Photon Bursts

The statistics of the detected photon bursts from a dilute sample of cliromophores can be used to count, and to some degree characterize, individual molecules passing tlirough the illumination and detection volume. This can be achieved either by flowing the sample rapidly through a narrow fluid stream that intersects the focused excitation beam or by allowing individual cliromophores to diffuse into and out of the beam. If the sample is sufficiently dilute that... 

Fig. 11.13. Photon burst detection of single molecules in a focused laser beam.

Confocal microscopes (see Section 11.2.1.1) are well suited to the detection of single molecules. A photon burst is emitted when the molecule diffuses through the excitation volume (0.1-1 fL). An example is given in Figure 11.16. 

Recently the fluorescence of IR-132 has been determined using single-photon timing at detection levels down to photon bursts from single molecules in 1 picoliter of a 25 fM solution of the dye.(14) At these extremely low concentration levels solvent... 

S. A. Soper, Q. L. Mattingly and P. Vegunta, Photon burst detection of single near-infrared fluorescent molecules, Anal. Chem. 65, 740-747,(1993). 

The method is based on the fact that one can excite coherently a set of overlapping resonances such that their decay exhibits a steplike behavior the system starts in a quiescent period in which no spontaneous emission occurs, followed by a photon burst in which spontaneous emission is greatly accelerated, followed by another quiescent period, and so on. The quiescent period (and subsequent photon bursts) is due to destructive and constructive interferences between the overlapping resonances. The reason it is impossible to suppress the decay over all times in this... 

Fig. 13. Single molecule detection of Dil at the dodecane-water interface by fluorescence microscopy (left). Short photon burst in the SDS systems and (right) long burst in the DMPC systems.

FIGURE 10.7. Total internal reflection fluorescence microscopy of the micro-two-phase system of dode-cane/water. (a) Continuous photons with an average of 11 molecules in the observation area, (b) photon burst with an average of 0.02 molecules in the observation area and (c) extension of the photon burst upon the addition of DMPC. A model for the photon burst upon the observation using single molecule diffusion (d). 

If in atoms a transition /) ) can be selected, which represents a true two level system (i.e., the fluorescence from A ) terminates only in /)), the atom may be excited many times while it flies through the laser beam. At a spontaneous lifetime r and a travel time T through the laser beam, a maximum of n = Tl 2x) excitation-fluorescence cycles can be achieved (photon burst). With T — 10 s and r = 10 s... 

If the spectroscopic detection of atoms can be performed on transitions that represent a true two-level system (Sect. 9.1.5), atoms with the radiative lifetime r may undergo up to T/2r absorption-emission cycles during their transit time T through the laser beam (photon burst). If the atoms are detected in carrier gases at higher pressures, the mean free path A becomes small A d) and T is only limited by the diffusion time. Although quenching collisions may decrease the fluorescence... 

Sect. 6.3), the ratio Tjlx becomes larger and the magnitude of the photon bursts may still increase inspite the decreasing quantum yield. 

Example 10.2 For gases at low pressures where the mean free path A is larger than the diameter d of the laser beam, we obtain the typical value T = d/v=lO ps for = 5 mm and u = 5 x 10 m/s. For an upper-state lifetime of T = 10 ns the atom emits 500 fluorescence photons (photon burst), allowing the detection of single atoms. With noble gas pressures of 1 mbar the mean-free path is 0.03 mm and the diffusion time through the laser beam may become 100 times longer. Although the lifetime is quenched to 5 ns, which means a fluorescence quantum yield of 0.5, this increases the photon burst to 5 X 10" photons. 

Figure 1. Single-molecule detection. A molecule flows through the focused laser beam, generating a photon burst, which is shown as the time dependent signal at the bottom of the figure. The burst must be detected above the noise in the background signal in a single molecule detection experiment.

Another type of error occurs when the signal from a molecule does not exceed the threshold, generating a miss. The detection efficiency is the ratio of detected molecules to total analyte molecules in the sample. Detection efficiency is degraded in three ways. First, if the detection threshold is set too high, then only a small fraction of the molecules will generate detectable photon bursts. Second, at high concentrations, several molecules may be present in the probe volume at the same time. The simple threshold counter is not able to resolve multiple molecules and the detection efficiency drops compared to low analyte concentration data. Third, if only a small portion of the sample passes through the detection volume, then most molecules can... 

In Hirschfeld s data, the background signal was about 100 photocounts, while the photon burst amplitude was about 400 photocoimts larger. That is, ii = 100 and = 400. Also, the probability that a molecule was present in the laser beam looks to be about Pq = 0.2 by definition, the probability that a molecule was not present is Pq = 0.8. Using the equations from above, the optimum threshold, in a Bayes sense, is given by... 

Figure 2.5 PCH from a photon burst trace of Fluorescein 27 in 50 mM sodium phosphate buffer at pH 7.0 (circles). A simple Poisson function, with an average of = 0.82 (black line, equal tothe mean number of photon counts in the recorded dataset) does not describe the data. The data are fit with a single species PCH (solid grey line) with /V = 0.13 ande = 123800 cpspm with, = 2.4. A total of 131072 data points were collected. Such data (grey) are referred to as super-Poissonian.

---