Laser time-correlated single photon counting

Figure 4.6 shows an apparatus for the fluorescence depolarization measurement. The linearly polarized excitation pulse from a mode-locked Ti-Sapphire laser illuminated a polymer brush sample through a microscope objective. The fluorescence from a specimen was collected by the same objective and input to a polarizing beam splitter to detect 7 and I by photomultipliers (PMTs). The photon signal from the PMT was fed to a time-correlated single photon counting electronics to obtain the time profiles of 7 and I simultaneously. The experimental data of the fluorescence anisotropy was fitted to a double exponential function. 

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

Time-Correlated Single-Photon Counting. For the application of TCSPC in the picosecond time domain, lasers with pulses whose half-widths are 20 ps or less are used. For better time resolution, the combination of a microchan-nel plate photomultiplier tube (MCP-PMT) and a fast constant fraction discriminator (CFD) are used instead of a conventional photomultiplier tube (PMT). A TCSPC system with a time response as short as 40 ps has at its core a Nd YLF (neodymium yttrium lithium fluoride) laser generating 70-ps, 1053-nm pulses at... 

Figure 2.16 shows an example for such a biexponential decay measured with time correlated single-photon counting.78,75 Several picosecond laser experiments have explored this early time behavior of the equilibration... 

Figure 8.9 Time-resolved fluorescent lifetime analysis of Cy3 attached to double-stranded DNA (Iqbal et al., 2008b). Fluorescent decay curve for Cy3 attached to a 16 bp DNA duplex, showing the experimental data and the instrument response function (IRF), and the fit to three exponential functions (line). The decay curve was generated using time-correlated single-photon counting, after excitation by 200 fs pulses from a titanium sapphire laser at 4.7 MHz.

Most of the time-resolved emission spectroscopy setups are home made in the sense that they are built from individual devices (laser, detection system,. ..) hence they are not of a plug and press type, so that their exact characteristics may vary from one installation to the other. Some of these differences have no impact on the overall capabilities of the system but some have a drastic influence on the way the collected data are processed and analysed. This aspect will be detailed in the next section, while this section deals with a general description of the apparatus. The most basic type of apparatus will be described, with no reference to sophisticated techniques such as Time Correlated Single Photon Counting or Circularly Polarized Luminescence devices. 

Time correlated single photon counting is a well-established technique that has been used to measure fluorescence lifetimes since the mid-1960 s. These early experiments, which used a variety of flashlamps and gaseous gap-discharge arcs as the excitation source, were reviewed by Ware [47, 48] in 1971. The traditional light sources have been replaced by laser sources in recent experiments, thus markedly extending the range of applications of this technique. Particularly well suited excitation sources for this method are the mode-locked lasers and synchronously pumped dye lasers which are capable of operation at MHz repetition rates. 

Time-correlated single photon counting A technique for the measurement of the time histogram of a sequence of photons with respect to a periodic event, e.g. a flash from a repetitive nanosecond lamp or a CW operated laser mode-locked laser). The essential part is a time-to-amplitude-converter (TAG) which transforms the arrival time between a start and a stop pulse into a voltage. Sometimes called single photon timing. 

The rotational reorientation times of the sample in several solvents at room temperature were measured by picosecond time-resolved fluorescence and absorption depolarization spectroscopy. Details of our experimental setups were described elsewhere. For the time-correlated single photon counting measurement of which the response time is a ut 40 ps, the sample solution was excited with a second harmonics of a femtosecond Ti sapphire laser (370 nm) and the fluorescence polarized parallel and perpendicular to the direction of the excitation pulse polarization as well as the magic angle one were monitored. The second harmonics of the rhodamine-640 dye laser (313 nm 10 ps FWHM) was used to raesisure the polarized transient absorption spectra. The synthesis of the sample is given elsewhere. All the solvents of spectro-grade were used without further purification. 

Zewail acknowledged early on that he was inspired to work in the dynamics area by amongst others, George Porter s development of fast reaction techniques, viz. Flash Photolysis which is reported elsewhere in this volume. In the early experiments outlined in the present paper, three detection techniques were employed time-correlated single photon counting, with 30-50 ps time resolution streak camera detection of fluorescence, with 10 ps resolution, and multiphoton ionisation with resolution determined by the pulse width of the laser, 1 or 15 ps. 

Steady-state emission spectra were recorded with a fluorescence spectrophotometer (Model 850, Hitachi Ltd.). Emission lifetime measurements were carried out using laser excitation pulses and a time-correlated single-photon counting system as described elsewhere. 

Fluorescence lifetimes were measured by time-correlated single photon counting using a mode-locked, synchronously pumped, cavity-dumped pyridine I dye laser (343 nm) or Rhodamine 6G dye laser (290 nm). Emissive photons were collected at 90° with respect to the excitation beam and passed through a monochromator to a Hamamatsu Model R2809U microchannel plate. Data analysis was made after deconvolution (18) of the instrument response function (FWHM 80 ps). 

In order to overcome this obstacle, we used a synchronously pumped, mode-locked dye laser, cavity-dumped at 4 MHz and time-correlated single-photon counting detection (18). Because of the higher sensitivity of this experimental system we were able to work at low e, using aqueous rhodamine B solutions with concentrations down to 10" M. To examine the dependence of the fluorescence decays on we chose to work with surface-solution interfaces, so as to minimize the problems associated with inhomogeneous surface coverage, which arise with dry surfaces... 

Time-resolved fluorimetry is also useful for the elimination of interferences from stray light due to Rayleigh and Raman scatter. The latter phenomena occur on a time scale of l(r14-l(T13 s and, as they have a much shorter duration than lamp or laser pulses, the light associated with them can be eliminated from the signal that ultimately reaches the detector. Time-correlated single-photon counting is superior in its ability to resolve multiple fluorescence from the same solution. 

If the spectral properties of the molecules are too similar for spectral identification, they may have different fluorescence lifetimes such that they could be identified in the time domain. By using pulsed laser excitation and time-correlated single-photon counting, which is the standard method for measuring fluorescence lifetimes [47], the researchers at Los Alamos have... 

Time-resolved PL measurements were also performed using time-correlated single-photon counting (TCSPC) and photoluminescence upconversion (PLUC) spectroscopies. Descriptions of the setups can be found in refs. [14, 65], respectively. All measurements were taken in continuous-flow He cryostats (Oxford Instruments OptistatCF) under inert conditions. Finally, PL efficiency measurements were performed on simple polymer thin films spin coated on Spectrosil substrates using an integrating sphere coupled to an Oriel InstaSpec IV spectrograph and excitation with the same Ar+ laser as above. 

Fluorescence lifetimes have been measmed directly by time-correlated single-photon counting for pentacene in p-terphenyl [100]. This experiment requires careful selection of the laser pulse eharaeteristies sueh that the pulse duration is short enough to resolve the 23 ns decay time, yet has a bandwidth narrow enough to allow speetral selection of individual molecules. Four different molecules had the same lifetime to within experimental uncertainty, indicating that the principal contributions to the Sj state decay (radiation and internal conversion to Sq) are not strongly sensitive to the local environment in this relatively homogeneous crystalline matrix. 

---