Photon burst detection

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

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 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... 

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... 

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.

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... 

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... 

Figure 3. Diffusion analysis of freely diffusing molecules in solution. Fluorescently labeled molecules produce brief, intense photon bursts such as the ones shown in (a) on the photon detectors. Molecules labeled with different colors can be spectrally separated and detected on different detectors, which results in colocalized emission to both detectors in the case of molecular reaction events, such as e.g. antibody-antigen binding. Such binding events also change the diffusion time of the molecules through the excitation spot, which can be measured by the burst width or, more quantitatively by an autocorrelation analysis of an entire diffusion trace, which results in an average diffusion time as shown on (b).

Sequencing using synthetic DNA stripped off as it passes through a nanopore, detected as a shortlived photon bursts... 

The point is now to estimate the maximum number of photons that can be detected from a burst. The maximum rate at which a molecule can emit is roughly the reciprocal of the excited-state lifetime. Therefore, the maximum number of photons emitted in a burst is approximately equal to the transit time divided by the excited-state lifetime. For a transit time of 1 ms and a lifetime of 1 ns, the maximum number is 106. However, photobleaching limits this number to about 105 photons for the most stable fluorescent molecules. The detection efficiency of specially designed optical systems with high numerical aperture being about 1%, we cannot expect to detect more than 1000 photons per burst. The background can be minimized by careful dean-up of the solvent and by using small excitation volumes ( 1 pL in hydrodynamically focused sample streams, 1 fL in confocal exdtation and detection with one- and two-photon excitation, and even smaller volumes with near-field excitation). 

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