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New light microscopy techniques with esoteric names such as MERFISH, STORM, ORCA, OPS, and SIM are rapidly emerging. Each one has its own technical refinements and application niche. It is perhaps surprising then that just two multiline laser illuminators, Lumencor’s ZIVA and CELESTA Light Engines, can fulfill the lighting requirements of all of these demanding techniques.
Dr. Bogdan Bintu at the University of California San Diego is well-placed to make this assessment, both from his current research in applying MERFISH to study neurogenesis in aged mammalian brains (Figure 1) and his previous experience in Dr. Xiaowei Zhuang’s laboratory at Harvard University.
Light microscopy requires an illuminator. For modern “alphabet soup” microscopy designed to provide high-resolution spatial mapping of multiple RNA, DNA, or proteins over large fields of view, that source is an array of lasers with discrete outputs. The temporal and spatial coordination of the laser outputs is critical to the end results. ZIVA and CELESTA Light Engines generate light of various wavelengths which remain spatially and temporally consistent. They offer a compact, integrated, turnkey box, providing the robust, stable, and maintenance-free illumination necessary to foster routine data acquisition that can proliferate to many end users.
STORM (stochastic optical reconstruction microscopy), invented in Xiaowei Zhuang’s Harvard University laboratory, is a super-resolution microscopy technique enabling resolution of objects below the diffraction limit of 200 nm. High irradiance illumination drives stochastic activation of fluorescent molecules to spatially distinguish them from their transiently dark neighbors. Bintu and colleagues implement STORM imaging using a CELESTA Light Engine for fields of view of up to 100 µm x 100 µm. Although the nuances of super-resolution microscopy may not be self-evident, the new insights offered to investigators certainly are (Figure 2).
Like STORM, MERFISH also originated in Zhuang’s laboratory. MERFISH (multiplexed error-robust fluorescence in situ hybridization) is a massively multiplexed single-molecule imaging technique. It is capable of measuring the copy number and spatial distribution of hundreds to thousands of target RNA transcripts in single cells [2]. In turn, single-cell transcriptomic characterization allows in situ identification and spatial mapping of cells in complex tissues (Figure 1). MERFISH requires high-intensity, spatially uniform light matched to typical sCMOS camera sensor dimensions. CELESTA Light Engine can provide 1,000–10,000 mW/mm2 at the sample plane. 477 nm, 637 nm, and 748 nm lasers identify DNA probes hybridized to RNA transcripts; 405 nm and 545 nm outputs illuminate DAPI-stained nuclei.
Only with bright, stable illumination which embodies spatial and temporal robustness can the capabilities and impact of the newest light microscopy techniques be realized. Pushing past traditional imaging hardware limitations and offering the highly multiplexed mapping capabilities required for spatial resolution techniques demands bright, well-behaved light. Turnkey Light Engines are a critical component in pushing past historical diffraction limitations to enable such groundbreaking imaging.
For additional information: lumencor.com.
