Hi,

Using a 'simple' epifluorescence microscope, with a high NA objective, a sensitive camera, and a laser powerful enough to provide the required excitation intensity, you should be able to see individual fluorescent particles. You should make sure that you have a very low background, since all out-of-focus fluorescence will also be captured by this setup. If you then see single-step photobleaching, you are sure that you were looking at an individual fluorescent particle. Hope this answers your question.

Laurent

Since you are concerned with individual fluorescent proteins, you will inevitably fall under the "conventional" optical resolution of optical microscopes. Therefore, you should first figure out how dense your proteins are, and assuming they are pretty dense, when detecting particles in your image you will never know whether you are detecting one or a bunch or particles. To solve this problem, have a look at super-resolution approaches like PALM-STORM, which use conventional optical setups, but use photo-activable (in case of PALM) or naturally blinking (in case of STORM) fluorophores that will statistically never light up at the same instant, allowing you precise localization (and ultimately allow you to reconstruct a super-resolved image if you combined thousands of consecutive images of the same sample). Depending on your surface protein, a number of such fluorophores may already be available (this review might be a good starting point for techniques and protocols: Henriques et al. (2011) PALM and STORM: Unlocking live-cell super-resolution. Biopolymers 95(5))

If you need photostable and/or photoactivable fluorophores, you may look for them by www.abberior.com

Hello Matthew,

For sure you can use classical fluorescent microscope to visualize single particle, this technique is called SPT for single particle tracking (Sergé et all Nature methods Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes). The TIRF illumination mode isn't necessary this just remove the noise bring by dyes which are not in proximity of the cover glass (200 nm).

For the fluorescence you can use Q-Dots, which are fluorescents nanocristals (20nm), this solution is the brightest and the stablest one, but they are controversial due to their big dimensions and toxicity for living cells. But if you want to use organic Dyes as Alexa or ATTO directly coupled to an antibody or an Fab fragment, I suggest you to use Alex 647 and ATTO 647N, they are the best in my opinion (danger with ATTO647N which is electro negatively charged) . But for these dyes you need 633 or 640 excitation, this mean a laser because classical Hg lights are really low at this wavelength. You need a low density of dyes to be able to see single particles.

Finally you need an high aperture objective with a sensitive camera, an EMCCD for example.

Technique called PALM or STORM also work on single molecule régime but using different modalities

vincent

The "ordinary" light microscope does not seem to be the limiting factor here. Provided you have an objective with a reasonable NA and good transmission, the critical factors are:

(1) quite obviously the type of fluorophore, camera and quality of your fluorescent filter

(2) whether you will be able to immobilize the proteins on the surface. If so, you could "simulate" a TIRF approach by removing labelled but unbound molecules, work with longer illumination times and, consequently, less powerful light sources.

(3) whether you protect your chemical fluorophore (most alexa dyes should definitely work here) with oxygen scavenging solutions.

I agree with all the previous answers, you should be able to capture the signal from an individual fluorophore, as long as you have a sensitive detector (EM-CCD or scientific CMOS cameras are the most widely used sensors for single molecule detection), an high NA objective (better if 1.4 NA or higher) and a method to suppress out of focus background (such as extensive washing the unabsorbed molecules).

I would also take a look at the filters that you have mounted on the scope: often a notch filter in the emission pathway is very useful to suppress unwanted reflections of your excitation source.

Regarding the choice of the light source, lasers are the most commonly used sources for single molecule detection. However a fluorescent (Hg arc, for example) lamp might work as well, and has been used in the past.

Dyes such as Alexas and Attos are all bright enough to provide sufficient photons for single molecule detection.

Thank you to everyone for your responses, especially considering it's the end of the world today and all. All really useful; reassuring to know that it could still be done with the tools I have available (which sadly doesn't include anything as fancy as PALM/STORM or STED!). Haven't been able to see nice photobleaching steps in what should be, and what appear to be single fluorescent puncta. But this could be that they are actually small aggregates; or I need to use a different fluorophore. Cheers!

A mathematical approach is another avenue to consider. If you can measure the point-spread functions of known single sub-diffraction particles (eg. quantum dots, fluorescent beads) you can deconvolve images of your samples to recreate sub-diffraction resolution. http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1002671

Hi Matthew,

sorry for this late reply but I have to mention that there is a collaborative centre at the university of Edinburgh where you can find a PALM microscope. This is called COSMIC and you can find it in the department of physics. If you or your PI get in contact with Dr J. Arlt, you will be able to obtain more details about accessibility.

Cheers,

Mathieu

one of the main problems in protein-protein complexes in single-molecule experiments is they tend to dissociate at the very high dilutions used. A simple low tech way around this is to encapsulate the proteins in liposomes before diluting and attaching to the surface. This has the added advantage of lifting the protein off the vesicle surface and reducing the autofluorescence.