RESUMEN
Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.
Asunto(s)
Técnicas Citológicas/métodos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Animales , Bacteriófagos/ultraestructura , Vesículas Cubiertas por Proteínas de Revestimiento/ultraestructura , Técnicas Citológicas/instrumentación , Aparato de Golgi/ultraestructura , Masculino , Ratones , Microscopía Fluorescente/instrumentación , Imagen Individual de Molécula/instrumentación , Espermatocitos/ultraestructura , Complejo Sinaptonémico/ultraestructuraRESUMEN
Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.
Asunto(s)
Algoritmos , Aumento de la Imagen/instrumentación , Microscopía por Video/instrumentación , Imagen Molecular/instrumentación , Nanotecnología/instrumentación , Reconocimiento de Normas Patrones Automatizadas/métodos , Semiconductores , Diseño de Equipo , Análisis de Falla de Equipo , Procesamiento de Señales Asistido por Computador/instrumentaciónRESUMEN
Stimulated emission depletion (STED) microscopy achieves diffraction-unlimited resolution in far-field fluorescence microscopy well below 100 nm. As common for (single-lens) far-field microscopy techniques, the lateral resolution is better than the axial sectioning capabilities. Here we present the first implementation of total internal reflection (TIR) illumination into STED microscopy which limits fluorophore excitation to ~70 nm in the vicinity of the cover slip while simultaneously providing ~50 nm lateral resolution. We demonstrate the performance of this new microscope technique with fluorescent bead test samples as well as immuno-stained microtubules. Total internal reflection STED microscopy provides superior axial sectioning capabilities with the potential to reduce photo-bleaching and photo-damage in live cell imaging.