FLIM & FRET

What is FLIM?

If a short pulsed light source is used for excitation in fluorescence microscopy, a decay of the fluorescence intensity will usually be observed (Fig 1). The exponential decay of the fluorescence over time is characteristic of each fluorescent molecular species.

Unlike intensity-based measurements of fluorescence, the lifetime is constant even if concentration, path length or illumination is varied in the sample. However, very subtle changes in the local environment of the molecule - such as pH, ion concentration, polarity and viscosity - produce changes in the lifetime. Molecular non-radiative energy transfers (FRET) will also produce easily detectable lifetime changes.

FLIM - Flourescence Lifetime Imaging - extends this powerful technique into the microscopy domain. Structural, biochemical and photodynamic processes can be visualised even in living cells (Fig 2). FLIM also provides an approach to FRET studies that avoids many of the pitfalls of intensity-based measurements.

Hamamatsu has combined the space and time dimensions of a streak camera image with the resolution of a microscope to open new horizons in biological imaging.

Hamamatsu Streak Camera

The Hamamatsu C9136 FLIM System uses a streak camera. In this, incident light releases photoelectrons from the photocathode in the streak tube, and these electrons are swept across the detector by deflection electrodes. This process resolves the time of light arrival into a varying position on the detector, and from this data the fluorescence decay curve can be constructed (for each single pixel), and the fluorescence lifetime determined.

Hamamatsu C9136 FLIM System

To build up a 2D microscopy image, the excitation laser is scanned in both X- and Y-directions. Pixels on an X-axis scan are captured by one frame from the streak camera, which has time as one dimension of its image and the X-scan as the other. This x-t image is then analysed for fluoresence lifetime values, and scan lines are repeated to build up the Y-direction of the final fluoresence lifetime image.

• Effective pixel resolution: 656 pixels on X-axis, 601 lines on Y-axis
• Effective field of view: 48 x 45 µm (with 60X objective)
• Spatial resolution: 0.2 µm
• Acquisition time: approx 3 s total (5 ms per line)
• Temporal resolution of fluorescence lifetime: less than 20 ps