High-resolution real-time dual-view imaging with multiple point of view microscopy

Abstract

Most methods to observe three-dimensional processes in living samples are based on imaging a single plane that is sequentially scanned through the sample. Sequential scanning is inherently slow, which can make it difficult to capture objects moving quickly in three dimensions. Here we present a novel method, multiple point-of-view microscopy (MPoVM), that allows simultaneous capturing of the front and side views of a sample with high resolution. MPoVM can be implemented in most fluorescence microscopes, offering new opportunities in the study of dynamic biological processes in three dimensions. 2. R. Tomer, K. Khairy, F. Amat, and P. J. Keller, "Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy," Nat. Methods 9(7), 755-763 (2012). 3. U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, "Multiview light-sheet microscope for rapid in toto imaging," Nat. Methods 9(7), 730-733 (2012) imaging using single-objective SPIM," Nat. Methods 12(7), 641-644 (2015). 34. S.-I. Chang and J.-B. Yoon, "Shape-controlled, high fill-factor microlens arrays fabricated by a 3D diffuser lithography and plastic replication method," Opt. Express 12(25), 6366-6371 (2004).

Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

“…d) In tilted mirror‐based axial SRFM, the front view and side view of the sample could be acquired at the same time because of the existence of the tilted mirror. Reproduced with permission . Copyright 2016, Optical Society of America.…”

“…If this technique is used to observe a larger field of view, the images from different areas overlap, yielding decreased resolution. In addition, by using the refocusing technique rather than multi‐plane detection, it is also possible to obtain front and side views of the sample; this approach has been used to capture living C. elegans with super resolution under the condition of sparse labeling . In general, the sample position relative to the mirror during implementation is significant, as it may affect the resolution …”

“…Small field-of-view 3D SPL, SPT, SRI, Live-cell [272,273] Defocus-based axial SPT [286,288] Four-focus illumination feedback Point-scanning 90 LP…”

“…In addition, by using the refocusing technique rather than multi-plane detection, it is also possible to obtain front and side views of the sample; this approach has been used to capture living C. elegans with super resolution under the condition of sparse labeling. [273] In general, the sample position relative to the mirror during implementation is significant, as it may affect the resolution. [274] Mirror-based axial SRFM techniques have a common primary advantage, i.e., they have improved fluorescence collection efficiency and can therefore, be applied to photon-limited systems.…”

Reflective imaging improves resolution, speed, and collection efficiency in light sheet microscopy

“…Reflection has previously been used to introduce LSFM excitation to the sample, cleverly permitting the use of high-NA detection objectives in single-view imaging [9][10][11] . Although reflection has also been used in epifluorescence imaging to collect additional specimen views 12,13 (albeit without view fusion), we are unaware of any similar efforts in LSFM. We reasoned that if samples were placed on reflective substrates, and light-sheet illumination and detection optics introduced appropriately (e.g.…”