456
D. P. Singh et al.
3.2 Best Way to Measure Steady-State and Time-Resolved
Fluorescence
Oxygen dissolved in the solvent shows a strong quenching effect. The intensity of
fluorescence can be increased by passing bubbling nitrogen or any inert gas through
the solution. The temperature regulation is also important during the fluorescence
measurements as it could cause a 1–2% of the reduction in fluorescence intensity
per degree of temperature rise. If the investigated material is photosensitive, it can
be degraded into different fluorescent products giving rise to different fluorescence
lifetimes. During the steady state measurements, appropriate settings of excitation
wavelength, integration time, resolution, excitation and emission slit widths, etc. are
important. Normally slit width 5 nm can be taken; however, it should be changed, if
the emission spectrum is getting saturated. Wider slit width offers more light to be
hitting the sample; thus wider emission spectrum and more light hitting the detector.
Therefore, we obtain a greater signal-to-noise ratio but a lesser resolution. In the timeresolved measurements, the decays should be collected at 10,000 photons in the peak
maximum over 1024 channels for better results. The magic angle (≈54.7
◦ ) should be
perfectly fixed and instrumental response function (IRF) should be incorporated in the
measurements to avoid the errors. From the deconvoluted fluorescence lifetime data,
the average fluorescence lifetime is calculated using Eq. (21). The acceptability of
the fits should be checked from the χ
2 criterion and the residual of the fitted functions
to the actual data.
3.3 Time-Correlated Single Photon Counting (TCSPC)
Most of the time-domain measurements are carried out by using the time-correlated
single-photon counting. In this technique, the sample is excited with an ultrashort
light-pulse, resulting in the waveform that will be visible during the excitation of
fluorophores and subsequent emission of photons. The detection rate of TCSPC is
typically 1 photon per 100 excitation pulses and the time is measured between the
excitation pulse and the observed photon. When the sample is excited, the exciting
signal is passed through a constant function discriminator (CFD) which measures the
incident time of the pulse. This signal passage through a time-to-amplitude converter
(TAC) that generates a voltage ramp which increases linearly as a function of time
on the nanosecond timescale. Meanwhile, the second channel detects the pulse from
the single detected photon. Now, the TAC contains a voltage that is proportional
to the time delay (t) between the excitation and emission signals. The voltage is
amplified by a programmable gain amplifier (PGA) and converted to a numerical
value by the analog-to-digital converter (ADC). If the signal is beyond the range, we
use a window discriminator (WD) to suppress the range. Then, voltage is converted
into a digital value that can be stored as a single event with the measured time delay.
A histogram of the decay is measured by repeating this process.
D. P. Singh et al.
3.2 Best Way to Measure Steady-State and Time-Resolved
Fluorescence
Oxygen dissolved in the solvent shows a strong quenching effect. The intensity of
fluorescence can be increased by passing bubbling nitrogen or any inert gas through
the solution. The temperature regulation is also important during the fluorescence
measurements as it could cause a 1–2% of the reduction in fluorescence intensity
per degree of temperature rise. If the investigated material is photosensitive, it can
be degraded into different fluorescent products giving rise to different fluorescence
lifetimes. During the steady state measurements, appropriate settings of excitation
wavelength, integration time, resolution, excitation and emission slit widths, etc. are
important. Normally slit width 5 nm can be taken; however, it should be changed, if
the emission spectrum is getting saturated. Wider slit width offers more light to be
hitting the sample; thus wider emission spectrum and more light hitting the detector.
Therefore, we obtain a greater signal-to-noise ratio but a lesser resolution. In the timeresolved measurements, the decays should be collected at 10,000 photons in the peak
maximum over 1024 channels for better results. The magic angle (≈54.7
◦ ) should be
perfectly fixed and instrumental response function (IRF) should be incorporated in the
measurements to avoid the errors. From the deconvoluted fluorescence lifetime data,
the average fluorescence lifetime is calculated using Eq. (21). The acceptability of
the fits should be checked from the χ
2 criterion and the residual of the fitted functions
to the actual data.
3.3 Time-Correlated Single Photon Counting (TCSPC)
Most of the time-domain measurements are carried out by using the time-correlated
single-photon counting. In this technique, the sample is excited with an ultrashort
light-pulse, resulting in the waveform that will be visible during the excitation of
fluorophores and subsequent emission of photons. The detection rate of TCSPC is
typically 1 photon per 100 excitation pulses and the time is measured between the
excitation pulse and the observed photon. When the sample is excited, the exciting
signal is passed through a constant function discriminator (CFD) which measures the
incident time of the pulse. This signal passage through a time-to-amplitude converter
(TAC) that generates a voltage ramp which increases linearly as a function of time
on the nanosecond timescale. Meanwhile, the second channel detects the pulse from
the single detected photon. Now, the TAC contains a voltage that is proportional
to the time delay (t) between the excitation and emission signals. The voltage is
amplified by a programmable gain amplifier (PGA) and converted to a numerical
value by the analog-to-digital converter (ADC). If the signal is beyond the range, we
use a window discriminator (WD) to suppress the range. Then, voltage is converted
into a digital value that can be stored as a single event with the measured time delay.
A histogram of the decay is measured by repeating this process.