The smfBox is an open-source platform for single-molecule FRET

Abstract

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET’s broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules

“…Here, we focus on the assembly of a prismTIRF microscope. Resources regarding the construction of an objective-based TIRF configuration [1,7,15,16], including the cost-effective smfBox [8] which is capable of single-molecule Förster resonance energy transfer (smFRET) experimentation, are available at the associated references.…”

“…Prism-based single-molecule total internal reflection fluorescence (prismTIRF) microscopy is becoming ever-more popular due to its wide variety of applications. Multiple publications are available which outline the various capabilities of prismTIRF microscopes [1,2], the rationale behind fluorophore selection [3][4][5], the general assembly of a two-color prismTIRF microscope [6][7][8], and the basics of how to perform such experiments [3,[9][10][11][12]. Although two-color TIRF microscopes are powerful tools, three-color TIRF microscopes allow more experimental flexibility by allowing users to perform three different combinations of dual fluorophore experiments.…”

Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules

“…We focus here on the assembly of a prismTIRF microscope (figure 1). Resources regarding the construction of an objective-based TIRF configuration [1,7,15], including the cost-effective smfBox [8] which is capable of single-molecule Förster Resonance Energy Transfer (smFRET) experimentation, are available at the associated references.…”

“…Prism-based single-molecule total internal reflection fluorescence (prismTIRF) microscopy is becoming ever-more popular due to its wide variety of applications. Multiple publications are available which outline the various capabilities of prismTIRF microscopes [1,2], the rationale behind fluorophore selection [3][4][5], the general assembly of a 2color prismTIRF microscope [6][7][8], and the basics of how to perform such experiments [3,[9][10][11][12]. However, even with the currently available guides, building and operating a prismTIRF microscope without expert assistance can be a challenge.…”

Single molecule imaging of protein aggregation in Dementia: Methods, insights and prospects

“…The distinct advantage of FCS over SMLM is its ability to quantify molecular dynamics in real-time and in solution or inside the tubular and lamellar organelles of the cell ( J R et al, 2009 ; Unsay et al, 2018 ) and using a wide array of labelling and immunostaining techniques. Combined with its cost-effectiveness and ease of assembly ( Ambrose et al, 2020 ), confocal FCS is a viable solution for answering some questions related to the kinetics of aggregation.…”