Once the fields of view have been aligned using the correlograms, each molecule is detected by a few pixels of the CCD camera. After each incorporation reaction, the presence of a labeled molecule is detected by the intensity, shape and location of the fluorescence signal at that spot. According to this signal, it can be decided automatically whether or not a nucleotide has been incorporated. The data collection of the fluorescence signal depends of the sequencing scheme. In real-time methods, a continuous stream of data on the millisecond timescale is needed. In cyclic sequencing schemes, a single or a few exposures are needed with integration times of about 100 milliseconds to determine the presence of a fluorescent molecule. The optimal detection integration time is influenced by factors such as bleaching time of the molecules and signal to noise. The goal is to observe the molecule in as short a time as possible to reduce the thousands of field-imaging times, without bleaching the molecule and while keeping the signal to noise high, by extracting the maximum numbers of photons from a molecule. In the next section we will elaborate on the example of single DNA molecule signals in sequencing experiments that use FRET to determine incorporation events (Braslavsky et al, 2003).
4.2.1. Intensity Traces
In this section we will describe the signal collection from a FRET experiment with some additional details. As discussed previously, the background noise can be suppressed by the use of single-pair FRET as a highly localized excitation source to monitor the incorporation of nucleotides in the templates. The first labeled nucleotide to be incorporated contains a donor fluorophore (Cy3), and successive nucleotides are labeled with an acceptor fluorophore (Cy5). The acceptor fluorescence is detected by exciting the donor, and the acceptors thus fluoresce only if they are in the vicinity of a donor. The noise from a nonspecific attachment of labeled nucleotides to the surface becomes very small, because the effective illumination region is only a few nanometers. In this example, the fluorescence dyes are not cleavable, hence photobleaching is used to null the acceptor fluorescence. After each incubation and FRET signal detection, the surface is illuminated with the acceptor specific excitation laser to bleach the acceptor but leave the donor unharmed. To efficiently visualize
this process throughout the whole sequencing experiment, the authors used intensity traces at the primer locations for both Cy3 and Cy5 signals to calculate the FRET efficiency (Figure 8).
Figure 8. Sequencing single DNA molecules with FRET. (A) Intensity trace from a single template
molecule through the entire session. The green and red lines represent the intensity of the Cy3 and Cy5 channels, respectively. The label at each column indicates the last nucleotide to be incubated, and successful incorporation events are marked with an arrow. (B) FRET efficiency as a function of the experimental epoch. Reprinted from Braslavsky, I., Hebert, B. Kartalov, E. and Quake, S. R. (2003). Sequence information can be obtained from single DNA molecules. Proc. Natl. Acad. Sci. USA. 100, 3960-3964. Copyright (2003), reprinted with permission from National Academy of Sciences (USA).
Alternate illumination can also be used to compare the signal from FRET to the signal from the Cy5 fluorophore directly. Some other uses of alternate illumination have been described in the literature (Kapanidis et al, 2005). Since the field of view shifts slightly between each reagent exchange, one has to be careful to shift the location of the intensity trace for each image set according to the peak of the correlation function. Also, because of the uneven illumination field from TIRM, one has to subtract a local background as opposed to a general noise subtraction for the whole field of view. In essence, the average intensity
over a 3x3 pixel region around the location of the primer constitutes the raw signal from the single molecule, and from that is subtracted an average over a 5x5 pixel region (excluding the central 3x3 region) which constitutes the local background. Here it is assumed that the density of the DNA templates is low enough that the 5x5 region around the primer location does not contain another DNA molecule.
The FRET efficiency is calculated as Ia/(Ia+Id), where Id and Ia are the average
intensities of the donor (Cy3) and the acceptor (Cy5), respectively. The FRET efficiency has a higher signal to noise than quantitation of either channel alone because it combines information from both fluorophores while simultaneously normalizing the relative intensities. The particular trace shown in Figure 8 reads out the correct sequence fingerprint for the template used (AAGAGA). Note the skip after the first G. This demonstrates that the sequencing scheme is asynchronous, an important feature that distinguishes sequencing at the single molecule level from the ensemble averaging inherent in macroscopic schemes. Thus, when an incorporation reaction is incomplete on a particular template molecule, it can be successfully completed in a later cycle without producing false information, or interfering with data from other DNA templates in the field of view. While using a complete trace is very useful to determine the sequence content of the template, it has a few drawbacks. For example, long illumination times in the FRET trace mode increase the risk of bleaching, even in the presence of an oxygen scavenger, which complicates the data analysis. A simpler method, relying on the information that is deduced from the trace mode, is discussed next.
4.2.2. Single Image Data Collection
After careful characterization of the single molecule signal in the experiments, one can assess what the detection probability of a molecule in one exposure will be compared to a more elaborate scheme of detection. This single image scheme can be implemented as a simple and fast method of detection, since the digital readouts of single-color sequencing (presence, or absence of a fluorescent molecule) are much simpler to analyze. Recent experiments have shown that such a collection mode is efficient and results in a reliable reading with a fast and simple data collection. (Harris et al, to be published).