Journal: bioRxiv

Article Title: Automated 3D multi-color single-molecule localization microscopy

doi: 10.1101/2023.10.23.563122

Figure Lengend Snippet: The system features a custom microscope body (magenta), emission path with 3D and synchronous multi-channel/color imaging capabilities (yellow) and focus lock path (red). Multiple illumination sources are available: single-mode illumination path with fiber-coupled laser engine source (cyan) ii) single-mode booster laser (blue), multi-mode illumination path (green). The configuration shown includes all optional modules. For scale, the underlying optical breadboard measures 1.5 × 0.9 m.

Article Snippet: Others may have other preferences, for example, the maximum illumination field is a relatively modest 77 μm in line with comparable commercial systems (e.g., Nikon N-STORM, ONI NanoImager, Bruker Vutara, Zeiss Elyra), whereas several reports have described SMLMs, with larger fields [ , , ].

Techniques: Microscopy, Imaging

Journal: bioRxiv

Article Title: Automated 3D multi-color single-molecule localization microscopy

doi: 10.1101/2023.10.23.563122

Figure Lengend Snippet: A) Single-mode illumination path(s) (blue): The single-mode laser(s) are spatially filtered, expanded and homogenized. The resulting flat-top intensity profile is further expanded by exchangeable telescopes to control the size of the illumination field. Setting the angle for TIRF/HILO illumination is accomplished by positioning of a motorized mirror. The laser power is monitored in real time by measuring the <1% transmission of the laser lines at the illumination dichroic mirror. Emission path (green): A primary image is produced by a tube lens, restricted in spatial extent by a slit and relayed in a 4f configuration via a spectrally discriminative image splitter to form two secondary images on an sCMOS camera. A variable astigmatic lens (cylindrical lens pair) can be removed/inserted for 2D/3D imaging, while a Bertrand lens allows viewing of the objective back focal plane. Focus lock path (red): The focus lock laser is separated from emitted fluorescence by the emission dichroic mirror, focused to the objective back focal plane and launched obliquely to return under the TIRF condition allowing axial drift of the coverslip relative to the objective lens to be measured via a quadrant photodiode and corrected via the control loop of the piezo flexure stage mounting the objective. B) For clarity the multi-mode laser illumination path (orange) has been shown separately but all modules are co-compatible. A magnified image of the tip of a square core multimode fiber (coupled to a multi-mode laser engine) is produced and re-imaged onto the sample via a tube lens in a retrofocus arrangement and the objective lens. The multi-mode laser is coupled into the microscope by translation of the motorized mirror (HILO/TIRF) fully out of the beam path

Article Snippet: Others may have other preferences, for example, the maximum illumination field is a relatively modest 77 μm in line with comparable commercial systems (e.g., Nikon N-STORM, ONI NanoImager, Bruker Vutara, Zeiss Elyra), whereas several reports have described SMLMs, with larger fields [ , , ].

Techniques: Control, Transmission Assay, Produced, Imaging, Fluorescence, Microscopy

Journal: The Journal of Comparative Neurology

Article Title: Glucagon‐Like Peptide‐1 Targets in the Human Nodose Ganglion

doi: 10.1002/cne.70135

Figure Lengend Snippet: Lipofuscin autofluorescence artifact is reduced by high‐power illumination. Images were acquired using confocal microscopy (Zeiss LSM980) on samples that were not processed for RNAscope unless otherwise noted. (A–D) Native (unstained) tissue sections exhibited strong autofluorescence due to lipofuscin, a pigment that accumulates in neurons and appears as crescent‐shaped structures in the cytoplasm. Lipofuscin emitted across all excitation wavelengths. (E–H) Pretreatment with illumination using a TiYO system for 30 min prior to DAPI counterstaining reduced lipofuscin autofluorescence intensity, although some signal remained detectable. (I) Quantification of mean gray levels (± SEM, three samples per group) across channels showed that illumination effectively reduced autofluorescence in all wavelengths except the DAPI channel. (J) RNAscope detection of Snap25 mRNA (yellow puncta) clearly labeled neuronal cytoplasm. Residual lipofuscin was still visible under RNAscope signals as pale, crescent‐shaped structures (outlined with a dotted line), but its intensity remained lower than that of the RNAscope signal. Scale bar in (A) applies to all panels.

Article Snippet: To minimize lipofuscin autofluorescence commonly observed in aging sensory neurons (Sapio et al. ; Shiers et al. ), slides were exposed for 30 min to high‐power illumination using the TiYo quenching system (Nepa Gene Co. Ltd, Chiba, Japan).

Techniques: Confocal Microscopy, RNAscope, Labeling