FLIMX - Toolbox for fluorescence lifetime analysis



FLIMX is a MATLAB software package to determine and analyze the fluorescence lifetime in time-resolved fluorescence data. Its main application is fluorescence lifetime ophthalmology (FLIO), but FLIMX has also been used for fluorescence lifetime imaging microscopy (FLIM) data. FLIMX offers a variety of software tools to process and analyse FLIO / FLIM data.
FLIMX is an open source project and can be used under the open source BSD–license.

FLIMX fluorescence lifetime approximation in an AMD patient.

How to cite FLIMX

Data analysis was performed with FLIMX (Klemm et al. 2015), which is documented and freely available for download online under the open source BSD–license (http://www.flimx.de).

Please cite the following reference in your publications if you have used our software for your data analyses:
Klemm M, Schweitzer D, Peters S, Sauer L, Hammer M, Haueisen J (2015) FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye. PLoS ONE 10(7): e0131640. doi:10.1371/journal.pone.0131640


import of time-resolved fluorescence measurements from:

organize the imported data:

  • multiple spectral channels per subject
  • multiple subjects per group (study)
  • define subgroups based an additional information (e.g. age, gender, disease state, …)
  • copy / move subjects between studies

pre-process the TCSPC data:

determine the fluorescence lifetime using:

define boundaries for each parameters of the fluorescence lifetime approximation

  • batch process multiple subjects / studies, which may have different parameters for the fluorescence lifetime approximation
  • create simulated TCSPC data based on an unrestricted number of exponential functions with custom parameters and an arbitrary number of photons
  • visualize FLIM / FLIO data in 1D / 2D / 3D and in scatter plots
  • combine multiple FLIM parameters into a single plot

define an infinite number of regions of interest (ROI):

  • ETDRS grid
  • rectangles
  • circles
  • polygons

combine multiple ROIs into groups (even of different ROI types)

  • calculate and export descriptive statistics of ROI and ROI groups from subjects and studies
  • statistically compare two groups / studies using paired t-test, unpaired t-test, Wilcoxon singed rank test, Wilcoxon rank sum test, a Holm-Bonferroni method
  • import masks from another e.g. imaging modality or an expert highlighting certain parts of an image (e.g. drusen in a FLIO measurement) for image segmentation
  • generate arithmetic images, e.g. to generate a mask based on thresholds; combine masks and FLIM parameters; calculate ratios of FLIM parameters; …

all data is saved by FLIMX for later re-use, refinement of the analysis or documentation:

  • imported measurements
  • approximated fluorescence lifetimes and the used parameters
  • simulation parameters
  • ROI definitions
  • color scaling


FLIMX Source Code

The latest version of the code can be downloaded from:

Requires MATLAB R2018b or later. We recommend to use the lastest MATLAB version.

Changelog: https://github.com/M-Klemm/FLIMX/commits/master


The FLIMX User Guide can be found here:
FLIMX User Guide

Related Publications

A complete list of research citing FLIMX can be found on Google Scholar


Sauer L, Vitale AS, Milliken CM, Modersitzki NK, Blount JD, Bernstein PS. Autofluorescence Lifetimes Measured with Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) Are Affected by Age, but Not by Pigmentation or Gender. Transl Vis Sci Technol. 2020;9(9):2.

Sauer L, Vitale AS, Andersen KM, Hart B, Bernstein PS. Fluorescence Lifetime Imaging Ophthalmoscopy (Flio) Patterns in Clinically Unaffected Children of Macular Telangiectasia Type 2 (Mactel) Patients. Retina. 2020;40(4):695-704.

Brauer JL, Schultz R, Klemm M, Hammer M. Influence of Lens Fluorescence on Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) Fundus Imaging and Strategies for Its Compensation. Transl Vis Sci Technol. 2020;9(8):13.

Vitale AS, Sauer L, Modersitzki NK, Bernstein PS. Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) in Patients with Choroideremia. Transl Vis Sci Technol. 2020;9(10):33.

Schweitzer D, Haueisen J, Brauer J, Klemm M. Comparison of algorithms to suppressartifacts from the natural lens in fluorescencelifetime imaging ophthalmoscopy (FLIO). Biomed Opt Express. 2020;11(10): 5586-5602.


Sauer L, Komanski CB, Vitale AS, Hansen ED, Bernstein PS. Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) in Eyes With Pigment Epithelial Detachments Due to Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci. 2019;60(8):3054-63.

Bernstein P, Dysli C, Fischer J, Hammer M, Katayama Y, Sauer L, et al. Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO). In: Bille JF, editor. High Resolution Imaging in Microscopy and Ophthalmology: New Frontiers in Biomedical Optics. Cham: Springer International Publishing; 2019. p. 213-35.

