TY - JOUR
T1 - Construction of a three-color prism-based tirf microscope to study the interactions and dynamics of macromolecules
AU - Fairlamb, Max S.
AU - Whitaker, Amy M.
AU - Bain, Fletcher E.
AU - Spies, Maria
AU - Freudenthal, Bret D.
N1 - Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021/5/23
Y1 - 2021/5/23
N2 - Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope which can be utilized for the study of macromolecular complexes, including the multi-component protein–DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology.
AB - Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope which can be utilized for the study of macromolecular complexes, including the multi-component protein–DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology.
KW - Construction guide
KW - DNA repair complexes
KW - Prism
KW - Protein– DNA complexes
KW - Single-molecule FRET
KW - Single-molecule TIRF
KW - SmFRET
KW - Transcription complexes
UR - http://www.scopus.com/inward/record.url?scp=85109144541&partnerID=8YFLogxK
U2 - 10.3390/biology10070571
DO - 10.3390/biology10070571
M3 - Article
C2 - 34201434
AN - SCOPUS:85109144541
SN - 2079-7737
VL - 10
JO - Biology
JF - Biology
IS - 7
M1 - 571
ER -