Abstract
Multiple signal classification algorithm (MUSICAL) exploits temporal fluctuations in fluorescence intensity to perform super-resolution microscopy by computing the value of a super-resolving indicator function across a fine sample grid. A key step in the algorithm is the separation of the measurements into signal and noise subspaces, based on a single user-specified parameter called the threshold. The resulting image is strongly sensitive to this parameter and the subjectivity arising from multiple practical factors makes it difficult to determine the right rule of selection. We address this issue by proposing soft thresholding schemes derived from a new generalized framework for indicator function design. We show that the new schemes significantly alleviate the subjectivity and sensitivity of hard thresholding while retaining the super-resolution ability. We also evaluate the trade-off between resolution and contrast and the out-of-focus light rejection using the various indicator functions. Through this, we create significant new insights into the use and further optimization of MUSICAL for a wide range of practical scenarios.
Highlights
Conventional optical microscopy is limited in resolution due to diffraction of light
A stack of images taken of such sample from a diffraction-limited system over T time-steps is expressed in a matrix form as A = [a(1) . . . a(T)], where each column vector a(t) contains the intensity measured by a set of sensing elements at time step t
Sn(t) is the number of photons produced by the nth emitter during the time step t, which is a random variable resulting from the photokinetics of the fluorophore
Summary
Conventional optical microscopy is limited in resolution due to diffraction of light. The need to overcome this limit has given rise to super-resolution microscopy techniques, called optical nanoscopy Among these techniques, structured illumination microscopy (SIM) [1] allows a lateral resolution enhancement by a factor of 2 over the optical resolution limit, stimulated emission depletion (STED) microscopy [2] and single molecule localization (SML) [3,4,5] can achieve resolutions close to 20 nm, and MINFLUX [6] which combines concepts of SML and STED to achieve even 2 nm resolution. Every single image is a linear combination of the columns of G, weighted by the photon emissions of emitters
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
