# Literature/202212291648 optical methods for single molecule analysis

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The earliest method to get single-molecule data is to interrogate a very small volume. For example, if we consider a $1\mu m^3=1\,\textrm{fl}$ volume with only one molecule inside, we get a concentration of $100\, \textrm{nM}$. However, at a concentration of $1\ \textrm{fM}$, we have 600 molecules per $10^9$ femto-liter volumes.

Therefore, flow-methods (like Flow Cytometry) operate a concentration regimes close to pico-molar: concentrated enough to detect a statistically significant number of molecules, and diluted enough to neglect coincidental detections (known as swarming in flow cytometers).

Low concentrations are important, for example see: literature/202212291643 Errors in amino acid sequences, why pushing sensitivity of assays.

• Many methods rely on pre-concentration steps, to limit the volume that must be scanned. Examples:

• Some methods are truly single-molecule sensitive: