Extending Whole Slide Imaging: Color Darkfield Internal Reflection Illumination (DIRI) for Biological Applications

RESEARCH ARTICLE Extending Whole Slide Imaging: Color Darkfield Internal Reflection Illumination (DIRI) for Biological Applications Yoshihiro Kawano1,2*, Kana Namiki3, Atsushi Miyawaki3, Takuji Ishikawa1,4 1 The Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Miyagi, Japan, 2 Olympus Corporation, Shinjuku-Ku, Tokyo, Japan, 3 Cell Function & Dynamics, Brain Science Institute, RIKEN, Wako, Saitama, Japan, 4 Department of Finemechanics, Tohoku University, Sendai, Miyagi, Japan a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Kawano Y, Namiki K, Miyawaki A, Ishikawa T (2017) Extending Whole Slide Imaging: Color Darkfield Internal Reflection Illumination (DIRI) for Biological Applications. PLoS ONE 12(1): e0167774. doi:10.1371/journal.pone.0167774 Editor: Constantino Carlos Reyes-Aldasoro, City University London, UNITED KINGDOM Received: July 6, 2016 Accepted: November 21, 2016 * Abstract Whole slide imaging (WSI) is a useful tool for multi-modal imaging, and in our work, we have often combined WSI with darkfield microscopy. However, traditional darkfield microscopy cannot use a single condenser to support high- and low-numerical-aperture objectives, which limits the modality of WSI. To overcome this limitation, we previously developed a darkfield internal reflection illumination (DIRI) microscope using white light-emitting diodes (LEDs). Although the developed DIRI is useful for biological applications, substantial problems remain to be resolved. In this study, we propose a novel illumination technique called color DIRI. The use of three-color LEDs dramatically improves the capability of the system, such that color DIRI (1) enables optimization of the illumination color; (2) can be combined with an oil objective lens; (3) can produce fluorescence excitation illumination; (4) can adjust the wavelength of light to avoid cell damage or reactions; and (5) can be used as a photostimulator. These results clearly illustrate that the proposed color DIRI can significantly extend WSI modalities for biological applications. Published: January 13, 2017 Copyright: © 2017 Kawano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by a Grant-inAid for Scientific Research (A), No. 26242039 (http://www.jsps.go.jp/english/e-grants/index. html). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Olympus Corporation provided several microscope parts, but did not fund this study nor influence its outcome. Introduction The complexity of the human body makes it difficult to understand. For example, it consists of between 50 and 75 trillion cells, and its genes are capable of creating over 20,000 proteins. Optical microscopy is the main tool used to investigate the structure and functions of cells, tissues, and organs in the human body. Scientists have a wide variety of imaging techniques and tools at their disposal to better understand complicated biological environments. Whole slide imaging (WSI) or so call vertual slide technology is one of the core imaging techniques, which enables automated imaging and allows scientists to observe large samples. Microscopes performing WSI can easily be connected to the Internet, raising the prospect of Internet of Things applications. The observation area of WSI is not limited to the field of view of an objective lens [1–5]. PLOS ONE | DOI:10.1371/journal.pone.0167774 January 13, 2017 1 / 18 Color Darkfield Internal Reflection Illumination (DIRI) for Biological Applications Competing Interests: Brendan Brinkman and Yoshihiro Kawano are employed by a commercial company: Olympus Corporation. Lorne D. Davies, Olympus America Inc. and Kawano have filed a US patent application entitled: WIDE FIELD MICROSCOPIC IMAGING SYSTEM AND METHOD (publication number US2012/0092477 published on April 19, 2012, date of filing October 18, 2010). This does not alter our adherence to PLOS ONE policies on sharing data and materials. Darkfield microscopy combined with WSI is often used to observe the details of tissue and cell structures. The main advantage of darkfield microscopy is that unstained samples can be observed due to a mismatch in the refractive index, which generates image contrast, although staining with an appropriate color is often required [6]. The typical set-up of transmitted darkfield illumination consists of a halogen burner bulb with either a central stop for darkfield in a standard brightfield condenser or a dedicated substage darkfield condenser. Central axial illumination from the halogen burner bulb is blocked by the darkfield central stop. Peripheral illumination obliquely illuminates a specimen contained on a slide from the bottom. Thus, only forward-scattered light or refracted light from the specimen enters the objective lens (Fig 1a), which creates darkfield images. We show the advantages of darkfield microscopy using real samples. Fig 1b shows a brightfield image of cheek cells using UPlanSApo 60× oil, numerical aperture (NA) 1.35 (Olympus, Tokyo, Japan), with an extended magnification lens of 2.5×. The human cheek cell (epithelial cell) sample was freshly taken using Q-chip and diluted in phosphate-buffered solution (PBS). The image contrast was low, making it difficult to observe the cells. A darkfield image of the cheek cells is shown in Fig 1c; in this image, the background is very dark, which results in the granules inside the cells being clearly resolved. Conventional darkfield units are commercially available for a standard microscope. There are two types of darkfield condensers: dry and oil. A dry darkfield condenser cannot be used with objective lenses that have a NA > 0.8, because the illumination cone passes directly through the objective lens and eliminates the dark background of the image. An oil darkfield condenser, on the other hand, can accept such a high-NA lens. However, if a user needs to switch from an oil lens to a dry lens, the condenser also must be switched back to the dry darkfield condenser. This necessitates the disruptive and somewhat laborious process of removing the oil from the slide, which is incompatible with automated WSI. To overcome this problem, we previously developed darkfield internal reflection illumination (DIRI) with white light-emitting diodes (LEDs) [7, 8]. DIRI eliminates sources of light from the top and bottom of a sample using a side-illuminated darkfield, which is an improved version of a Hausmann’s darkfield illuminator [9], occasionally used for brain imaging [10–13]. Although the developed DIRI is useful [7, 8], it has substan (...truncated)