Introduction

STORM (Stochastic Optical Reconstruction Microscopy) is a technique to create image that resolve objects spaced closer than the diffraction limit of the optical system. In traditional optical microscopy, the quality of images is fundamentally limited by diffraction of light through an aperture. However, STORM is able to overcome this limit by fluorophore position data from many sequential images of the same sample. Because it relies on position measurements of the fluorophores, which can be determined with much higher accuracy than the diffraction limit of light, it is possible to create images that distinguish objects spaced below the diffraction limit. By imaging fluorophores asynchronously, the positions of fluorophores spaced closer can be determined independently. As a result, neighboring fluorophores can be distinguished when their positions are recorded in the compiled image

Light originating from a point from a point particle diffracts as it passes through an aperture, resulting in an Airy disc diffraction pattern in the image. According to the Rayleigh criterion, assuming maximal numerical aperture, the best resolution for a light microscope is given by the equation

$ R = \frac{0.61\lambda})/({NA}) $,

(2)E_{photon}=\frac{hc}{\lambda}

where λ is the wavelength of light and NA is the numerical aperture. Given realistic values for the numerical aperture and the wavelength of visible light, it is not possible to attain a resolution smaller than a couple hundred nanometers.

STORM overcomes this fundamental resolution limit however by activating only a small number of fluorophores in a sample at a given time. This is accomplished by using switchable fluorophores that activate and inactivate using different wavelengths of light. By choosing fluorophores that switch between an excited state and a dark state, it is possible to only activate a small percentage of the fluorophores in a sample at once. Thus even through the fluorophores may be spaced closer than the resolution limit, at a given time the activated fluorophores will be on average spaced apart more than the resolution limit. At each time point, the position of each fluorophore imaged can be approximated by finding the centroid of the airy disk created by each fluorophore. The position data from all sequential images can then be compiled to create a single image of the sample.

While STORM technology has already been developed, each setup is often prohibitively expensive. The Nikon STORM microscope costs anywhere between $500,000 and $750,000. We set out to adapt this technology to be able to be easily constructed in a teaching optics laboratory for less than $20,000. The system we developed costs less than $10,000.