Suppression of photobleaching-induced artifacts in frequency-domain FLIM by permutation of the recording order

Background: Photobleaching can lead to significant errors in frequency-domain fluorescence lifetime imaging microscopy (FLIM). Existing correction methods for photobleaching require additional recordings and processing time and can result in additional noise. A method is introduced that suppresses the effects of photobleaching without the need for extra recordings or processing. Methods: Existing bleach correction methods and the method introduced in this report whereby the recording order of the phases is permuted were compared using numerical simulations. Results: Certain orders were found to make measurements virtually insensitive to photobleaching. At 12 recordings, errors in measured phase and modulation depth decreased by a factor 512 and 393, respectively, compared to recordings using sequential recording order. The optitual order is independent of modulation depth, phase, and extent of photobleaching. Thus, the same order can be used for practically all Situations. Application of the method in FLIM measurements of EYFP-transfected HeLa cells was found effectively to suppress photobleaching induced artifacts. Conclusions: In view of the ease of implementation, its inherent robustness, and the possibility to still apply existing correction methods afterward, there is no good reason not to use the permuted recording order presented in this report instead of a sequential order. (C) 2004 Wiley-Liss, Inc.