Abstract
Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.
Highlights
During interphase, eukaryotic chromatin is organized into a remarkably complex and dynamic assembly of large-scale (30– 300 nm) domains built from a basic nucleosomal motif [1,2]
Many biochemical studies and electron microscopy observations have suggested the existence of chromatin fibers ranging from 30 to 300 nm corresponding to intermediate folding levels [5,6,7,8]
While there have been numerous efforts to minimize these factors during EM observations [6,9,10,11,12,13], none of the studies have fully resolved the lack of specific labeling methods or concerns of perturbations induced during the harsh sample preparation procedures required for electron microscopy
Summary
Eukaryotic chromatin is organized into a remarkably complex and dynamic assembly of large-scale (30– 300 nm) domains built from a basic nucleosomal motif [1,2]. As the cell cycle progresses, interphase chromatin is further condensed to become the maximally dense, structurally reproducible mitotic chromosome structure. Many biochemical studies and electron microscopy observations have suggested the existence of chromatin fibers ranging from 30 to 300 nm corresponding to intermediate folding levels [5,6,7,8]. Detailed structural observations have been hindered by the extraordinary sensitivity of chromatin organization to environmental factors and the remarkably high density of mitotic chromosomes. While there have been numerous efforts to minimize these factors during EM observations [6,9,10,11,12,13], none of the studies have fully resolved the lack of specific labeling methods or concerns of perturbations induced during the harsh sample preparation procedures required for electron microscopy. Developing alternative strategies for the direct high-resolution observation of chromatin structure under nondisturbing conditions and ideally with the potential for specific labeling is a high priority
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