ABSTRACT

A basic goal of biophysics is to quantitatively understand cellular function as the consequence of the fundamental laws of physics governing a system of extreme complexity. For this, the knowledge of its molecules a nd b iochemical re actions i s o f ut most i mportance. I n add ition, however, more de tailed spatial information about nanostructures involved is necessary. A serious problem to analyze cellular nanostructures by far- eld light microscopy is the conventional optical resolution restricted to about 200 nm laterally and 600 nm a xially. Various recently introduced laseroptical “nanoscopy” methods such as SI-, 4Pi-, and STED microscopy have allowed a signi cant improvement of the spatial analysis far beyond t hese l imits. Here, t he focus w ill be on a c omplementary approach, “spectrally a ssigned localization m icroscopy” (SALM). SA LM i s ba sed on l abeling “point-like” objects (e.g., si ngle molecules) with di erent spectral signatures (see the following text), spectrally selective registration and high-precision l ocalization m onitoring by f ar- eld uorescence m icroscopy. e ba sic c ondition i s that in a given observation volume de ned, e.g., by the full-width-at-half-maximum (FWHM) of the point-spread-function (PSF) of the optical system used, at a g iven t ime and for a g iven spectral registration mode, only one such object (e.g., a si ngle molecule) i s registered. According to t he t ype of uorophores used (e.g., photostable, or photoconvertable, or stochastically convertible), various SALM procedures have been described (e.g., FPALM, PALM, PALMIRA, RPM, SPDM, STORM, dSTORM) and a lateral spatial resolution in a few tens of nm range has been realized. In particular, in this chapter a SALM procedure will be described, which a llows “nanoimaging” of large numbers of molecules at high intracellular densities by spectral precision distance/position determination microscopy (SPDM) and in c ombination w ith w idely u sed uorescent p roteins a nd s ynthetic dye s i n s tandard me dia, including physiological conditions. e technique called SPDMPhymod (SPDM with physically modi ed uorophores) is based on excitation intensity-dependent reversible photobleaching and the induction of “ uorescent bursts” of the excited molecules at s tochastic “onset” times. In this case, the “spectral signature” can be de ned as the time duration between the start of the exciting illumination and the time of t he si ngle mole cule uorescence bursts i nduced (“onset t ime”). Si nce suc h onset t imes c an be s tochastically d istributed over a p eriods of many seconds up to t he range of minutes, i n a g iven specimen, e.g., a cell, thousands of di erent spectral signatures can be created. Presently, SPDMPhymod techniques (also called reversible photobleaching microscopy, RPM) have been used to determine the intracellular spatial location of single molecules at a density up to ca. 1000 molecules/µm2 of the same type with an estimated best localization precision of 2 nm (at an excitation wavelength of λexc = 488 nm). Distances in the range of 10-30 nm were nanoscopically resolved between such individual uorescent molecules. In combination with structured illumination, a 3D e ective optical resolution of 40-50 nm was realized (ca. one-tenth of the exciting wavelength). As original applications, nanoimaging of the distribution of tubulin proteins in human interphase broblasts and histone proteins in mitotic HeLa cells are reported.