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Method Article
Anne L van de Ven
The Methodist Hospital Research Institute, 6670 Bertner Ave, Houston, TX 77030
Corresponding Author
Seok Hyun Yun
Wellman Center for Photomedicine and Harvard Medical School, Massachusetts General Hospital, 40 Blossom St, Boston, MA 02114
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Intravital microscopy is emerging as an important experimental tool for the research and development of multi-functional therapeutic nanoconstructs. The direct visualization of nanoparticle dynamics within live animals provides invaluable insights into the mechanisms that regulate nanotherapeutics transport and cell-particle interactions. Here we present a protocol to image the dynamics of nanoparticles within the liver and tumors of live mice immediately following systemic injection using a high-speed (30-400 fps) confocal or multi-photon laser-scanning fluorescence microscope. Techniques for quantifying the real-time accumulation and cellular association of individual particles with a size ranging from several tens of nanometers to micrometers are described, as well as an experimental strategy for labeling Kupffer cells in the liver in vivo. Experimental design considerations and controls are provided, as well as minimum equipment requirements. The entire protocol takes approximately 4-8 hours and yields quantitative information. These techniques can serve to study a wide range of kinetic parameters that drive nanotherapeutics delivery, uptake, and treatment response.
Keywords
nanotherapeutics, drug delivery, optical microscopy, mononuclear phagocyte system, Kupffer cells
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The authors declare no competing financial interests.
This is a list of supplementary files associated with this preprint. Click to download.
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Video 1 Real-time accumulation of plateloid 600×200 nm particles in the liver microvasculature (t ==~== 5 sec). <iframe width="560" height="315" src="http://www.youtube.com/embed/TKpzXATYXck?rel=0" frameborder="0" allowfullscreen></iframe> 600×200 nm plateloid silicon particles (red) were imaged in the liver vasculature (green) of Tie2-GFP+ mice from the time of injection. The flow of red blood cells (blue, moving cells) and the presence of Kupffer cells (blue, static stellate cells) were monitored simultaneously. Particle accumulation appeared rapid and increased steadily with time. The video was recorded at 30 fps over a 250×250 μm field-of-view.
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Video 2 Real-time accumulation of plateloid 600×200 nm particles in the liver microvasculature (t ==~== 30 min). <iframe width="560" height="315" src="http://www.youtube.com/embed/lY62FCXbtlk?rel=0" frameborder="0" allowfullscreen></iframe> 600×200 nm plateloid silicon particles (red) were imaged in the liver vasculature (green) of Tie2-GFP+ mice from the time of injection. The flow of red blood cells (blue, moving cells) and the presence of Kupffer cells (blue, static stellate cells) were monitored simultaneously. Thirty minutes after injection, the accumulated particles were distributed in clusters that co-localized with the Kupffer cells. The video was recorded at 30 fps over a 250×250 μm field-of-view.
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Video 3 Time-lapse video of 600×200 nm particle uptake by Kupffer cells. <iframe width="420" height="315" src="http://www.youtube.com/embed/oTVfb6J2MHg?rel=0" frameborder="0" allowfullscreen></iframe> 600×200 nm plateloid silicon particles (red) were imaged in the liver vasculature (green) of Tie2-GFP+ mice from the time of injection. The flow of red blood cells (blue, moving cells) and the presence of Kupffer cells (blue, static stellate cells) were monitored simultaneously. Particle accumulation appeared rapid and increased steadily with time. Frames were acquired at 30 fps over a 200×200 μm field-of-view, averaged using a 4-frame rolling average, and extracted at 1 sec intervals.
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Supplementary Figure 4 Nanoparticle preparation and injection. (1) Resuspend 5×10^8^ nanoparticles in 60 μl PBS and sonicate until monodisperse. (2) Load the nanoparticle solution in a 28G ½ syringe. Remove air and carefully recap. (3) Briefly dip capped syringe in a sonicating bath several times immediately before injection. (4) Retro-orbital particle injection is readily performed by holding the syringe horizontally while gently bracing the head from the opposite side.
