SPC‑160 Delivers Linear Intensities in High
Count Rate FLIM Applications
Wolfgang
Becker, Stefan Smietana, Becker & Hickl GmbH
At high
count rates the photon numbers recorded in TCSPC FLIM are impaired by counting
loss effects. The photon numbers in the pixels therefore becomes a nonlinear
function of the pixel intensity. Consequently, the images are more and more losing
contrast with increasing count rate. In the new SPC‑160 TCSPC FLIM
modules we therefore implemented a parallel counter channel that delivers pixel
photon number with virtually no counting loss. FLIM images built up from
lifetimes of the TCSPC data and intensities from the parallel counter show
substantially improved contrast compared to conventional FLIM images.
FLIM at High Count Rates
In the last five years, the optical
efficiency of FLIM systems has been improved considerably. The optical systems
of laser scanning microscopes have become more efficient, better detectors have
been introduced [2], and tuneable excitation sources allow the excitation
wavelength to be matched to the absorption band of the fluorophores [1].
Consequently, FLIM experiments are performed at increasingly higher count
rates. High count rates help keep the acquisition times of megapixel FLIM
applications [3] in reasonable limits, and help obtain higher lifetime accuracy
in lifetime-transient recordings [1, 6].
Interestingly, the problem of TCSPC FLIM at
high count rates is not the pile-up effect, as commonly believed. The error in
the recorded lifetime remains less than 5% up to a count rate of 20% of the
excitation pulse frequency [1]. That means, for 80 MHz laser repetition
rate count rates of more than 10 MHz can be used. More annoying than
pile-up is the nonlinearity of the intensity scale by counting loss in the dead
time of the photon timing electronics [1]. TCSPC FLIM images recorded at
extremely high count rates thus lack intensity contrast. The images appear
flat and unappealing, see Fig. 1, left (page 2).
For most FLIM applications the nonlinearity
of the intensity scale is a purely aesthetic problem: Information is derived
from the shape of the decay curves, not from the intensities in the pixels.
However, intensities can be important if the fluorescence signals of several
fluorophores have to be unmixed from data recorded in several wavelength
channels, or if two-dimensional pixel histograms of decay data and intensities
are used to separate different fluorescent species.
Parallel Counter Channel
A radical way to avoid intensity nonlinearity
is to use a parallel counter channel that does not go through the timing
electronics of the TCSPC board. This counter and the required data pathway have
been integrated in the new SPC‑160 TCSPC modules [4].
The input pulses for the counter come
directly from the input constant-fraction discriminator (CFD) of the TCSPC
device. The CFDs of the bh TCSPC modules have about 5 ns dead time. With
PMTs or hybrid detectors (which have virtually no dead time) the counter
delivers intensities unaffected by counting loss. The photon numbers recorded
in the parallel counter are transferred into the computer on a pixel-by pixel
basis and stored together with the FLIM data from the timing electronics. The SPCImage
data analysis [15] builds up the FLIM images by using the fluorescence
lifetimes from the TCSPC channel and the intensities from the parallel counter
channel. The improvement in image contrast over conventional FLIM data is substantial,
compare Fig. 1, left and right.
Fig. 1: Mouse kidney sample recorded at
an average count rate of 5 MHz. Left: Intensity from TCSPC channel. Right:
Intensity from parallel counter channel. bh SPC-160 TCSPC FLIM module [4],
DCS-120 confocal scanner [5], SPCImage FLIM data analysis [1, 5]
The detector peak count rates in the bright
areas of the images are exceeding 10 MHz. Nevertheless, no differences in the
recorded lifetimes can be seen between the two images.
References
1.
W. Becker, The bh TCSPC handbook. Becker &
Hickl GmbH, 5th Edition (2012), www.becker-hickl.com, printed copies available.
2.
W. Becker, B. Su, K. Weisshart, O. Holub, FLIM
and FCS Detection in Laser-Scanning Microscopes: Increased Efficiency by GaAsP
Hybrid Detectors. Micr. Res. Tech. 74, 804-811
(2011)
3. Megapixel FLIM. Application note, available on www.becker-hickl.com.
4. SPC‑160 TCSPC module. Data sheet, available on
www.becker-hickl.com
5.
Becker & Hickl GmbH, DCS-120 Confocal
Scanning FLIM Systems, user handbook. www.becker-hickl.com, printed copies available.
6. W. Becker, V. Shcheslavkiy, S. Frere, I. Slutsky, Spatially Resolved
Recording of Transient Fluorescence-Lifetime Effects by Line-Scanning TCSPC. Microsc. Res. Techn. 77, 216-224 (2014).
