Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of internal organs in small animals

Nature Protocols volume 7, pages 1456–1469 (2012)Cite this article

Subjects

Abstract

Intravital fluorescence microscopy has emerged as a powerful technique to visualize cellular processes in vivo. However, owing to their size, the objective lenses required have limited physical accessibility to various tissue sites in the internal organs of small animals. The use of small-diameter probes using graded-index (GRIN) lenses expands the capabilities of conventional intravital microscopes to minimally invasive imaging of internal organs. In this protocol, we describe the detailed steps for the fabrication of front- and side-view GRIN probes and the integration and operation of the probes in a confocal microscope to enable visualization of fluorescent cells and microvasculature in various mouse organs. Some experience in building an optical setup is required to complete the protocol. We also present longitudinal imaging of immune cells in renal allografts and tumor development in the colon. Fabrication and integration can be completed in 5–7 h, and a typical in vivo imaging session takes 1–2 h.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic of GRIN optical probes.
Figure 2: Probe assembly.
Figure 3: Front-view probe mount attachment.
Figure 4: Side-view probe mount attachment.
Figure 5: Optical characterization of a fabricated probe.
Figure 6: Integration of the probe into a commercial microscope.
Figure 7: In vivo fluorescence images obtained by a front-view probe.
Figure 8: Time-lapse imaging of kidney graft with front-view probe.
Figure 9: In vivo fluorescence images by side-view probe.
Figure 10: Time-lapse imaging of colorectal tumorigenesis with side-view probe.

Similar content being viewed by others

References

  1. Flusberg, B.A. et al. Fiber-optic fluorescence imaging. Nat. Meth. 2, 941–950 (2005).

    Article  CAS  Google Scholar 

  2. Fan, Z. et al. In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response. Nat. Med. 16, 718–722 (2010).

    Article  CAS  Google Scholar 

  3. Kim, P. et al. In vivo wide-area cellular imaging by side-view endomicroscopy. Nat. Meth. 7, 303–305 (2010).

    Article  CAS  Google Scholar 

  4. Kiesslich, R., Goetz, M., Vieth, M., Galle, P.R. & Neurath, M.F. Technology insight: confocal laser endoscopy for in vivo diagnosis of colorectal cancer. Nat. Clin. Pract. Oncol. 4, 480–490 (2007).

    Article  Google Scholar 

  5. Becker, C., Fantini, M.C. & Neurath, M.F. High resolution colonoscopy in live mice. Nat. Protoc. 1, 2900–2904 (2006).

    Article  CAS  Google Scholar 

  6. Hsiung, P.L. et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat. Med. 14, 454–458 (2008).

    Article  CAS  Google Scholar 

  7. Dela Cruz, J.M., McMullen, J.D., Williams, R.M. & Zipfel, W.R. Feasibility of using multiphoton excited tissue autofluorescence for in vivo human histopathology. Biomed. Opt. Express 1, 1320–1330 (2010).

    Article  Google Scholar 

  8. Waldner, M.J., Wirtz, S., Neufert, C., Becker, C. & Neurath, M.F. Confocal laser endomicroscopy and narrow-band imaging-aided endoscopy for in vivo imaging of colitis and colon cancer in mice. Nat. Protoc. 6, 1471–1481 (2011).

    Article  CAS  Google Scholar 

  9. Barretto, R.P. et al. Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy. Nat. Med. 17, 223–228 (2011).

    Article  CAS  Google Scholar 

  10. Jung, J.C. & Schnitzer, M.J. Multiphoton endoscopy. Opt. Lett. 28, 902–904 (2003).

    Article  Google Scholar 

  11. Kim, P., Puoris'haag, M., Cote, D., Lin, C.P. & Yun, S.H. In vivo confocal and multiphoton microendoscopy. J. Biomed. Optics 13, 010501 (2008).

    Article  Google Scholar 

  12. Miyajima, M. et al. Early acceptance of renal allografts in mice is dependent on Foxp3(+) cells. Am. J. Pathol. 178, 1635–1645 (2011).

    Article  CAS  Google Scholar 

  13. Cole, R.W., Jinadasa, T. & Brown, C.M. Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nat. Protoc. 6, 1929–1941 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Colvin and C. Chase for providing the mice with renal transplantation; R. Kucherlapati and K. Hung for the mouse model of spontaneous colorectal tumor; H. Ploegh for the MHCII-GFP mice; C. Lin, D. Cote, L. Kaplan, M. Ferrari and G.Y. Koh for discussions; and D. Stevenson for proofreading of the manuscript. This work was supported by grants from the US National Institutes of Health (R21AI081010, RC1DK086242, RC2DK088661, U54CA143837, R01AI081734, P41EB015903), the US Department of Defense (FA9550-10-1-0537) and the National Research Foundation of Korea (WCU R31-2008-000-10071-0, 2009-352-C00042, 2011-0009503, WCI 2011-001).

