Fluorescence lifetime imaging microscopy

What is fluorescence lifetime imaging microscopy?
Features
Imaging principle
Example images
Deep tissue of a fruit fly
Liver of chronically alcohol-intoxicated mice
Highlighted product: HPDs (hybrid photodetectors)
Other imaging techniques

What is fluorescence lifetime imaging microscopy?

FLIM stands for fluorescence lifetime imaging microscopy. FLIM is an imaging technique that leverages the intrinsic fluorescence lifetime properties of fluorescent dyes. It creates microscopic images by mapping based on the length of fluorescence lifetimes.

Fluorescence lifetime refers to the time it takes for molecules (such as fluorescent dyes) to emit photons and return to their ground state after being excited by light. In the simplest terms, it is defined as the time it takes for fluorescence intensity to decrease to 1/e of its initial value after excitation (see Figure 1).

Definition of fluorescence lifetime

Figure 1: Definition of fluorescence lifetime

Features

Measurement method not dependent on fluorescence intensity

In conventional fluorescence imaging, there is a disadvantage where the fluorescence intensity changes due to factors such as the concentration of fluorescent reagents or cell thickness.

However, in fluorescence lifetime microscopy (FLIM), we measure lifetime instead of intensity. This means that changes in fluorescence intensity due to factors like reagent concentration or cell thickness do not affect FLIM images.

Easy preparation of optics and measurements

In conventional fluorescence imaging, when you want to observe molecules or organelles within cells separately, you need to use appropriate reagents for each and capture images using multiple wavelengths.

However, with fluorescence lifetime microscopy (FLIM), you can distinguish differences in molecules or organelles based on their fluorescence lifetime duration. This means there is no need to change excitation light or filters, potentially simplifying the user’s measurement preparation.

Imaging principle

In FLIM, imaging is typically performed by combining the optical system of a laser microscope, such as a confocal microscopy, with a fluorescence lifetime measurement method called time-correlated single photon counting (TCSPC).

The imaging process involves exciting the observed sample using pulsed light while acquiring the fluorescence lifetime at each scan point using TCSPC. This procedure is repeated for multiple scan points, and each point contributes to a 2D image where each pixel contains fluorescence lifetime data. During image visualization, color coding based on the fluorescence lifetime duration at each point allows for mapping and visualization of fluorescence lifetime data.

Figure 2: Principle of fluorescence lifetime microscopy

Example images

Example images provided by Becker & Hickl GmbH

Deep tissue of a fruit fly

3D image composed of 200 slices

Phasor plot mapping（FLIM-phasor）

Hamamatsu detector used: R10467U-40

An imaging example obtained from a 3D image containing information on the photon count and fluorescence lifetime for each pixel, along with mapping data of fluorescence lifetime characteristics.

Liver of chronically alcohol-intoxicated mice

Confirmed liver tissue damage in the orange-colored area

Hamamatsu detector used: R10467U-40

Imaging example taking advantage of the shorter fluorescence lifetime of damaged liver tissue compared to other areas.

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Highlighted product: HPDs (hybrid photodetectors)

An HPD (hybrid photodetector) is a new photomultiplier tube that incorporates a semiconductor element into an electron tube (vacuum tube). It multiplies photoelectrons directly into the semiconductor by injecting them from the photoelectric surface.

For measuring fluorescence lifetimes, detectors require high sensitivity and high time resolution to capture the rapid decay of fluorescence intensity. An HPD achieves these necessary characteristics at a high level through its unique internal structure. By using an HPD in FLIM, it contributes to highly sensitive and accurate fluorescence lifetime measurements at each scanning point, enabling precise fluorescence lifetime imaging.

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