Sauer L, Calvo CM, Vitale AS, Henrie N, Milliken CM, Bernstein PS. Imaging of Hydroxychloroquine Toxicity with Fluorescence Lifetime Imaging Ophthalmoscopy. Ophthalmol Retina. 2019;3(10):814-25.

Klemm M, Sauer L, Klee S, Link D, Peters S, Hammer M, et al. Bleaching effects and fluorescence lifetime imaging ophthalmoscopy. Biomed Opt Express. 2019;10(3):1446-61.


Andersen KM, Sauer L, Gensure RH, Hammer M, Bernstein PS. Characterization of Retinitis Pigmentosa Using Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO). Translational Vision Science & Technology. 2018;7(3).

Hammer M, Sauer L, Klemm M, Peters S, Schultz R, Haueisen J. Fundus autofluorescence beyond lipofuscin: lesson learned from ex vivo fluorescence lifetime imaging in porcine eyes. Biomed Opt Express. 2018;9(7):3078-91.

Sauer L, Andersen KM, Dysli C, Zinkernagel MS, Bernstein PS, Hammer M. Review of clinical approaches in fluorescence lifetime imaging ophthalmoscopy. J Biomed Opt. 2018;23(9):1-20.

Sauer L, Andersen KM, Li BX, Gensure RH, Hammer M, Bernstein PS. Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) of Macular Pigment. Invest Ophth Vis Sci. 2018;59(7):3094-103.

Sauer L, Gensure RH, Andersen KM, Kreilkamp L, Hageman GS, Hammer M, et al. Patterns of Fundus Autofluorescence Lifetimes In Eyes of Individuals With Nonexudative Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci. 2018;59(4):AMD65-AMD77.

Sauer L, Gensure RH, Hammer M, Bernstein PS. Fluorescence Lifetime Imaging Ophthalmoscopy: A Novel Way to Assess Macular Telangiectasia Type 2. Ophthalmol Retina. 2018;2(6):587-98.

Brown CN, Green BD, Thompson RB, den Hollander AI, Lengyel I, consortium E-R. Metabolomics and Age-Related Macular Degeneration. Metabolites. 2018;9(1).


Dysli C, Wolf S, Berezin MY, Sauer L, Hammer M, Zinkernagel MS. Fluorescence lifetime imaging ophthalmoscopy. Prog Retin Eye Res. 2017;60:120-43.

Sauer L, Klemm M, Peters S, Schweitzer D, Schmidt J, Kreilkamp L, et al. Monitoring foveal sparing in geographic atrophy with fluorescence lifetime imaging ophthalmoscopy - a novel approach. Acta ophthalmologica. 2017.

Peters S, Griebsch M, Klemm M, Haueisen J, Hammer M. Hydrogen peroxide modulates energy metabolism and oxidative stress in cultures of permanent human Muller cells MIO-M1. J Biophotonics. 2017;10(9):1180-8.

Feeks JA, Hunter JJ. Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice. Biomed Opt Express. 2017;8(5):2483-95.

Le Marois A, Suhling K. Quantitative Live Cell FLIM Imaging in Three Dimensions. Adv Exp Med Biol. 2017;1035:31-48.

Shapovalov KA, Salmin VV, Lazarenko VI, Gar′kavenko VV. Modeling of the Autofluorescence Spectra of the Crystalline Lens with Cataract Taking into Account Light Scattering. J Appl Spectrosc (USA). 2017;84(2):278-83.


Sauer L, Peters S, Schmidt J, Schweitzer D, Klemm M, Ramm L, et al. Monitoring macular pigment changes in macular holes using fluorescence lifetime imaging ophthalmoscopy. Acta ophthalmologica. 2016.

Klemm M, Blum J, Link D, Hammer M, Haueisen J, Schweitzer D. Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model. Biomed Opt Express. 2016;7(9):3198-210.

Klemm M, Nagel E, Schweitzer D, Schramm S, Haueisen J, editors. Effects of short term changes in the blood glucose level on the autofluorescence lifetime of the human retina in healthy volunteers. Ophthalmic Technologies XXVI; 2016; SPIE Photonics West, San Francisco.


Klemm M, Nagel E, Dietzel A, Lai KW, Supriyanto E, Schweitzer D. Agreement Between Eyes in Wide-Field Fluorescence Lifetime Imaging Ophthalmoscopy Measurements at the Human Retina in Healthy Volunteers. In: Ibrahim F, Usman J, Mohktar MS, Ahmad MY, editors. International Conference for Innovation in Biomedical Engineering and Life Sciences. IFMBE Proceedings. 56: Springer Singapore; 2015. p. 298-301.

Klemm M, Schweitzer D, Peters S, Sauer L, Hammer M, Haueisen J. FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye. Plos One. 2015;10(7).