Virtual Nanoscopy: Ultra-high resolution electron microscopy with IIPImage

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Guest Post by Frank Faas & Raimond Ravelli, Department of Molecular Cell Biology, Leiden University Medical Center

The Leiden University Medical Centre in The Netherlands conducts a wide range of scientific research ranging from pure fundamental medical research to applied clinical research. Recently, our group in the Department of Molecular Cell Biology, published a paper entitled Virtual nanoscopy: Generation of ultra-large high resolution electron microscopy maps in the Journal of Cell Biology about a pipeline to acquire, combine and visualize electron microscopy images as panoramas of virtually unlimited size. The technique, named Virtual Nanoscopy, can reveal large macromolecules, organelles, cells, tissue, up to entire animal cross-sections.

Virtual slide of a sagittal section of a Zebrafish, imaged at 1.6 nm/pixel resolution over an area of 1.5 x 0.6 mm2

As a proof of principle we have put online a 281 gigapixel ultrastructural map of a 5 days old zebrafish embryo, which is a commonly used vertebrate model organism. The virtual slide was recorded at 120 kV with a magnification at the detector plane of 9460. A total of 26,434 unbinned 4k × 4k images was collected with a FEI Eagle CCD camera (>8 s readout time full frame) in 4.5 d. The sample was maintained at −1 µm defocus throughout the whole data collection. The resulting slide of 1,461 × 604 µm2 in size consists of 921,600 × 380,928 pixels of 1.6 nm square each.

The image shows the cartilage in slate blue, the eye in sienna, the brain in forest green, the muscles in salmon pink, the liver in indian red, the intestine in dark khaki, the pancreas in plum, the pronephric duct in yellow, the olfactory pit in lime green and the yolk in turquoise. Other examples can be found in the data section of our website.

Virtual nanoscopy has changed the way electron microscopy is carried out in our laboratory. Instead of collecting just a snapshot of part of a cell, we now routinely collect entire cross-sections which provides a much better context of the observed phenomena. Afterwards the data can be browsed as if behind a microscope at nanometer scale, hence the name virtual nanoscopy.

To visualise these images on the web we use the IIPImage server in combination with IIPMooviewer, both of which we adapted slightly to our needs. The images are stored as TIFF tiled image pyramids using the BigTIFF extensions available in libtiff 4.0 with a tile size of 256×256 pixels.

We can only recommend IIPImage and greatly appreciate the quick feedback from the developers. Many thanks to the developers of IIPImage for sharing their creation with the world!

by Frank Faas & Raimond Ravelli, Department of Molecular Cell Biology, Leiden University Medical Center

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