Communication to the Editor
Microfluidic Device for a Rapid Immobilization of Zebrafish Larvae in Environmental Scanning Electron Microscopy
Abstract Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip-based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold–palladium deposition. This process is inherently low-throughput and can introduce many analytical artifacts. This work describes a proof-of-concept microfluidic chip-based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user-friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab-on-a-chip device was fabricated using a high-speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip-based device within few minutes for ESEM imaging. VC 2014 International Society for Advancement of Cytometry
Key terms Danio rerio; environmental scanning electron microscopy; imaging; immobilization; lab-on-a-chip; larvae; microfluidics; Zebrafish
SMALL animal models, for example small vertebrate such as zebrafish (Danio rerio) and clawed African frog (Xenopus laevis), are widely used in biomedical, pharmacological and environmental studies (1). This popularity stems from the fact that both zebrafish and Xenopus provide some unique investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, and/or tissue samples (2–5). The main advantage is the potential for miniaturisation and automation of experiments, while still allowing for effects under study to occur in the context of intact physiological milieu, with all its intercellular, multiorgan, and multisystem interactions (6,7). Therefore, they bridge the existing gap between the traditional high-throughput cell-based in vitro assays and low-throughput rodent in vivo tests. To facilitate high-throughput studies using small model organisms, a new generation of miniaturized lab-on-a-chip (LOC) devices have recently been developed for both zebrafish and Xenopus embryo on-chip culture and pharmacological interrogation (1,6,7). Only recently, Akagi et al. reported on applications of microfluidic living embryo arrays for hydrodynamic trapping of single living zebrafish embryos and larvae (1,6–8). These specimens are intrinsically challenging to image due to immobilisation difficulties and rapid dislodgement related to high momentums of inertia. Despite a large body of evidence using optical imaging for the characterization of zebrafish and Xenopus embryos on chip-based devices, no attempts have been so far made to interface microfabricated chip-based technologies with scanning electron microscopy (SEM). Because the SEM imaging is based on electron scattering at the surface rather than electron transmission, the analysis is fast and large amounts of samples can be analysed with a
Grant sponsor: Australian Research Council DECRA, Grant number: DE130101046.
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)
Received 29 October 2014; Revision Received7 November 2014; Accepted 20 November 2014
DOI: 10.1002/cyto.a.22603
*Correspondence to: Donald Wlodkowic; School of Applied Sciences, RMIT University, Melbourne, VIC 3083, Australia. E-mail: [email protected]
Cytometry Part A 00: 00 00, 2014
C 2014 International Society for Advancement of Cytometry V
Communication to the Editor great depth of view (9,10). Such images are particularly appealing for small model organisms’ morphological analysis by providing detailed 3D surface topographical information and yielding more information than the flat or reconstructed 3D images usually produced by confocal imaging. Conventional SEM imaging requires, however, high vacuum environments, and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with, for example, gold–palladium deposition (9,10). This greatly impedes higher throughput phenotype-based analysis and screening of morphological features using SEM imaging. In contrast to conventional SEM, environmental scanning electron microscopy (ESEM) imaging is performed in in gaseous atmosphere under low vacuum mode (11). One of the main advantages of the modern ESEM is the opportunity to work on biological samples without complex and artefact generating manipulations such as staining (11,12). Gaseous/vapor detection enables images to be obtained in secondary (SE) mode. This signal is mixed with the backscattered electron image to provide strong material and morphological contrast (11). In principle, the resolution of the ESEM depends primarily on a gas pressure inside the chamber, accelerating voltage and electron beam current. With the reduction of pressure inside the chamber to very low vacuum level, ESEM imaging will become a typical SEM mode of operation. Very low vacuum is, however, disadvantageous as it will lead to rapid evaporation of water from biological tissues, thus negating any advantages of ESEM. The main disadvantage of ESEM relates to gas pressure required to maintain hydrated specimens and at such pressures undoubtedly a lower resolution, a lower quality of images and a lower magnification than the comparable SEM imaging is achieved (11,12). 