If you can look at the spectra of the fluorescence you should see clearly what is your sample and what is impurity. It is not likely that both would have the same spectrum. Additionally, samples in single molecule detection are usually highly fluorescent while the impurity are not likely to be. To identify if you have one complex emitting you could look at the time of arrival of the photon to the detector. In short, one emitter can emit only one photon at the time. Other thing that could suggest that you observe more than one complex is the intensity of fluorescence. In case you have two unconnected emitters you would have twice as much fluorescence than in case of one molecule.

I have to agree with Michal here. If you are doing single molecule detection then it is very likely that you are using a highly fluorescent protein molecule (for example) which has well-characterised excitation and emission peaks.

As well as this, it is important to know exactly what is in your sample and be aware of the possibility of impurities and whether they have fluorescence properties of their own.

To answer the second part of your question, it would be possible to do this by knowing the concentration of your sample and from that you will be able to estimate the number of molecules you have in a sample- it would then be possible to dilute the sample to the point where one could say there is only a single molecule of the target present. Fluorescence measurements could then be performed on this sample, alternatively, I'm sure the method proposed by Michal would also work.

I agree with the answers of Michal and Derek. BUT PLEASE NOTE AND ANSWER:

- Is it an organic molecule or biomolecule?? For organic molecules e.g.

- Prior to publication, you MUST HAVE a TLC pure compound with a good elemental analysis and a NMR, MS related to this molecule.

- The impurity (ies) can interact with the pure compound and will lead to artefacts when you solve the impure drug in a solvent in order to calculate the concentration prior to measure UV-Vis and single molecule detection.

PLEASE REWRITE your question because it is a bad formulated question and give what a kind of molecule (e.g. bio or dyes or fluorochrome) and what a kind of fluorescence microscop or microspectralphotometer .

YOU DON ´ T GIVE no details about your sample (slide or microspheres...) when you write fliorescence microscopy.

please see this link and the part copied for you:

http://www.ingentaconnect.com/content/asp/jnn/2013/00000013/00000002/art00105?crawler=true

Single-molecule force spectroscopy has revolutionized our ability to probe the details of molecular structures and interactions, but the numbers of individual measurements required for achieving a statistically reliable result can sometimes prove daunting. To overcome this problem, a number of instruments have recently been developed that are capable of monitoring the behavior of tens of individual biomolecules simultaneously. In this work, we have constructed a novel electromagnetic apparatus for multiplex single molecule force measurements utilizing magnetic microspheres. In this system, the magnetic field is generated with an electron-lens of an electron microscope mated with a high voltage flash light circuit to rapidly attain a stable magnetic field. We show that this instrument can generate a uniform magnetic force of up to ∼20 pN within 5 ms, over a region spanning 1 mm. The successful application of this apparatus to the force-dependent extension of dsDNA fully validates this approach. Furthermore, the lens-like design of the pole piece is fully compatible with optical imaging, thus allowing for the integration of single molecule fluorescence capabilities that should make this system a particularly powerful apparatus for multi-dimensional characterization of fast processes within interacting single molecules.

Rewrite and purify and purify...

JRG