Fluorescence-lifetime imaging microscopy

The fluorescence lifetime microscopy (English fluorescence lifetime imaging microscopy, FLIM ) is a fluorescence -based imaging technique of microscopy. In contrast to other fluorescence microscopy techniques, fluorescence lifetime microscopy, is not based on a measurement of fluorescence intensity, but in the measurement of the lifetimes of the excited states of different fluorescent molecules. The fluorescence lifetime microscopy is used in particular in conjunction with the confocal and multi-photon microscopy.

Physics

The fluorescence lifetime microscopy, based on a measurement of the fluorescence lifetime of excited fluorescent molecules. The fluorescence lifetime is the average time that a molecule remains in the excited state, before returning with the release of a photon to its ground state. The fluorescence reduction is reflected in an exponential decrease in fluorescence intensity with time t resist:

Simultaneously, the fluorescence lifetime is inversely proportional to the sum of the decay rates k radiant and non-radiative processes.

The fluorescence lifetime of a dye depends, among other things, of its identity and its chemical environment. She is influenced by energy transfer mechanisms, such as Förster resonance energy transfer. However, the fluorescence lifetime is independent of the initial fluorescent intensity.

Measurement

The fluorescence lifetime microscopy, provides images with the fluorescence lifetime of each pixel of the image. Measuring the fluorescence lifetime of the fluorescence lifetime microscopy, based on either a pulsatile excitation and measurement of fluorescence decay time, or on an intensity-modulated excitation and measurement of the phase shift.

Pulsed excitation

The theoretically simplest method to determine the fluorescence lifetime is the count of released photons using the time-correlated single photon counting ( TCSPs ), according to a periodic excitation with short light pulses in the picosecond range, which is significantly shorter than typical fluorescence lifetimes ( nanosecond range ). At sufficiently short excitation can for most samples, the time-dependent exponential decay of the fluorescence intensity can be observed, from which the fluorescence lifetime can be determined. For this purpose, the excitation intensity is reduced until that per excitation pulse, only about one photon is detected. For this can be the time between excitation pulse and photon with an accuracy of a few picoseconds measure. A histogram is created from many such individual measurements then, from which the fluorescence lifetime can be determined directly. This method also has the advantage that it is independent of fluctuations in the excitation intensity.

Phase modulation

Using Phasenfluorimetrie the fluorescence lifetime can be determined from the phase shift of the fluorescence intensity after a modulated excitation. Here, the excitation intensity is sinusoidally modulated, for example by means of an acousto-optical modulator:

In this case, ω is the angular frequency of the modulation, and ME is the modulation amplitude.

The fluorescence signal follows the excitation signal time offset:

Herein, the location of detection, ie the pixel x, y. The offset ( a phase? F ) reflects the temporary retention of the fluorescent dye in its excited state. In addition, the modulation amplitude of the signal is reduced mF. The fluorescence lifetime can be determined in two ways:

For the detection of image sensors ( CCD cameras, avalanche photodiodes fields) can be used here, where the time in which they are sensitive and can be finely controlled. This is done in CCD cameras, for example, about from microchannel plate image intensifier, the gain of which can be modulated by the same signal which is used for controlling the lighting ( Gated CCD). There are then made recordings in which the detection and excitation are different phase with each other. From this an image of the fluorescence lifetimes is then reconstructed.