Three-Dimensional Printing of Super-Resolution Microscopy Images
Emily M. Mace , 1 * Jesse Moon , 2 and Jordan S. Orange 1 1 Center for Human Immunobiology , Texas Children’s Hospital and Baylor College of Medicine , Houston , TX 77030 2 Freelance Industrial Designer , Austin , TX
*
mace@bcm.edu Introduction
Human beings are tactile creatures, and the ability to handle and observe an object from multiple dimensions is a powerful tool for us. As microscopists we oſt en extract quan- titative data from the reconstructions of cellular structures observed in micrographs. Within the past 200 years, technology has exploded at such a rate that we now have the ability to visualize subcellular features with resolution that is no longer limited by the diffraction barrier of light, and we are able to resolve complex structures, sometimes with double-digit nanometer resolution. In addition, time-resolved imaging in three dimensions is increasingly feasible and informative.
Recently the commercialization of desktop three- dimensional (3D) printers has provided opportunities for institutions and businesses to exploit this technology. It is now in the hands of scientists and medical doctors for use in the bioengineering and medical fi elds. Cell biology is no exception. With the ability to model cellular structures, we are able to more fully enter the world that holds us so fascinated. While the uses of 3D printing and nanofabrication for bioengi- neering purposes are extensive, this article will specifi cally describe the use of 3D printing to model non-diff raction- limited stimulated emission depletion (STED) microscopy images [ 1 , 2 ].
Materials and Methods Printers . 3D printers are available in a range of prices and
features. T ese are dictated in part by the technologies used: stereolithography, fused deposition modeling, selective laser sintering, selective laser melting, electron beam melting, and laminated object manufacturing (a comprehensive review of 3D printing technology can be found at http://3dprinting
industry.com/3d-printing-basics-free-beginners-guide/ technology ). Most of the entry- level consumer models are based on the fused deposition model- ing principle, in which thermo- plastics are melted and deposited using Cartesian coordinate ( X , Y , Z ) driven motors to convert a 3D computer graphics model into a printed object. In this case we have
used a MakerBot® Replicator 2X, in which the XY stage is fi xed and the extruders move in the XYZ plane to deposit fi lament. T is requires the rendering of the 3D image into a suitable fi le format
26
and subsequently importing it into the MakerBot soſt ware (Makerware), followed by printing with acrylonitrile butadiene styrene (ABS) fi lament. Natural killer cells . Our purpose was to model the F-actin cytoskeleton of natural killer (NK) cells [ 3 ]. NK cells are innate immune effector cells that recognize and kill virally infected and tumorigenic targets. As such, they are a critical component of human host defense [ 4 ]. Their deadly yet specific function relies on the directed secretion of specialized lysosomes, termed lytic granules, which contain perforin and granzymes. These lytic granules, which range in size from 100–750 nm, are secreted through clearances in a pervasive F-actin network at the immunological synapse formed between the NK cell and its target [ 5 , 6 ]. The regulation of F-actin is an important area of interest in the cell biology of this specialized type of secretion. In order to study the structure and regulation of F-actin, we used a technique in which we activated NK cells on glass coated with antibody to activating receptors (NKp30 and CD18), which induces F-actin rearrangement and ultimately lytic granule degranulation. Cells were fixed, permeabilized, and stained with phalloidin Alexa Fluor 488 to visualize the F-actin. STED microscopy was done on a Leica SP8 STED, with tunable pulsed white light laser and HyD detectors. Images were acquired using LASAF software and deconvolved using Huygens software (Scientific Volume Imaging), which resulted in a final resolution of 40 nm in the XY planes and ~200 nm in the Z plane.
Results
Following acquisition of the microscopy image and deconvolution with Huygens, we imported the 3D image as .ICS and .IDS fi les to Imaris 7.4 (Bitplane). In Imaris, we used the sur- faces feature to convert the raw data to a computer-generated
Figure 1 : Surface rendering of a STED microscopy image of F-actin in a human NK cell. Images were acquired in XYZ on a Leica SP8 STED microscope and subsequently exported to Huygens software for deconvolution (A). Following deconvolution, images were surface rendered in Imaris (Bitplane) (B, C). Scale bar=5 μ m.
doi: 10.1017/S1551929515000607
www.microscopy-today.com • 2015 July
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