PySTACHIO: Python Single-molecule TrAcking stoiCHiometry Intensity and simulatiOn, a flexible, extensible, beginner-friendly and optimized program for analysis of single-molecule microscopy data

Abstract

Graphical abstract

Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET

“…The two sensors are identical except for the fluorescent proteins used, with crGE2.3 making use of mEGFP and mScarletI as donor and acceptor respectively to make use of the greater single-protein intensities compared to mCerulean3 and mCitrine ( Mouton et al, 2020 ), with the CrGE2.3 thus being suitable for single-molecule tracking. Here we used our redeveloped single molecule tracking code PySTACHIO ( Shepherd, Higgins, et al, 2021b ) which performs two-channel tracking as well as colocalization analysis using overlap integrals alongside straightforward distance cutoffs as described in the Methods section. The localization was used to calculate the normalized fret NFRET which is possible for this FRET pair due to low spectral overlap and cross-excitation.…”

“…Data was analyzed using PySTACHIO ( Shepherd, Higgins, et al, 2021b ) with snr_min_threshold=0.5 and struct_disk_radius=9 in Alternating Laser Excitation (ALEX) mode. We relaxed our usual constraints on trajectory length because ALEX mode with 10 ms exposure allows considerable diffusion between successive captures in one channel and thus trajectory linking is compromised in this single-molecule regime.…”

“…The excitation intensity is strongly increased as a result compared to conventional epifluorescence excitation, up to ~100 times comparing to widefield image intensities, since the area targeted is smaller by approximatively a factor 10. This feature enables single-molecule detection at very high speeds, helped by precision localization through iterative Gaussian masking of detected bright spots ( Shepherd, Higgins, et al, 2021b ). Typically, a stack of images is acquired, first the intense illumination excites typically a large number of fluorophores.…”

“…We additionally present a new imaging method with combining crowding sensing with mCerulean3-mCitrine FRET sensor and FM4-64, a fluorescent dye that labels endolysosomal lipids ( Vida & Emr, 1995 ), allowing simultaneous visualization of the vacuole membrane. Finally, we present our most recent data in which we have successfully quantified single molecule FRET in vivo using the crGE2.3 crowding sensor and our super-resolution image analysis software suite PySTACHIO ( Shepherd, Higgins, et al, 2021a ).…”

MPTHub: An Open-Source Software for Characterizing the Transport of Particles in Biorelevant Media

“…While the ability to analyze motion at the single-particle level provides a valuable body of information that is not possible to attain with purely ensemble techniques, manual handling of generated large datasets constitutes a time-consuming and prone to error process [ 24 ]. Various available software has been shown useful in aiding this task [ 25 , 26 , 27 , 28 , 29 , 30 ]. However, these solutions offer limited possibilities for data processing customization, need intermediate to advanced computing skills for installing and use, or require payment of a license (self or that of an ancillary software such as MATLAB).…”