Commonly, three criteria can be used to support the observation of single molecules. These are digital bleaching, blinking, and intensity calibrations. If a signal of a particular intensity disappears in one step (digital, meaning either on or off), the source of the signal must have been a single emitter. Digital bleaching of single fluorophores occurs in one step. Blinking has been used to argue for the observation of single quantum dots, where fluorescent emissions cease temporarily before being observed again. Working with single emitters in fixed cells, blinking and/or bleaching will provide valuable evidence supporting the observation of single molecules. Enhancing the signal by multiple labeling is a powerful strategy to improve signal intensities for imaging. Here, bleaching can still provide good support if multiple irreversible stepwise decreases of fluorescence intensity are observed. Observing a subsequent stepwise bleaching of immobilized fluorophores known quantities of multiple labels is a strong indicator for observing single molecules. When multiply labeling, autoquenching of dyes is possible, and, if so, the total steps observed for a given molecule might not represent the total labeling ratio.
These criteria are challenging to meet during live observation, because a particle that leaves the focal plane will leave the same signature as a bleached emitter. A moving emitter that blinks will not be seen while in the dark state and cannot be analyzed. To calibrate mobile particles, intensity calibrations can be used to identify single molecules. For calibration of fixed samples, such as described in the FISH section, individual probes can be immobilized to measure the average intensity, and then the average intensity of this signal is used to generate a fluorescence intensity standard curve against which to compare mobile molecules. A slightly lower intensity for these molecules during live imaging is expected, because their movement will blur their signals during image acquisition. With one or few fluorophores, this intensity calibration approach works well as in FISH quantification. However, the use of many fluorophores introduces much more variation to the intensity averages of single particles and, therefore, presents challenges for calibrating the genetically encoded MCP-MBS particles. In this case, an intensity distribution–based argument can be made. Digital photobleaching is accepted as support for observing single dyes. If the intensity distribution of all the observed objects in a field shows discrete steps separated by uniform integral intensity differences, this argues for the presence of a fundamental base unit, and the steps then most likely represent multiples of this base unit.
Can this be used to argue for the observation of single molecules with a uniform multiplexed label? If sensitivity does not allow detection of single molecules, the observed base intensity (the lowest intensity value of all observed objects) could be any multiple of the true single molecule. However, the least complex possibility is that the lowest intensity particles are, in fact, single molecules when sensitivity is sufficient to detect them. If this is true, objects observed in the same frame or movie with an intensity that is an integral multiple of the lowest intensity found must be complexes of the exact stoichiometry that agrees with this integral difference. In plain English if the lowest intensity objects are singles, then objects twofold this intensity are dimers, threefold this intensity are trimers, etc. With multimeric single molecules (e.g., proteins that exist constitutively as multimeric) then this property will obviously be inherently present in the value of the base unit. Nonetheless, all such objects will still be equivalent in intensity. When the nature of the molecule under study is unknown, then this possibility merely leads to some uncertainty about the nature of the single molecule, not that it is a single molecule. mRNA does not multimerize nonspecifically, although complementary sequence can lead to dimerization in some cases. FISH studies have confirmed that an individual mRNA species does not generally multimerize (Femino et al., 1998
). Therefore, there is precedent that the base unit for MCP-MBS labeled mRNA is, in fact, single species.
A potential weakness of the argument with integral multiples comes from the imaging method. Brightly labeled single molecules by multiplexing its labels increases the focal depth that the particle is observable in a far-field microscope. Bright complexes that are out of the focal plane could have the same or any intermediate intensity. As a consequence, z-sections and photon reassignment could be used to minimize artifacts as in deconvolution for FISH (Femino et al., 1998
). Alternately, 3D tracking (Levin and Gratton, 2007) or optical sectioning would help. The use of confocal or TIRF microscopes could provide such sectioning (different microscopes for single molecule detection will be described later). By use of an epiilluminated microscope, blurring of out-of-focus signal may be used to define a threshold for a diffraction-limited signal that has a higher probability of originating from the optical plane of the objective. Theoretically, it is even possible to analyze diffraction patterns to determine the exact 3D position (Speidel et al., 2003
The imaging, and especially the dynamic imaging of single molecules, requires a critical reevaluation of the way most biological imaging is performed. How many molecules can be tracked at the same time? How fast do they exchange? How precisely can their positions be determined? How much signal must be integrated? How long can a single molecule be imaged before it bleaches? What time resolution is needed to track them? What signal sampling is required? There are no uniform answers to these questions, because each experiment will require optimization of these parameters. To keep the tone of this chapter consistent, we summarize a descriptive framework as an appendix to this chapter that should help researchers in finding reasonable start parameters on the basis of established principles. The appendix also contains a separate section on suitable controls for microscope function and data acquisition, which are important, because microscope stability can have a large impact on the results of single molecule experiments.