundamentals of TCSPC‑based FLIM acquisition.

2. Explains the operational principles, advantages, and limitations of different FLIM hardware configurations, including pulsed diode lasers, white‑light lasers, two‑photon excitation, and SPAD/HyD/GaAsP detectors.

3. Summarizes the biological and biophysical applications of FLIM, including NAD(P)H metabolic imaging, FLIM‑FRET for protein interactions, and membrane‑environment sensing using Laurdan or Flipper‑TR probes.

Practical and Technical Skills

1. Operates multiple FLIM systems (e.g., Leica SP8, Zeiss LSM880, Abberior Infinity Line, Stellaris FALCON) to acquire fluorescence lifetime data under supervised laboratory conditions.

2. Performs FLIM measurements using a range of probes and sensors, including intracellular pH indicators, NAD(P)H autofluorescence, and lipid‑packing reporters.

3. Applies appropriate FLIM data formats, acquisition settings, and detector configurations to ensure reliable and reproducible lifetime measurements.

Data Analysis and Interpretation

1. Analyzes FLIM datasets using commercial and open‑source software (e.g., SymPhoTime64, NIS Elements, additional open‑access tools) and interprets lifetime distributions, decay curves, and fitting models.

2. Identifies and evaluates common acquisition artefacts, instrumental biases, and interpretation pitfalls, and proposes strategies to avoid or correct them.

3. Implements image segmentation, object‑based analysis, and statistical evaluation to extract quantitative biological information from multi‑parametric FLIM images.

Critical Thinking and Integration

1. Assesses the suitability of specific FLIM approaches (e.g., FLIM‑FRET, NAD(P)H, environmental sensing) for answering defined biological or biophysical research questions.

2. Integrates fluorescence lifetime data with complementary imaging or biochemical information to draw evidence‑based conclusions about cellular processes or material properties.

Communication and Research Competence

1. Communicates FLIM‑based findings clearly and professionally, demonstrating the ability to interpret results, justify methodological choices, and articulate limitations in a research‑oriented context.

Last update: Benda Aleš, Mgr., Ph.D. (04.02.2026)

Course registration requirements

The course is intended for magister and doctoral students.

Last update: Benda Aleš, Mgr., Ph.D. (12.09.2018)