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Supplementary Figure 2 Custom coverslip holder with 3 degrees of positioning freedom. (A) Assembled coverslip holder and rail system for liver imaging. The white arrows indicate directions of positioning freedom. (B) Individual Thor Labs Mini-series hardware components with parts numbers, as well as other required components. (C) The partially assembled coverslip holder is mounted on two 3” posts (MS3R) placed end-to-end. Note that spacing between the 2” posts (MS2R) may be adjusted for different coverslips. We also find it useful to extend the 2” posts to their full length in order to increase the working space around the objective. The second horizontal post (MS3R) is easily attached to either end of the coverslip holder, allowing the coverslip orientation to be flipped without disassembly.
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Supplementary Figure 1 Step-by-step preparation of the liver for dynamic intravital imaging. (1) Shave the surgical site. (2) Cover the shaved region with a thick layer of hair removal cream. (3) After 1-2 minutes, wipe-off the cream and hair using sterile gauze followed by alcohol prep pads. (4) Sterilize the surgical site with a Chlorhexidine swabstick followed by (5) a 70% isopropyl alcohol scrub. (6) Surgically open the skin and underlying tissues using small midline incisions. Bupivicaine may be topically applied to the exposed subcutaneous tissue before the second incision is made. (7) Position the mouse face-up on the microscope stage, placing 2×2 or 4×4 inch rolled gauze (see arrow) underneath the back to expose the liver. Restrain limbs using Transpore surgical tape. (8) Use pre-moistened cotton swabsticks to gently position the liver and incision. (9) Custom retractors can be made by bending the tip of a 30G ½ needle with forceps. (10) Place the needle-based retractors using forceps, being careful to position them in such a way that they cannot accidently puncture the liver. Retractors may be taped in place as needed. (11) Drape the surrounding tissue with moistened gauze and disposable paper drape (not shown). (12) Carefully place an intraperitoneal line filled with PBS in the lower abdomen. Add a 1 ml or 3 ml syringe loaded with PBS. Placing the syringe on the heated microscope stage helps warm the saline prior to injection. (13) Gently position a coverslip against the exposed liver, adjusting the post clamp and slider knobs as necessary to achieve proper positioning. (14) A small ring of Vetbond (cyanoacrylate glue) may be used to seal the liver against the coverslip. (15) Position the microscope objective and adjust stage height to bring the liver in focus.
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Supplementary Figure 3 Step-by-step preparation of the custom coverslip holder and slider. (1) Attach washers, nuts, and screws (HW-KIT5) to the end of two 2” posts (MS2R). (2) Slide the post clamps (MSRA90) to the end and tighten. Attach the remaining washers and nuts. (3) Attach the 3” posts (MS3R) end-to-end. Slide the assembled 2” posts on one end and tighten them 50 mm apart. Note that the 2” posts should be mounted below the 6” horizontal post. (4) Use 2 small pieces of scotch tape to attach a coverslip to the underside of the coverslip holder. The coverslip may be aligned against the washers for added stability. (5) Assembled coverslip holder shown with correct orientation. (6) Mount a 2” post (MS2R) to the slider (RC2) using an adaptor (MSA8). (7) Mount the rail (RLA1200) to the microscope stage using ¼”-20 screws. (8) Position the slider on the rail. (9) Mount the coverslip holder to the slider using a post clamp (MSRA90). The coverslip height and x,y positions may be adjusted by loosening and repositioning the appropriate tension screws (see arrows).
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Video 5 Real-time accumulation of plateloid 1000×400 nm particles in regions of RBC capture (t ==~== 20 sec). <iframe width="420" height="315" src="http://www.youtube.com/embed/xlEzOIluSaY?rel=0" frameborder="0" allowfullscreen></iframe> Autologous RBCs freshly labeled with Vybrant® DiD (blue, ~3-5% by number) were allowed to circulate in liver vasculature (green) of Tie2-GFP+ mice. Plateloid 1000×400 particles (red), injected 15 minutes after the RBCs, appeared to accumulate in regions of RBC capture. The video was recorded at 30 fps over a 250×250 μm field-of-view.
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Video 7 Time-lapse microscopy of plateloid 1000x400 nm particles in breast tumor vasculature. <iframe width="420" height="315" src="http://www.youtube.com/embed/flNy8cd34qE?rel=0" frameborder="0" allowfullscreen></iframe> 1000×400 nm plateloid silicon particles (red) were imaged in MDA-MB-231 tumors from the time of injection. The vasculature (green) was delineated using 40kDA FITC-dextran (1µM). Particle accumulation was primarily observed at early time-points, when particle influx was highest. Images were recorded at 45-second intervals over a 1060×530 μm field-of-view.
