“衍射极限”实际上不是一个真正的障碍,除非处理远场和定位精度。这种衍射障碍并不是坚不可摧的,可以利用一些智能技术来突破光学衍射极限。讨论了四种技术,近场扫描光学显微镜(NSOM)法,受激发射损耗(STED)显微镜法,光激活定位显微镜(PALM)法或随机光学重建显微镜(STORM)法和结构照明显微镜(SIM)法,并且介绍了各自的基本原则与优劣。NSOM利用纳米级探测器检测通过光纤的极小汇聚光斑,从而获得单个像素的分辨率;PALM和STORM利用荧光探针,实现暗场和荧光的转换,从而观察到极小的荧光团;SIM则是利用栅格图案与样品叠加成像来实现。其中,STORM具有相对较高的潜力,能够更为有效地突破衍射极限。

Notorious term 'diffraction limit' is not actually a true barrier unless we are dealing with far field and localization precision.This diffraction barrier is not impenetrable and can be broken with some intelligent techniques.We discuss here four powerful techniques,near-field scanning optical microscopy(NSOM),stimulated emission depletion(STED) microscopy,photo-activated localization microscopy(PALM) & stochastic optical reconstruction microscopy(STORM) and structured illumination microscopy(SIM),along with their underlying principles together with pros and cons.NSOM uses a nanometer scale detector or source which compels the light to pass through the tiny tip of a fiber while keeping the distance between the tip and sample less than λ.At any given moment in a STED microscope,laser light is focused into a small spot by the objective and as a result,all fluorophores within this focused spot radiate fluorescence,which is then gathered by the objective and headed to the detector where it forms a single pixel.Fluorescent probes are employed by STORM/PALM,which are able to toggle between dark states and fluorescent so that with every snapshot taken,only a tiny,optically resolvable portion of the fluorophores is observed.Structured illumination is a wide field technique in which a grid pattern is produced by the interference of diffraction orders which are superimposed on the sample while taking images.STORM has the relatively high potential to effectively break the conventional diffraction barrier with fewer hurdles.