As the name suggests, single-molecule techniques are those that enable investigation of properties of individual molecules as opposed to bulk techniques where readouts/measurements result from averaging of properties of multiple molecules. For the longest time, the biggest hurdle for single-molecule techniques was the ability to detect signal from a desired single molecule, i.e., the signal was too weak, especially in a biological system. But since the late 90s, the advent of powerful cameras, new microscopy techniques, development and characterization of extremely bright dyes (fluorophores) have enabled detection from single molecules in biological systems.

Why single-molecule?

1. Capture heterogeneity of a biological system:
Biological systems are incredibly complex involving interaction between many components, e.g. DNA/RNA, proteins, and metabolites. And even the weakest interactions can have critical outcomes. Therefore, to understand the system as a whole, it is imperative that we take all different interactions into account.  Bulk measurements can only report on average of these interactions thus masking their intrinsic heterogeneity. Single-molecule measurements allow you to investigate each interaction separately thus helping us to unmask the inherent heterogeneity of a system.

2. Analyze molecular mechanism of important processes:
Biological processes are a combination of many stochastic events i.e. there is no synchronization between behaviors of multiple DNA/RNA or protein molecules (biomolecules) that make up the biological process. This combination makes investigating the process and its steps very difficult via bulk measurements because they only report on the averaging of the multiple stochastic events. E.g., if we relied solely on scorelines to judge tennis players, we could make a reasonably good assessment that Roger Federer is a great tennis player. But only the scorelines obscures many critical aspects of his greatness, like his technique, footwork, etc. which are essential information because they not only serve entertainment aspect of tennis but is also used as a model for upcoming players and overall development of the sport. In a way, the scoreline can be thought of as a read of a bulk measurement, i.e., the average of multiple different shots (events) that Federer hit, with no information about quality and nature of the shots.

If we can watch single molecule(s) of that entire process or a system, we can understand how various parts of the system work individually and combinatorially. But capturing single biomolecules directly in action is almost impossible because their sizes are extremely small and are thus beyond fundamental optical limits of microscopes. So, single molecule detection for biological systems is typically achieved by attaching an extremely bright fluorescent dye on the biomolecule of interest which makes it 'fluorescently visualizable' as it emits light of a certain wavelength when illuminated with a light source (laser) of a particular wavelength (color). Apart from just being able to localize and track the motion of the biomolecule via the fluorescent dye attached to it, there are many photophysical properties of these dyes that can be utilized to serve the variety of purposes. One such example is FRET, which involves transfer of fluorescent 'energy' from one dye (donor) molecule to other (acceptor) molecule. So if a donor dye molecule is being illuminated with a bright laser for its fluorescent visualization via its emission of a certain wavelength or color. A part of its 'energy' can end up being donated to an acceptor dye molecule in its vicinity, which would emit the 'donated energy' via its emission of a different wavelength or color. The transfer of energy is dependent on the distance between the dye molecules, so intensity and wavelength of the emission from these two dye molecules can act as a reporter of distance between them. Implementation of FRET technique in a single-molecule setup is commonly referred to as single-molecule FRET (smFRET), which can be adapted to investigate any biological process and its steps.

E.g., if Federer had been extremely small like a protein molecule then we could watch the internal shape changes of the protein/Federer as it does its job/play tennis by attaching a donor and an acceptor molecule to specific locations (e.g., hands of Federer) and illuminate them with a laser light of certain wavelength. The real-time output emission from the protein/Federer ( combination of both donor+acceptor emission) will report on real-time changes in the distance between two specific points in protein/ hands of Federer. This information will help you understand and analyze the molecular mechanism of the protein/Federer.

Fig.1 Implementation of smFRET to investigate real-time changes in distance between Federer's hands during play.

3. Localization of single molecules for super-resolution imaging:
Biological structures (cells, tissue, etc.) are made up of thousands of constituent biomolecules (protein, DNA, RNA, etc.). Visualization and localization of each of the constituent biomolecules, in singulo, can be used to build up the entire biological structure giving us a high quality & high-resolution image of it. Design and implementation of this idea were one of the subjects of 2014 Nobel prize in Chemistry.

4. Adaptation of single molecule technique in sequencing and other applications:
If multiple single molecules can be detected simultaneously, then multiple reactions can be performed simultaneously and investigated separately. This idea has been most successfully implemented in DNA sequencing applications. Previous methods of DNA sequencing relied on series of individual experiments that had to be painstakingly performed separately for each specific region in the genome to be sequenced. Data was then collected from multiple such individual experiments for final analysis.  But the ability to detect the readout of sequencing from a single (or few) DNA molecule(s), performed in parallel but analyzed separately, has revolutionized biology. The point I want to emphasize here is that the ability of massively parallel single-molecule detection can be repurposed to analyze a large number of parallelly performed reactions separately.

5. There are many other advantages of single-molecule investigations, some of which I have discussed in other sections of my research descriptions.