Low Efficiency Upconversion Nanoparticles for High-Resolution Coalignment of Near-Infrared and Visible Light Paths on a Light Microscope

Abstract

The combination of near infrared (NIR) and visible wavelengths in light microscopy for biological studies is increasingly common. For example, many fields of biology are developing the use of NIR for optogenetics, in which an NIR laser induces a change in gene expression and/or protein function. One major technical barrier in working with both NIR and visible light on an optical microscope is obtaining their precise coalignment at the imaging plane position. Photon upconverting particles (UCPs) can bridge this gap as they are excited by NIR light but emit in the visible range via an anti-Stokes luminescence mechanism. Here, two different UCPs have been identified, high-efficiency micro540-UCPs and lower efficiency nano545-UCPs, that respond to NIR light and emit visible light with high photostability even at very high NIR power densities (>25,000 Suns). Both of these UCPs can be rapidly and reversibly excited by visible and NIR light and emit light at visible wavelengths detectable with standard emission settings used for Green Fluorescent Protein (GFP), a commonly used genetically-encoded fluorophore. However, the high efficiency micro540-UCPs were suboptimal for NIR and visible light coalignment, due to their larger size and spatial broadening from particle-to-particle energy transfer consistent with a long lived excited state and saturated power dependence. In contrast, the lower efficiency nano-UCPs were superior for precise coalignment of the NIR beam with the visible light path (~2 µm versus ~8 µm beam broadening respectively) consistent with limited particle-to-particle energy transfer, superlinear power dependence for emission, and much smaller particle size. Furthermore, the nano-UCPs were superior to a traditional two-camera method for NIR and visible light path alignment in an in vivo Infrared-Laser-Evoked Gene Operator (IR-LEGO) optogenetics assay in the budding yeast S. cerevisiae. In summary, nano-UCPs are powerful new tools for coaligning NIR and visible light paths on a light microscope.

Functional midbody assembly in the absence of a central spindle

“…Embryos in Fig. S4 C were imaged on an inverted microscope (Ti; Nikon) using a spinning-disk confocal unit (CSU-10; Yokogawa) with Borealis (Spectral Applied Research), two 150-mW excitation lasers at 491 and 561 nm (Cairn), and an emission filter wheel (Sutter Instruments) with bandpass filters corresponding to 525/50 nm and 620/50 nm (Chroma) as described previously ( Hirsch et al, 2018 ; Sundaramoorthy et al, 2017 ).…”

Patterning, regulation, and role of FoxO/DAF-16 in the early embryo

“…For experiments in Figure S3A , images were acquired on an inverted Nikon Eclipse Ti microscope (modified for compatibility with near-infrared light, as in (Hirsch et al, 2018; Sundaramoorthy et al, 2017)). The microscope was equipped with a spinning disc confocal unit (Yokogawa, CSU-10 with Borealis upgrade (Spectral Applied Research)), an Orca-R2 CCD camera (Hamamatsu Photonics), and a Piezo-driven (ASI) motorized stage for Z-sectioning.…”

FLIRT: fast local infrared thermogenetics for subcellular control of protein function

“…During all FLIRT experiments, focus was maintained relative to the coverslip using the CRISP auto focus system (Applied Scientific Instrumentation) before each image acquisition. Sunstone upconverting nanoparticles (also called Sunstone UCPs or nano 545 -UCPs, Sigma Aldrich #90992) mounted in H 2 O between a slide and coverslip were used for IR laser co-alignment with the visible light image path at the beginning of each experimental session as previously described 21 .…”

“…The IR laser is focused at the beginning of each imaging session using photon upconverting nanoparticles (UCPs) (Supplementary Figure 1b), which are excited by IR light but emit visible light at wavelengths similar to commonly used fluorophores ( e.g. GFP) via a 3-photon anti-Stokes luminescence mechanism, allowing precise focus of the laser-targeted region onto the specimen plane 8 . A microfluidic temperature control system maintains specimen temperature and functions as a heat sink.…”