Artifact‐free objective‐type multicolor total internal reflection fluorescence microscopy with light‐emitting diode light sources—Part I

Abstract

Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective-type or prism-less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light-emitting diode (LED)-based implementation of objective-type TIRF (LED-TIRF). The proposed LED-TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF-epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED-TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non-coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser-TIRF. Unlike azimuthal spinning laser-TIRF, LED-TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short-lived, localized translocation events of a Ca 2+ -sensitive protein kinase C α fusion protein are demonstrated. K E Y W O R D Slight-emitting diodes, multicolor cell imaging, objective-type prismless TIRF microscopy, protein kinase C translocation, total internal reflection fluorescence excitation

Theoretical background of light‐emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane‐associated proteins—Part II

“…A photograph of such a LED‐TIRF illuminator along with a scheme of the beampath is given in Figure . Several extensions of this beampath, including combination of LED with various wavelengths, combination of epifluorescence excitation into the beampath have been described . To control and change the range of applied AOI, a zoomable option of the second projection can be applied.…”

“…It facilitates axial alignment of the system, and ensures a telecentric BFP illumination. The assembled and preadjusted rail was mounted onto a joystick‐controlled, motorized three‐axis translation stage (Eppendorf 5171, Hamburg, Germany), and adjusted as described earlier .…”

KS0365, a novel activator of the transient receptor potential vanilloid 3 (TRPV3) channel, accelerates keratinocyte migration

“…To assess the localization of TRPV3 in keratinocytes, stably transfected m308k mTRPV3‐YFP cells were plated on poly‐ l ‐lysine‐coated (0.02 mg·ml −1 ) glass coverslips and imaged after 48 h at 50%–60% confluence using a custom‐made light‐emitting diode (LED)‐TIRF microscope as previously described by Kogel et al (2019). Ten minutes before imaging, cell nuclei of m308k mTRPV3‐YFP cells were counterstained using Hoechst33342 (10 mg·ml −1 stock solution in H 2 O) diluted 1:2000 in HBS.…”

“…To assess the localization of TRPV3 in keratinocytes, stably transfected m308k mTRPV3-YFP cells were plated on poly-L-lysine-coated (0.02 mgÁml À1 ) glass coverslips and imaged after 48 h at 50%-60% confluence using a custom-made light-emitting diode (LED)-TIRF microscope as previously described by Kogel et al (2019). Ten minutes before…”

Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

“…To significantly improve the optical stability of the platform, we devised a fabrication process for an advanced microstructure of the nanolitre well array, using a layer of amorphous fluorocarbon (AF) polymer for refractive index coordination, which was sandwiched with a glass substrate and microfabricated polydimethylsiloxane (PDMS). This structure allowed the use of TIRF illumination with incoherent light-emitting diodes (LED-TIRF) 34,35 in the LCI-S platform, ensuring the detection of sufficient fluorescence signal from the target at small incident light angles, while reducing the background signal from leakage light (Fig. 1c, d).…”