The photo-kinetics of fluorescent molecules have enabled the circumvention of the far-field optical diffraction limit.Despite its enormous potential,the necessity to label the sample may adversely influence the delica...The photo-kinetics of fluorescent molecules have enabled the circumvention of the far-field optical diffraction limit.Despite its enormous potential,the necessity to label the sample may adversely influence the delicate biology under investigation.Thus,continued development efforts are needed to surpass the far-field label-free diffraction barrier.The statistical similarity or finite coherence of the scattered light off the sample in label-free mode hinders the application of existing super-resolution methods based on incoherent fluorescence imaging.In this article,we present physics and propose a methodology to circumvent this challenge by exploiting the photoluminescence(PL)of silicon nitride waveguides for near-field illumination of unlabeled samples.The technique is abbreviated EPSLON,Evanescently decaying Photoluminescence Scattering enables Label-free Optical Nanoscopy.We demonstrate that such an illumination has properties that mimic the photo-kinetics of nano-sized fluorescent molecules,i.e.,such an illumination permits incoherence between the scattered fields from various locations on the sample plane.Thus,the illumination scheme enables the development of a far-field label-free incoherent imaging system that is linear in intensity and stable over time,thereby permitting the application of techniques like structured illumination microscopy(SIM)and intensity-fluctuation-based optical nanoscopy(IFON)in label-free mode to circumvent the diffraction limit.In this proof-of-concept work,we observed a two-point resolution of~180 nm on super-resolved nanobeads and resolution improvements between 1.9×to 2.8×over the diffraction limit,as quantified using Fourier Ring Correlation(FRC),on various biological samples.We believe EPSLON is a step forward within the field of incoherent far-field label-free super-resolution microscopy that holds a key to investigating biological systems in their natural state without the need for exogenous labels.展开更多
文摘The photo-kinetics of fluorescent molecules have enabled the circumvention of the far-field optical diffraction limit.Despite its enormous potential,the necessity to label the sample may adversely influence the delicate biology under investigation.Thus,continued development efforts are needed to surpass the far-field label-free diffraction barrier.The statistical similarity or finite coherence of the scattered light off the sample in label-free mode hinders the application of existing super-resolution methods based on incoherent fluorescence imaging.In this article,we present physics and propose a methodology to circumvent this challenge by exploiting the photoluminescence(PL)of silicon nitride waveguides for near-field illumination of unlabeled samples.The technique is abbreviated EPSLON,Evanescently decaying Photoluminescence Scattering enables Label-free Optical Nanoscopy.We demonstrate that such an illumination has properties that mimic the photo-kinetics of nano-sized fluorescent molecules,i.e.,such an illumination permits incoherence between the scattered fields from various locations on the sample plane.Thus,the illumination scheme enables the development of a far-field label-free incoherent imaging system that is linear in intensity and stable over time,thereby permitting the application of techniques like structured illumination microscopy(SIM)and intensity-fluctuation-based optical nanoscopy(IFON)in label-free mode to circumvent the diffraction limit.In this proof-of-concept work,we observed a two-point resolution of~180 nm on super-resolved nanobeads and resolution improvements between 1.9×to 2.8×over the diffraction limit,as quantified using Fourier Ring Correlation(FRC),on various biological samples.We believe EPSLON is a step forward within the field of incoherent far-field label-free super-resolution microscopy that holds a key to investigating biological systems in their natural state without the need for exogenous labels.