3D images obtained on ESEM can, however, yield adequate information about surface topography that warrants its applications in 3D topographical screening regimens (11,12). To fully exploit ESEM capabilities and deploy this noteworthy technique in small model organisms phonotypical analysis, specimens must be quickly and efficiently immobilized without any extensive preparative procedures. Microfluidic technologies are particularly appealing for this purpose (1,6,7). However, for interfacing LOC devices with ESEM they are required to be not only small, rapid to use and user friendly, but also provide an open surface with no water surrounding the embryo or larvae (1,12). Here, we for the first time provide evidence that a chip-based technology can be successfully interfaced with ESEM imaging to provide rapid immobilisation of zebrafish larvae for highdefinition imaging of morphological features. With the assistance of this device, multiple zebrafish larvae can be aligned and immobilised in the chip for ESEM imaging. Most importantly larvae remain viable during processing and are well retained during imaging. Our innovative approach avoids extensive preparative procedures, is easy to perform for non-specialised personnel and thus prospectively amenable for future automation. 2
MATERIALS AND METHODS Chip fabrication was performed in transparent poly(methyl methacrylate) thermoplastic sheet (PMMA; Acrylic; PSP Plastics, Auckland, New Zealand) using a noncontact, 30 W CO2 laser cutting system (VLS 3.50, Universal Laser Systems, Scottsdale, AZ; 6). Wilde type zebrafish AB line (Zebrafish International Resource Centre, Eugene, OR) was used as described (6,13). Forty-eight hours postfertilization (hpf) embryos were dechorionated and exposed to 0.2 mg/mL Tricaine mesylate (SigmaAldrich, New Zealand) in buffered E3 medium (embryo E3 medium consisting of: 146 mg/L NaCl; 6.3 mg/L KCl; 24.3 mg/L CaCl2; 40.7 mg/L MgSO4) prior to ESEM image acquisitions to temporarily anaesthetize the larvae and inhibit the intrinsic body twitching movements (6,13). Embryos where exposed to the anesthetic for 20 min. For the imaging of fixed zebrafish larvae, whole larvae were fixed in 4% paraformaldehyde (PFA) dissolved in phosphate buffered solution (PBS) at 4 C overnight in a small tube. The fixed larvae were washed with PBS and cooled in a vial that contained 100% methanol at 220 C. Animal research was conducted with approval from The University of Auckland Animal Ethics Committee (approval ID R903). High-resolution ESEM images were captured under low vacuum mode according to the manufacturer’s instructions using Quanta 200 ESEM FEG system (FEI Company, Hillsboro, OR) as described earlier (12). The ESEM microscope was equipped with a high performance thermal emission SEM column with dual-anode source emission geometry. Resolution of ESEM has been adjusted at 4.0 nm using 20 kV acceleration and
Microfluidic Device for a Rapid Immobilization of Zebrafish Larvae in Environmental Scanning Electron Microscopy
Abstract Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip-based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold–palladium deposition. This process is inherently low-throughput and can introduce many analytical artifacts. This work describes a proof-of-concept microfluidic chip-based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user-friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab-on-a-chip device was fabricated using a high-speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip-based device within few minutes for ESEM imaging. VC 2014 International Society for Advancement of Cytometry
Key terms Danio rerio; environmental scanning electron microscopy; imaging; immobilization; lab-on-a-chip; larvae; microfluidics; Zebrafish
SMALL animal models, for example small vertebrate such as zebrafish (Danio rerio) and clawed African frog (Xenopus laevis), are widely used in biomedical, pharmacological and environmental studies (1). This popularity stems from the fact that both zebrafish and Xenopus provide some unique investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, and/or tissue samples (2–5). The main advantage is the potential for miniaturisation and automation of experiments, while still allowing for effects under study to occur in the context of intact physiological milieu, with all its intercellular, multiorgan, and multisystem interactions (6,7). Therefore, they bridge the existing gap between the traditional high-throughput cell-based in vitro assays and low-throughput rodent in vivo tests. To facilitate high-throughput studies using small model organisms, a new generation of miniaturized lab-on-a-chip (LOC) devices have recently been developed for both zebrafish and Xenopus embryo on-chip culture and pharmacological interrogation (1,6,7). Only recently, Akagi et al. reported on applications of microfluidic living embryo arrays for hydrodynamic trapping of single living zebrafish embryos and larvae (1,6–8). These specimens are intrinsically challenging to image due to immobilisation difficulties and rapid dislodgement related to high momentums of inertia. Despite a large body of evidence using optical imaging for the characterization of zebrafish and Xenopus embryos on chip-based devices, no attempts have been so far made to interface microfabricated chip-based technologies with scanning electron microscopy (SEM). Because the SEM imaging is based on electron scattering at the surface rather than electron transmission, the analysis is fast and large amounts of samples can be analysed with a
Grant sponsor: Australian Research Council DECRA, Grant number: DE130101046.
