Lambert Instruments

Confocal imaging on a widefield fluorescence microscope can now be done in combination with frequency domain FLIM. The increased spatial resolution in the z-direction results in lifetime images with enhanced contrast as the detection of out-of-focus emission is reduced significantly. This allows you to see differences in fluorescence lifetime e.g. between the cell membrane and the cytoplasm. Read more about the technology of confocal imaging or view the LIFA confocal product with a photo of the set-up.

These data were obtained with the LIFA, either widefield (with LED light; 468nm peak) or confocal (with spinning disk CSU10 and 470nm-diode laser). The fluorescence lifetime images are generated at 2 different z-positions, z1 and z2. In the download more z-positions are shown, as well as avi movies through a lot of z-positions.

Confocal (Spinning Disk)	Widefield (LED)
		z1
		z2

These images show different pollen grains: the lifetime in pseudo colours and the intensity in grey scale. Because of the pinholes in the spinning disk, the exposure time is higher with confocal imaging. In this image 220msec exposure time per phase step was taken for the confocal image versus 195msec for the widefield (LED light). However, when a diode laser with higher power is used, the exposure time can be shortened.
These images are extracted from the download.

Confocal (Spinning Disk)	Widefield (LED)
		z1
		z2

These images show YFP-transfected mammalian cells and were taken with 12 phase steps of each 400msec for the confocal, versus 70msec for the widefield image. The calculated lifetimes are 2,34ns (z1) and 2,42ns (z2) in the confocal images, versus 2,58ns (z1) and 2,57ns (z2) in the widefield images.
These differences could be due to the fact that the diode laser has one excitation wavelength, exactly 470nm, while the LED has a range of wavelengths for which we used the emission band pass filter of 465-495nm.
These images are extracted from the download.