Fluorescence Lifetime Microscopy

Comparison of the conventional fluorescence microscopy (A, D) with the fluorescence lifetime microscopy (B, E) together with the associated distribution of the fluorescence lifetimes (C, F).
The images show stained cell nuclei .
The decrease in fluorescence lifetimes in the lower row, caused a further fluorescent dye by the addition, indicates a Förster resonance energy transfer through.

The fluorescence lifetime microscopy ( English fluorescence lifetime imaging microscopy ,  FLIM ) is a fluorescence -based imaging technique of microscopy . In contrast to other fluorescence microscopic methods, it is not based on a measurement of the fluorescence intensity, but on the measurement of the different lifetimes of the excited states of fluorescent molecules .

Fluorescence lifetime microscopy is used in particular in connection with confocal microscopy and multiphoton microscopy .

Physical basis

→ Main article : Fluorescence lifetime

Fluorescence lifetime microscopy is based on a measurement of the fluorescence lifetime of excited fluorescent molecules. The fluorescence lifetime is the mean time that a molecule remains in the excited state before it returns to its ground state by releasing a photon . The fluorescence degradation is reflected in an exponential decrease in fluorescence intensity over time  t : ${\displaystyle \tau }$ ${\displaystyle I}$

${\displaystyle I(t)=I_{0}\exp \left(-{\frac {t}{\tau }}\right)}$

With

• the initial fluorescence intensity at the time . ${\displaystyle I_{0}}$ ${\displaystyle t=0}$

At the same time, the fluorescence lifetime is inversely proportional to the sum of the decay rates  k for radiating and non-radiating processes:

${\displaystyle {\frac {1}{\tau }}=\sum k_{i}}$

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

Procedure

Fluorescence lifetime microscopy provides images with the fluorescence lifetime for each pixel of the image. The measurement of the fluorescence lifetime in fluorescence lifetime microscopy is based either on a pulsed excitation and a measurement of the fluorescence decrease over time, or on an intensity- modulated excitation and a measurement of the phase shift .

Pulsed stimulation

→ Main article : Time-correlated single photon counting

The theoretically simplest method for determining the fluorescence lifetime consists in counting released photons with the help of time-correlated single photon counting (TCSPC), after periodic excitation with short light pulses in the picosecond range , i.e. significantly shorter than typical fluorescence lifetimes ( nanosecond range ).

If the excitation is sufficiently short, the time-dependent exponential decrease in fluorescence intensity can then be observed for most samples, from which the fluorescence lifetime can be determined. For this purpose, the excitation intensity is reduced to such an extent that only about one photon is detected per excitation pulse. For this, the time between the excitation pulse and the photon can be measured with an accuracy of a few picoseconds. A histogram is then created from many such individual measurements, 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

With the help of phase fluorimetry , the fluorescence lifetime can be determined via the phase shift of the fluorescence after an intensity-modulated excitation. The excitation intensity (engl. Excitation : excitation) in this case is sinusoidally modulated, z. B. with the help of an acousto-optical modulator : ${\displaystyle E}$

${\displaystyle E(t)=E_{0}\cdot (1+m_{\text{E}}\cdot \sin(\omega t))}$

With

• the angular frequency  ω of the modulation
• the amplitude  m _{E of} the modulation.

The signal of the fluorescence intensity follows the excitation signal with a time delay: ${\displaystyle F}$

${\displaystyle F(t,{\vec {r}})=F_{0}({\vec {r}})\cdot {\bigl (}1+m_{\text{F}}({\vec {r}})\cdot \sin[\omega t+\varphi _{\text{F}}({\vec {r}})]{\bigr )}}$

With

• the location of the detection, i.e. the pixel  x, y ${\displaystyle {\vec {r}}={\binom {x}{y}}}$
• the offset (as phase  φ _F ), which describes the temporary retention of the fluorescent dye in the excited state
• the modulation amplitude  m _F , which is reduced compared to  m _E.

The fluorescence lifetime can then be determined in two ways:

• about the phase: ${\displaystyle \tau ({\vec {r}})={\frac {1}{\omega }}\cdot \tan {\varphi _{\text{F}}({\vec {r}})}}$
• on the amplitude change: . ${\displaystyle \tau ({\vec {r}})={\frac {1}{\omega }}\cdot {\sqrt {\left({\frac {m_{\text{E}}}{m_{\text{F}}}}\right)^{2}-1}}}$

For detection here image sensors (to CCD cameras , avalanche photodiodes fields ) are used, in which the time in which they are sensitive and can be finely controlled. With ICCD cameras this is done e.g. B. via image intensifiers made of microchannel plates , the amplification of which can be modulated by the same signal that is used to control the lighting ( gated CCD ). Then recordings are made in which detection and excitation are differently out of phase with one another; from these, an image of the fluorescence lifetimes is reconstructed.

literature

• Theodorus WJ Gadella: FRET and FLIM techniques . Elsevier, Amsterdam 2009, ISBN 978-0-08-054958-3 .
• A. Periasamy, RM Clegg: Flim Microscopy in Biology and Medicine . CRC Press, 2010, ISBN 978-1-4200-7890-9 .

Web links

• Applications in Confocal Microscopy: Fluorescence Lifetime Imaging Microscopy (FLIM). Olympus Corporation, archived from the original on December 20, 2011 ; accessed on April 3, 2010 (English). 
• Application Note: Two-photon fluorescence lifetime imaging (2P-FLIM) for ion sensing in living cells (PDF; 537 kB) PicoQuant GmbH 2008
• Application Note: Quantitative in vivo imaging of molecular distances using FLIM-FRET (PDF; 636 kB) PicoQuant GmbH 2009
• Principle of TCSPC FLIM (Becker & Hickl, website)
• Molecular Imaging with FLIM (Becker & Hickl, website)

Individual evidence

1. ↑ Day-Uei Li, Jochen Arlt, Justin Richardson, Richard Walker, Alex Buts, David Stoppa, Edoardo Charbon, Robert Henderson: Real-time fluorescence lifetime imaging system with a 32 × 32 0.13 µm CMOS low dark-count single-photon avalanche diode array . In: Optics Express . tape 18 , no. 10 , 2010, ISSN  1094-4087 , p. 10257-10269 , doi : 10.1364 / OE.18.010257 .
2. ^ Joseph R. Lakowicz , Klaus W. Berndt: Lifetime-selective fluorescence imaging using an rf phase-sensitive camera . In: Review of Scientific Instruments . tape 62 , no. 7 , 1991, ISSN  0034-6748 , pp. 1727 , doi : 10.1063 / 1.1142413 .