Abstract
BackgroundInnovative new techniques that aid in the visualization of microscopic anatomical structures have improved our understanding of organismal biology significantly. It is often challenging to observe internal 3D structures, despite the use of techniques such as confocal laser scanning microscopy (CLSM), micro-computed tomography (Micro-CT), magnetic resonance imaging (MRI), focused ion beam scanning electron microscopy (FIB-SEM) and others. In the current paper, we assess LED-SIM (DMD-based LED-illumination structured illumination microscopy), which facilitates the acquisition of nano- and micro-3D structures of small organisms in a high-resolution format (500 nm in the XY-plane and 930 nm along the Z-axis).ResultsWe compare other microstructural imaging techniques (involving conventional optical microscopy, CLSM and Micro-CT) with LED-SIM to assess the quality (e.g. resolution, penetration depth, etc.) of LED-SIM images, as well as to document the potential short-comings of LED-SIM. Based on these results we present an optimized set of protocols to ensure that LED-SIM arthropod and nematode samples with different cuticles or textures are prepared for analysis in an optimal manner. Six arthropod and nematode specimens were tested and shown to be suitable for LED-SIM imaging, which was found to yield high resolution 3D images.ConclusionsAlthough LED-SIM still must be thoroughly tested before it is widely accepted and the Z-axis resolution still requires improvement, this technique offers distinct high quality 3D images. LED-SIM can be highly effective and may provide high quality 3D images for zoological studies following the guidelines of sample preparation presented in the current paper.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-016-0158-9) contains supplementary material, which is available to authorized users.
Highlights
Innovative new techniques that aid in the visualization of microscopic anatomical structures have improved our understanding of organismal biology significantly
It derived from computed tomography (CT), which has been used as a medical diagnostic tool since the early 1970s [10, 11]
We found that LED-structured illumination microscopy (SIM) offered a distinct improvement over optical microscopes in terms of fast providing highquality 3D images, as well as advantages over other advanced methods due to its affordability and ease of use
Summary
Innovative new techniques that aid in the visualization of microscopic anatomical structures have improved our understanding of organismal biology significantly. It is often challenging to observe internal 3D structures, despite the use of techniques such as confocal laser scanning microscopy (CLSM), micro-computed tomography (Micro-CT), magnetic resonance imaging (MRI), focused ion beam scanning electron microscopy (FIB-SEM) and others. Non-optical strategies include micro-computed tomography (Micro-CT)/ nano-computed tomography (Nano-CT), magnetic resonance imaging (MRI), focus ion beam scanning electron microscopy (FIB-SEM)/serial block-face scanning electron microscopy (SBF-SEM) and so forth. Optical strategies involve confocal laser scanning microscopy (CLSM)/two-. With maximum spatial resolution of about 0.5 μm, it is routine to image hard and dry samples of approximately 1 ~ 200 mm in size. Other CT variants, such as Nano-CT, capable of finer resolution (approximately 0.1 μm) and faster scanning speed (generally less than 1 h, e.g., 10 ~ 30 min), the size of sample should be in hundreds of micrometers. Based on the principle of nuclear magnetic resonance (NMR), MRI is predominantly used to measure the distribution of hydrogen protons within a sample [12], which makes MRI a technique ideally suited
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