Conventional
fluorescence microscopy is performed by selectively staining the sample with
fluorescent molecules, either linked to antibodies as in
immunohistochemistry or using fluorescent proteins genetically fused to the genes of interest. Typically, the more concentrated the fluorophores, the better the contrast of the fluorescence image. A single fluorophore can be visualized under a microscope (or even under the naked eye) if the number of photons emitted is sufficiently high, and in contrast the background is low enough. The two dimensional image of a point source observed under a microscope is an extended spot, corresponding to the
Airy disk (a section of the
point spread function) of the imaging system. The ability to identify as two individual entities two closely spaced fluorophores is limited by the
diffraction of light. This is quantified by
Abbe's criterion, stating that the minimal distance d that allows resolving two point sources is given by d = \frac{\lambda}{2 NA} where \lambda is the
wavelength of the fluorescent emission and NA is the
numerical aperture of the microscope. The theoretical resolution limit at the shortest practical excitation wavelength is around 150 nm in the lateral dimension and approaching 400 nm in the axial dimension (if using an objective having a numerical aperture of 1.40 and the excitation wavelength is 400 nm). However, if the emission from the two neighboring fluorescent molecules is made distinguishable, i.e. the photons coming from each of the two can be identified, then it is possible to overcome the diffraction limit. Once a set of photons from a specific molecule is collected, it forms a diffraction-limited spot in the image plane of the microscope. The center of this spot can be found by fitting the observed emission profile to a known geometrical function, typically a
Gaussian function in two dimensions. The error that is made in localizing the center of a point emitter scales to a first approximation as the inverse square root of the number of emitted photons, and if enough photons are collected it is easy to obtain a localization error much smaller than the original point spread function. The two steps of identification and localization of individual fluorescent molecules in a dense environment where many are present are at the basis of PALM, STORM and their development. Although many approaches to molecular identification exist, the light-induced photochromism of selected fluorophores developed as the most promising approach to distinguish neighboring molecules by separating their fluorescent emission in time. By turning on stochastically sparse subsets of fluorophores with light of a specific wavelength, individual molecules can then be excited and imaged according to their spectra. To avoid the accumulation of active fluorophores in the sample, which would eventually degrade back to a diffraction-limited image, the spontaneously occurring phenomenon of
photobleaching is exploited in PALM, whereas reversible switching between a fluorescent on-state and a dark off-state of a dye is exploited in STORM. In summary, PALM and STORM are based on collecting under a fluorescent microscope a large number of images each containing just a few active isolated fluorophores. The imaging sequence allows for the many emission cycles necessary to stochastically activate each fluorophore from a non-emissive (or less emissive) state to a bright state, and back to a non-emissive or bleached state. During each cycle, the density of activated molecules is kept low enough that the molecular images of individual fluorophores do not typically overlap.
Localization of individual fluorophores In each image of the sequence, the position of a fluorophore is calculated with a precision typically greater than the diffraction limit - in the typical range of a few to tens of nm - and the resulting information of the position of the centers of all the localized molecules is used to build up the super-resolution PALM or STORM image. The localization precision \sigma can be calculated according to the formula: \sigma=\sqrt{\left(\frac{s_i^2 +\frac{a^2}{12}}{N}\right)\cdot\left(\frac{16}{9}+\frac{8\pi s_i^2 b^2}{a^2 N^2}\right)} where N is the number of collected photons, a is the pixel size of the imaging detector, b^2 is the average background signal and s_i is the standard deviation of the point spread function. The requirement of localizing at the same time multiple fluorophores simultaneously over an extended area determines the reason why these methods are wide-field, employing as a detector a
CCD, EMCCD or a
CMOS camera. The requirement for an enhanced
signal-to-noise ratio to maximize localization precision determines the frequent combination of this concept with widefield fluorescent microscopes allowing
optical sectioning, such as
total internal reflection fluorescence microscopes (TIRF) and
light sheet fluorescence microscopes. ==Super-resolution image==