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Video 6 Example of laser-induced phototoxity in the liver. <iframe width="420" height="315" src="http://www.youtube.com/embed/7x-vFDDaKdY?rel=0" frameborder="0" allowfullscreen></iframe> A Tie2-GFP mouse was systemically injected with a 2MDa dextran tracer (blue) and imaged continuously for 10 minutes using a polygon-based laser scanning system. The RBCs, which appear as dark spots within the dextran tracer, initially flow well and then suddenly appear to stop.
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Video 4 Real-time capture of RBCs by Kupffer cells. <iframe width="420" height="315" src="http://www.youtube.com/embed/9mUfZEzLaKI?rel=0" frameborder="0" allowfullscreen></iframe> Autologous RBCs freshly labeled with Vybrant® DiD (blue, ~3-5% by number) were imaged in the liver vasculature (green) of Tie2-GFP+ mice immediately after injection. RBC capture appeared rapid, with several cells accumulating within the field-of-view in under one minute. The presence of captured RBCs does not appear to impact blood flow through the vasculature. The video was recorded at 30 fps over a 250×250 μm field-of-view.
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Biological techniques
Associated Publications
Rapid tumoritropic accumulation of systemically injected plateloid particles and their biodistribution
by Anne L. van de Ven, Pilhan Kim, O'Hara Haley, +9
Journal Of Controlled Release (03 May, 2013)
Integrated intravital microscopy and mathematical modeling to optimize nanotherapeutics delivery to tumors
by Anne L. van de Ven, Min Wu, John Lowengrub, +5
AIP Advances (03 May, 2013)
Modeling of nanotherapeutics delivery based on tumor perfusion
by Anne L van de Ven, Behnaz Abdollahi, Carlos J Martinez, +5
New Journal Of Physics (09 May, 2013)
Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions
by Alessandro Parodi, Nicoletta Quattrocchi, Anne L. van de Ven, +10
Nature Nanotechnology (03 May, 2013)
Method Article
Anne L van de Ven
The Methodist Hospital Research Institute, 6670 Bertner Ave, Houston, TX 77030
Corresponding Author
Seok Hyun Yun
Wellman Center for Photomedicine and Harvard Medical School, Massachusetts General Hospital, 40 Blossom St, Boston, MA 02114
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 10 May, 2013
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Intravital microscopy is emerging as an important experimental tool for the research and development of multi-functional therapeutic nanoconstructs. The direct visualization of nanoparticle dynamics within live animals provides invaluable insights into the mechanisms that regulate nanotherapeutics transport and cell-particle interactions. Here we present a protocol to image the dynamics of nanoparticles within the liver and tumors of live mice immediately following systemic injection using a high-speed (30-400 fps) confocal or multi-photon laser-scanning fluorescence microscope. Techniques for quantifying the real-time accumulation and cellular association of individual particles with a size ranging from several tens of nanometers to micrometers are described, as well as an experimental strategy for labeling Kupffer cells in the liver in vivo. Experimental design considerations and controls are provided, as well as minimum equipment requirements. The entire protocol takes approximately 4-8 hours and yields quantitative information. These techniques can serve to study a wide range of kinetic parameters that drive nanotherapeutics delivery, uptake, and treatment response.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
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Associated Publications
Rapid tumoritropic accumulation of systemically injected plateloid particles and their biodistribution
by Anne L. van de Ven, Pilhan Kim, O'Hara Haley, +9
Journal Of Controlled Release (03 May, 2013)
Integrated intravital microscopy and mathematical modeling to optimize nanotherapeutics delivery to tumors
by Anne L. van de Ven, Min Wu, John Lowengrub, +5
AIP Advances (03 May, 2013)
Modeling of nanotherapeutics delivery based on tumor perfusion
by Anne L van de Ven, Behnaz Abdollahi, Carlos J Martinez, +5
New Journal Of Physics (09 May, 2013)
Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions
by Alessandro Parodi, Nicoletta Quattrocchi, Anne L. van de Ven, +10
Nature Nanotechnology (03 May, 2013)