A novel combined Structured Illumination and Single Molecule Localization Microscope and its application to Retinal Structures

Fluorescence microscopy methods have become an major imaging tool in biomedical lifescience. However, each method only addresses specific questions due to intrinsic limitations. A new, fully automated and user-friendly 'Combo'-microscope setup has been developed, which combines the two advanced high-resolution methods Structured Illumination and Localization Microscopy into one imaging system. As both methods complement one another, the 'Combo'-microscope will greatly extend the range of application in biomedical research. Moreover, artifacts, introduced in the course of the imaging and/or reconstruction processes, can be revealed and potential mis-interpretation of super-resolution data is limited. This work was motivated by the age-related macular degeneration (AMD), a disease, which is the leading cause of blindness in the Western world. Autofluorescent particles within age-related deposits (drusen) beneath the retinal pigment epithelium are studied on advanced resolution level. Furthermore, the newly built microscope is used for a dual-mode dual-color three-dimensional visualization of the axon initial segment, a crucial region for signal transduction in vision, of retinal ganglion cells. Finally, an in vitro study comparing the pharmaceuticals currently used for AMD-treatment is outlined

Autofluorescence imaging of human RPE cell granules using structured illumination microscopy

ABSTRACT Background/aims To characterise single autofluorescent (AF) granules in human retinal pigment epithelium (RPE) cells using structured illumination microscopy (SIM). Methods Morphological characteristics and autofluorescence behaviour of lipofuscin (LF) and melanolipofuscin (MLF) granules of macular RPE cells (66-year-old donor) were examined with SIM using three different laser light excitation wavelengths (488, 568 and 647 nm). High-resolution images were reconstructed and exported to Matlab R2009a (The Mathworks Inc, Natick, MA, USA) to determine accurate size and emission intensities of LF and MLF granules. Results SIM doubles lateral resolution compared with conventionally used wide-field microscopy and allows visualisation of intracellular structures down to 110 nm lateral resolution. AF patterns were examined in 133 LF and 27 MLF granules. LF granules (9686220 nm) were significantly smaller in diameter than MLF granules (10976110 nm; p<0.001). LF granules showed an inhomogeneous intragranular pattern, and the average intensity negatively correlated with the size of these granules when excited at 647 nm. The autofluorescence of MLF granules was more homogeneous, but shifted towards higher excitation wavelengths in the centre of the granules. Conclusion SIM is a useful tool for examining AF signals within single LF and MLF granules in RPE cells. This allows new insights into RPE autofluorescence patterns

4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues

Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample

Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with a mean size of approximately 14 RyRs per cluster, which is almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often <50 nm, suggesting that peripheral RyR clusters may exhibit strong intercluster signaling. The wide variation of cluster size, which follows a near-exponential distribution, is compatible with a stochastic cluster assembly process. We suggest that calcium sparks may be the result of the concerted activation of several RyR clusters forming a functional “supercluster” whose gating is controlled by both cytosolic and sarcoplasmic reticulum luminal calcium levels