Author information

Author notes

  1. Jun Ki Kim, Woei Ming Lee, Pilhan Kim and Myunghwan Choi: These authors contributed equally to this work.

Authors and Affiliations

  1. Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Jun Ki Kim, Woei Ming Lee, Pilhan Kim, Myunghwan Choi, Keehoon Jung, Seonghoon Kim & Seok Hyun Yun

  2. Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of Science and Technology, Daejeon, Korea

    Pilhan Kim, Myunghwan Choi, Seonghoon Kim & Seok Hyun Yun

  3. Harvard-MIT Health Sciences and Technology, Cambridge, Massachusetts, USA

    Seok Hyun Yun

Authors
  1. Jun Ki Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Woei Ming Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pilhan Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Myunghwan Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Keehoon Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Seonghoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Seok Hyun Yun
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.K.K., W.M.L., P.K., M.C. and S.H.Y. designed and wrote the protocol. K.J. and S.K. contributed figures.

Corresponding author

Correspondence to Seok Hyun Yun.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

GRIN lens polishing device. A photograph (a) and schematic (b) of the jig used for the polishing of GRIN lenses. This device helps position the GRIN lens perpendicular to the surface of the polishing film. Different ferrule diameters can be chosen according to the diameter of the GRIN lens. The lens is loaded into the ferrule and a pushpin is loaded on top of the lens. The user can press the pushpin by hand, applying the necessary downward pressure on the lens during grinding and smoothing. The pushpin has an elastic rubber tip so that damage on the top lens surface is avoided. During polishing, the weight of the pushpin aids in sustaining a constant pressure on the lens, ensuring a regular polishing rate. As the length of the GRIN lens gets close towards the desired length, the grade of lapping polishing film should be changed from 5 µm to 1 µm to reduce the roughness of the lens surface. (PDF 1647 kb)

Supplementary Fig. 2

Technical drawing of the front-view probe holder. The front-view probe holder is used to hold the probe using a setscrew. A small blob of UV epoxy is applied to the end of the setscrew and cured, ensuring a firm grab of the optical probe without exerting excessive stress. (PDF 113 kb)

Supplementary Fig. 3

Technical drawing of the side-view probe holder. The side-view probe holder consists of four parts: custom-made stainless bearing collar, ball bearing, custom-made stainless holder and rotation pulley. (PDF 186 kb)

Supplementary Fig. 4

Drawing for assembling the side-view probe mount. The side-view probe holder is connected to a second rotation pulley via a timing belt. A rotation shaft is held in position using a set-screw in the rotation pulley. This second pulley is attached onto a cage plate with ball bearings using the collar shaft. An additional cage with ball bearing and shaft collar is used to stabilize the rotation shaft during rotation control. The viewing angle or the orientation of the prism surface is controlled by rotating the rotation shaft. (PDF 168 kb)

Supplementary Fig. 5

Cell population and motility analysis. Images of a kidney graft transplanted from a wild-type donor mouse to a FoxP3-GFP recipient mouse. Green, FoxP3+ GFP+ cells (green); red, autofluorescence from kidney capsules. Time-lapse sequence was taken every 20 seconds for 6 minutes. Track of individual cells during 6 min is overlaid onto the image taken at the end of time-lapse acquisition (Trace). The lower panel shows images zoomed at the area marked by the square. A cell population is analyzed by counting the number of cells in each FOV, and cell motility can be traced by taking time-lapse imaging. These measurements allow various dynamic processes, such as infiltration, migration, and cell-cell interaction, to be analyzed quantitatively. Scale bars, 50 µm in top panels and 20 µm in lower panels. (PDF 3076 kb)

Supplementary Video 1

Mounting the GRIN probe into a microscope. This video shows the process of mounting a side-view probe into a microscope. (MOV 877 kb)

Supplementary Video 2

Visualizing fluorescent microspheres with a GRIN probe. This video shows the process of imaging fluorescent beads with a side-view probe. (MOV 910 kb)

Supplementary Video 3

Demonstration of in vivo vasculature imaging in the murine colon with a side-view GRIN probe. This video shows the process of imaging blood vessels in the descending colon. (MOV 3330 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, J., Lee, W., Kim, P. et al. Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of internal organs in small animals. Nat Protoc 7, 1456–1469 (2012). https://doi.org/10.1038/nprot.2012.078

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.078

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing