<title>Lifetime-resolved fluorescence imaging</title>

Abstract

Fluorescence lifetime imaging is a relatively new technique for acquiring directly the nanosecond temporal characteristics of the fluorescence emission of a spatially extended object, and for capturing the dynamic features at every pixel of an image simultaneously. In general, the applications of fluorescence lifetime imaging have been mainly in the microscope, but other diverse imaging situations can benefit from the technology. Our instrument employs periodically modulated excitation light, synchronous modulation of the amplification stages of a microchannel plate intensifier, and subsequent digital recording of the image with a charge-coupled device (CCD) camera. The digitized images can be subsequently analyzed with a variety of different ways. A short description of the lifetime resolved fluorescence imaging instrumentation is given together with typical applications depicting lifetime spatial distributions, multiple lifetime analysis, statistical analysis of the image data, and suppression or enhancement of particular fluorescent species. 1.2 Advantages of temporal resolution of the fluorescence emission. Conventional fluorescence microscopy measures a steady-state fluorescence signal. However, the O-8194-1432-8/94/$6.OO SPIE Vol. 2137 / 105 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/16/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx SPIEVo!. 2137/ 107

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

“…It is even possible to re-record the entire sequence in reverse order as in the linear approximation approach described above. By fitting an exponential, or another function, to the intensity loss as function of time between the recording of images, a more accurate estimate of the bleach rate is obtained than when only one extra image is used (13).…”

“…A linear bleach correction method that has been applied in the past is to repeat the measurement of the entire sequence in reverse order (12,13). Pixel values in corresponding image pairs are than averaged.…”

“…Many photobleaching correction algorithms are based on modeling photobleaching as a linear decay instead of an exponential decay (12,13). The advantages of these methods are that they are relatively easy to implement in the measurement procedure, and that they require less processing time then the exponential correction algorithms.…”

“…To compensate for these effects, usually some form of bleach correction is applied. Typically one or more images in the sequence are re-recorded and used to correct measured intensities for photobleaching on a pixel-by-pixel basis (12)(13)(14). A problem with some of these correction algorithms is that they can add noise.…”

Fluorescence lifetime determination for application in microscopy

“…In a fluorescence microscopy, filters and dichroic mirror are choosen so that the excitation light can reach the specimen through the objective lens and only the fluorescence light is seen through the eye-pieces or is detected by the camera (see figure 2). Fluorescence lifetime imaging is done using a normal fluorescence microscope equipped with a special illumination source and image sensor.…”