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)
Received 29 October 2014; Revision Received7 November 2014; Accepted 20 November 2014
DOI: 10.1002/cyto.a.22603
*Correspondence to: Donald Wlodkowic; School of Applied Sciences, RMIT University, Melbourne, VIC 3083, Australia. E-mail: [email protected]
Cytometry Part A 00: 00 00, 2014
C 2014 International Society for Advancement of Cytometry V
Communication to the Editor great depth of view (9,10). Such images are particularly appealing for small model organisms’ morphological analysis by providing detailed 3D surface topographical information and yielding more information than the flat or reconstructed 3D images usually produced by confocal imaging. Conventional SEM imaging requires, however, high vacuum environments, and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with, for example, gold–palladium deposition (9,10). This greatly impedes higher throughput phenotype-based analysis and screening of morphological features using SEM imaging. In contrast to conventional SEM, environmental scanning electron microscopy (ESEM) imaging is performed in in gaseous atmosphere under low vacuum mode (11). One of the main advantages of the modern ESEM is the opportunity to work on biological samples without complex and artefact generating manipulations such as staining (11,12). Gaseous/vapor detection enables images to be obtained in secondary (SE) mode. This signal is mixed with the backscattered electron image to provide strong material and morphological contrast (11). In principle, the resolution of the ESEM depends primarily on a gas pressure inside the chamber, accelerating voltage and electron beam current. With the reduction of pressure inside the chamber to very low vacuum level, ESEM imaging will become a typical SEM mode of operation. Very low vacuum is, however, disadvantageous as it will lead to rapid evaporation of water from biological tissues, thus negating any advantages of ESEM. The main disadvantage of ESEM relates to gas pressure required to maintain hydrated specimens and at such pressures undoubtedly a lower resolution, a lower quality of images and a lower magnification than the comparable SEM imaging is achieved (11,12). 3D images obtained on ESEM can, however, yield adequate information about surface topography that warrants its applications in 3D topographical screening regimens (11,12). To fully exploit ESEM capabilities and deploy this noteworthy technique in small model organisms phonotypical analysis, specimens must be quickly and efficiently immobilized without any extensive preparative procedures. Microfluidic technologies are particularly appealing for this purpose (1,6,7). However, for interfacing LOC devices with ESEM they are required to be not only small, rapid to use and user friendly, but also provide an open surface with no water surrounding the embryo or larvae (1,12). Here, we for the first time provide evidence that a chip-based technology can be successfully interfaced with ESEM imaging to provide rapid immobilisation of zebrafish larvae for highdefinition imaging of morphological features. With the assistance of this device, multiple zebrafish larvae can be aligned and immobilised in the chip for ESEM imaging. Most importantly larvae remain viable during processing and are well retained during imaging. Our innovative approach avoids extensive preparative procedures, is easy to perform for non-specialised personnel and thus prospectively amenable for future automation. 2
MATERIALS AND METHODS Chip fabrication was performed in transparent poly(methyl methacrylate) thermoplastic sheet (PMMA; Acrylic; PSP Plastics, Auckland, New Zealand) using a noncontact, 30 W CO2 laser cutting system (VLS 3.50, Universal Laser Systems, Scottsdale, AZ; 6). Wilde type zebrafish AB line (Zebrafish International Resource Centre, Eugene, OR) was used as described (6,13). Forty-eight hours postfertilization (hpf) embryos were dechorionated and exposed to 0.2 mg/mL Tricaine mesylate (SigmaAldrich, New Zealand) in buffered E3 medium (embryo E3 medium consisting of: 146 mg/L NaCl; 6.3 mg/L KCl; 24.3 mg/L CaCl2; 40.7 mg/L MgSO4) prior to ESEM image acquisitions to temporarily anaesthetize the larvae and inhibit the intrinsic body twitching movements (6,13). Embryos where exposed to the anesthetic for 20 min. For the imaging of fixed zebrafish larvae, whole larvae were fixed in 4% paraformaldehyde (PFA) dissolved in phosphate buffered solution (PBS) at 4 C overnight in a small tube. The fixed larvae were washed with PBS and cooled in a vial that contained 100% methanol at 220 C. Animal research was conducted with approval from The University of Auckland Animal Ethics Committee (approval ID R903). High-resolution ESEM images were captured under low vacuum mode according to the manufacturer’s instructions using Quanta 200 ESEM FEG system (FEI Company, Hillsboro, OR) as described earlier (12). The ESEM microscope was equipped with a high performance thermal emission SEM column with dual-anode source emission geometry. Resolution of ESEM has been adjusted at 4.0 nm using 20 kV acceleration and
