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Abstract Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.
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Autofocusing technologies for whole slide imaging and automated microscopy
Abstract Whole slide imaging (WSI) has moved digital pathology closer to diagnostic practice in recent years. Due to the inherent tissue topography variability, accurate autofocusing remains a critical challenge for WSI and automated microscopy systems. The traditional focus map surveying method is limited in its ability to acquire a high degree of focus points while still maintaining high throughput. Real‐time approaches decouple image acquisition from focusing, thus allowing for rapid scanning while maintaining continuous accurate focus. This work reviews the traditional focus map approach and discusses the choice of focus measure for focal plane determination. It also discusses various real‐time autofocusing approaches including reflective‐based triangulation, confocal pinhole detection, low‐coherence interferometry, tilted sensor approach, independent dual sensor scanning, beam splitter array, phase detection, dual‐LED illumination and deep‐learning approaches. The technical concepts, merits and limitations of these methods are explained and compared to those of a traditional WSI system. This review may provide new insights for the development of high‐throughput automated microscopy imaging systems that can be made broadly available and utilizable without loss of capacity.
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- PAR ID:
- 10379576
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Journal of Biophotonics
- Volume:
- 13
- Issue:
- 12
- ISSN:
- 1864-063X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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