custom total internal reflection fluorescence tirf microscope  (Nikon)

Journal: Nanophotonics

Article Title: Single-shot Stokes polarimetry of plasmon-coupled single-molecule fluorescence

doi: 10.1515/nanoph-2025-0352

Figure Lengend Snippet: DNA-PAINT experiment on single gold nanospheres. (a) Schematic of a gold nanosphere sample on a glass substrate excited with an epi-illumination fluorescence microscope. Fluorescent intensity time traces are recorded at 10 frames per second, showing bursts with an event typically lasting for 10 frames. Each fluorescent event, averaged over all frames above the threshold, provides a polarized PSF image. Four example events with the extracted α and DoLP indicate single-emitter binding to a particle at different locations. Scale bar of 200 nm applies to all PSFs. (b) Scatter plot of DoLP and α apparent for fluorescence events on six single nanospheres. (c) DoLP as a function of integrated fluorescence intensity showing that brighter events exhibit a higher DoLP because they occur at the equator of the particle.

Article Snippet: DNA-PAINT measurements were performed on an inverted total internal reflection fluorescence microscope (Nikon) with a 100×/NA 1.49 objective.

Techniques: Fluorescence, Microscopy, Binding Assay

Journal: Nature Communications

Article Title: Mechanism of Arp2/3 complex branch disassembly by human Coro7

doi: 10.1038/s41467-025-64787-z

Figure Lengend Snippet: a Time-lapse TIRF microscopy images of debranching events in the absence (left) or presence (right) of Coro7 FL. The flow rate is 200 µL min − 1 , corresponding to an average force of ~0.45 pN on an average ~2 µm-long branch . b Time courses of branch dissociation in the absence or presence of Coro7 FL. Solid lines through the data represent best fits to single (without Coro7 FL, black) or double (with Coro7 FL, blue shades) exponentials. c Debranching rate constants as a function of Coro7 FL concentration. The individual data points represent the observed debranching rate constants obtained from the best fits of the time courses shown in ( b ) to double exponentials. The teal and cyan horizontal lines represent the best global fits of the debranching time courses shown in ( b ) to a double exponential with a single rate constant for each phase (fast and slow). d Dependence of the fast-debranching phase amplitudes on Coro7 FL concentration. The blue circles represent the amplitudes obtained from individual fitting of the time courses shown in ( b ) to double exponentials, and the red circles represent the relative amplitudes obtained from global fitting. The solid line through the data represents the best fit to a rectangular hyperbola, yielding an apparent Coro7 FL binding affinity of 31 ± 1 nM for both individually and globally fitted parameters. e – g Time courses of branch dissociation in the absence or presence of various concentrations of Coro7 construct β2CA (solid lines indicate double-exponential fits) and constructs β1β2 and CA (solid lines indicate single-exponential fits). The graphs in ( b , e – g ) show averages; each condition was repeated two to four times, with an average of 43 fields of view, 113 mother filaments, and 155 branches analyzed per condition. In ( c and d ), the error bars represent standard deviation (SD) of the best-fit parameters from the time courses shown in ( b ), as calculated using Origin software. Source data are provided in the Source Data file.

Article Snippet: Direct visualization of actin filaments and branches was performed using a Nikon Eclipse Ti2 total internal reflection fluorescence (TIRF) microscope equipped with an SR HP Apo TIRF ×100 objective (Nikon) and an Andor iXon897 EMCCD camera.

Techniques: Microscopy, Concentration Assay, Binding Assay, Construct, Standard Deviation, Software