Mosaic FLIM acquires several (usually a large number of) TCSPC-FLIM recordings into a single, large FLIM data set. The data set can represent a spatial mosaic of images, a Z stack of images, a time series of images, or images recorded in different wavelength intervals of a multi-wavelength detector. The advantage of the technique is that no time for saving the individual images needs to be reserved, and that extremely fast time series can be recorded by triggered accumulation. We demonstrate the technique for a bh TCSPC FLIM system attached to a Zeiss LSM 710 NLO and a Zeiss LSM 7 NLO microscope.
Mosaic FLIM: New Dimensions in Fluorescence
Lifetime Imaging
Mosaic FLIM is based on bhs
multi-dimensional TCSPC FLIM technique [1]. In contrast to conventional TCSPC
FLIM, Mosaic FLIM acquires several FLIM recordings into a single large FLIM
data set. The results are displayed and saved as mosaics of FLIM images [2, 3].
The elements of a FLIM Mosaic can be recordings for
a) different
locations of the scan field in the sample [2, 3]
b) subsequent Z
planes [3, 4]
c) subsequent times
after a stimulation of the sample [2, 3]
d) different
wavelength [2, 3]
The results of Mosaic FLIM can be
considered photon distributions over the scan coordinates, x,y, of the
individual mosaic elements, the times, t, of the photons after the excitation
pulse, and additional parameters, such as the location of the scan field in the
sample (a), the depth of the image plane in the sample (b), the time after a
stimulation of the sample (c), or the wavelength of the photons (d). The
advantages of Mosaic FLIM over a conventional record-and-save procedure are:
- no readout and save time between
subsequent mosaic elements
- time-series data (c) can be accumulated
for repeated stimulation. This way, high lifetime accuracy is achieved even for
very fast recordings. Element time can be down to 50 ms for fast scanners.
- the entire data set can be analysed in a
single analysis run
- global fitting can be used over the
entire array of mosaic elements
Examples of FLIM Mosaic Recordings
Fig. 1: Spatial Mosaic FLIM of
plant tissue. 4x4 elements, each element has 512x512 pixels, with 256 time
channels per pixel. Total pixel number 2048x2048, total size of image area
2.5 x 2.5 mm. Recorded with Simple-Tau 150 FLIM system [1, 5] and
Zeiss LSM 710 in Tile-Imaging mode. Analysis by bh SPCImage [1, 5].
Fig. 2: Z-stack recorded by Mosaic FLIM. Pig skin stained with Indocyanin
Green. 16 planes from 0 to 60 µm from top of sample, each plane 512x512
pixels, 256 time channels. Amplitude-weighted lifetime of double-exponential
decay. Image of plane 8 shown magnified on the right. bh Simple-Tau 150 FLIM
system [1, 5] and Zeiss LSM 7MP OPO system [4].
Fig. 3: Time series recorded by temporal Mosaic FLIM. Non-photochemical
transients in chloroplasts of a moss leaf. 64 mosaic elements for consecutive
times after turn-on of excitation light. Acquisition time per element 1 second,
total time of sequence 64 seconds, image size of each element 128 x 128 pixels,
256 time channels. Time runs from lower left to upper right. bh DCS-120
confocal FLIM system [6] with SPC-150 TCSPC modules [1], SPCImage data analysis
software.
Fig. 4: Multi-wavelength Mosaic FLIM. bh MW-FLIM GaAsP 16-channel
detector, SPC-150 TCSPC FLIM module. 16 images with 512 x 512 pixels
and 256 time channels were recorded simultaneously [1], results displayed and
saved in Mosaic FLIM mode. Wavelength from upper left to lower right,
490 nm to 690 nm, 12.5 nm per image. bh DCS‑120 confocal
scanning FLIM system [6], Zeiss Axio Observer microscope, x20 NA=0.5 air lens.
System requirements
Mosaic FLIM requires a system computer with
64-bit architecture running Windows 7 or Windows 8, 64 bit. System memory
should be 16 Gigabytes or more. SPCM data acquisition software must be 64 bit,
version 9.60 or later.
