2. We have used a BH BDL-SMC-405 pulsed laser with 40 ps
pulse-width synchronized with the NKT supercontinuum laser
operating at 80 MHz. The two lasers were combined using a
fiber combiner (OzOptics, Canada). The supercontinuum laser
operated at the full 80 MHz as the “master” and was used to
trigger the BDL-SMC-405 in a “slave” mode with a 10-ns
delay. It is recommended to run the lasers at 40 MHz, i.e.,
25 ns between pulses to allow a full decay of the fluorescent
protein excited state before the next excitation pulse arrives.
Fluorescence emission was filtered using a 460/60 nm bandpass filter for CFP or mTurquoise2, or 515/30 nm filter for
GFP (Thorlabs) and focused onto the two hybrid detectors.
3. The laser beams are focused to a diffraction-limited spot using a
high numerical aperture oil or water-immersion objective (e.g.,
Nikon x60 VC NA 1.2) to illuminate specimens on the microscope stage. Fluorescence emission is collected by the same
objective in a standard confocal mode. Line, frame, and pixel
clock signals are generated and synchronized with two external
detectors in the form of two fast hybrid photomultiplier tubes
(PMT) or microchannel plate PMTs (i.e., Hamamatsu
R3809U or HPM-100). The raw FLIM data are generated by
linking these PMTs via two TCSPC PC modules. We used BH
SPC830 or SPC150 cards. A single TCSPC may be used
together with routing electronics, but this would lead to only
half of the intensity from each channel recorded.
4. Prior to FLIM data collection, expression levels of the fluorescent protein-tagged proteins of interest are verified by imaging
transfected cells using a confocal microscope. Here, an inverted
Nikon TE2000-U or Ti-E confocal microscope attached to a
Nikon C1 or C2 scanning unit was used with filter sets for GFP
(488 nm excitation) or mDsRed/mCherry/Alexa 555 (543 or
561 nm excitation) and mTurqouise2/Alexa405 (405 nm excitation). A 633-nm interference filter is used in the red channel
to significantly minimize the bleed-through of any GFP or
autofluorescence emission that would otherwise obscure the
mCherry emission. Ideally, equal expression levels for all proteins under investigation are selected to obtain optimal FRET
conditions (Fig. 4). Alternatively, higher levels of the acceptor
protein are desirable.
5. Prior to FLIM image data acquisition, the system performance
needs to be checked and verified. Standard dyes with wellcharacterized excitation and emission spectra and excitedstate lifetimes are commonly used (Table 5). We have routinely
used aqueous solutions (~50 μM) of 7-hydroxy-coumarin carboxylic acid (7-OH-CCA, 360–405 nm excitation, 420 nm
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Abdullah Ahmed et al.
pulse-width synchronized with the NKT supercontinuum laser
operating at 80 MHz. The two lasers were combined using a
fiber combiner (OzOptics, Canada). The supercontinuum laser
operated at the full 80 MHz as the “master” and was used to
trigger the BDL-SMC-405 in a “slave” mode with a 10-ns
delay. It is recommended to run the lasers at 40 MHz, i.e.,
25 ns between pulses to allow a full decay of the fluorescent
protein excited state before the next excitation pulse arrives.
Fluorescence emission was filtered using a 460/60 nm bandpass filter for CFP or mTurquoise2, or 515/30 nm filter for
GFP (Thorlabs) and focused onto the two hybrid detectors.
3. The laser beams are focused to a diffraction-limited spot using a
high numerical aperture oil or water-immersion objective (e.g.,
Nikon x60 VC NA 1.2) to illuminate specimens on the microscope stage. Fluorescence emission is collected by the same
objective in a standard confocal mode. Line, frame, and pixel
clock signals are generated and synchronized with two external
detectors in the form of two fast hybrid photomultiplier tubes
(PMT) or microchannel plate PMTs (i.e., Hamamatsu
R3809U or HPM-100). The raw FLIM data are generated by
linking these PMTs via two TCSPC PC modules. We used BH
SPC830 or SPC150 cards. A single TCSPC may be used
together with routing electronics, but this would lead to only
half of the intensity from each channel recorded.
4. Prior to FLIM data collection, expression levels of the fluorescent protein-tagged proteins of interest are verified by imaging
transfected cells using a confocal microscope. Here, an inverted
Nikon TE2000-U or Ti-E confocal microscope attached to a
Nikon C1 or C2 scanning unit was used with filter sets for GFP
(488 nm excitation) or mDsRed/mCherry/Alexa 555 (543 or
561 nm excitation) and mTurqouise2/Alexa405 (405 nm excitation). A 633-nm interference filter is used in the red channel
to significantly minimize the bleed-through of any GFP or
autofluorescence emission that would otherwise obscure the
mCherry emission. Ideally, equal expression levels for all proteins under investigation are selected to obtain optimal FRET
conditions (Fig. 4). Alternatively, higher levels of the acceptor
protein are desirable.
5. Prior to FLIM image data acquisition, the system performance
needs to be checked and verified. Standard dyes with wellcharacterized excitation and emission spectra and excitedstate lifetimes are commonly used (Table 5). We have routinely
used aqueous solutions (~50 μM) of 7-hydroxy-coumarin carboxylic acid (7-OH-CCA, 360–405 nm excitation, 420 nm
296
Abdullah Ahmed et al.