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OPT imaging platform

New 3D imaging infrastructure available at CRC

Optical Projection Tomography (OPT) is an optical imaging tool that creates high resolution 3D images of large biological samples in the range of 1mm - 30 mm across. It was created to fill the imaging gap between conventional microscopy and MRI. It is particularly suited to study fundamental biological processes using light emitted from inside the organ, via optical fluorescence. The main advantage of this imaging modality is that it avoids the need to physically section the sample.

opt animation

NOD mouse pancreas stained for insulin and quantified for different size categories

How does OPT work?

OPT uses visible light for illumination and in order to reduce scattering and absorption of light, the sample needs to be rendered transparent by the use of optical clearing techniques. Mouse embryos are ideal specimens for OPT as they are naturally transparent . The otherwise cleared sample is embedded in a block of agarose gel  and placed on a motor through a magnetic mount. It undergoes a complete rotation within the scanner through angular steps  of 0.9 degrees (ca. 400 images) while suspended in a quartz cuvette filled with the same solution used for optical clearing.

mouse liver
Vasculature in a mouse liver lobe stained for ASMA
mouse brain
Amyloid plaque deposition in a 5xFAD mouse brain
spinal cord
EAE mouse spinal cord, inflammation in red
optic nerve
Optic nerve

The most common clearing approach we use is matching the refractive indices within the organism by replacing intra and extracellular water with 1:2 mixture of benzyl alcohol and benzyl benzoate (BABB). The specimen in the OPT device is rotated about its vertical axis while under illumination in the IR-UV wavelengths. A series of optics focus the illumination onto the sample and then the fluorescent light to a CCD camera.  An image is acquired at a series of angles and tomographic reconstruction is performed using a back-projection algorithm, and this yields a 3D volumetric representation of the specimen.


Detailed information

Furthermore, OPT is able to take advantage of fluorescent dyes, and different wavelength channels can be used. This allows the observation of autofluorescence of the tissue (to inform on tissue structure), along with the mapping of gene and protein expression. The range of species successfully imaged so far includes human (1), mouse (2,3), chick (4), reptile species (5), zebrafish (6), Drosophila (7) and Arabidopsis (8). Successes with bigger specimens such as whole organs taken from the adult mouse have also now been reported, for brain (9), spinal cord (10), pancreas (11), kidney (11) and lungs (12). This opens up exciting new applications such as preclinical disease research, like whole pancreas imaging performed quantitatively to compare the mass of β-cell tissue in normal versus diabetic specimens, using the NOD mouse model (11) or imaging progressive inflammation of spinal cord and optical nerve in a mouse model of multiple sclerosis (10).

References

  1. Kerwin J, Scott M, Sharpe J, Puelles L, Robson SC, Martînez-de-la-Torre M, et al. 3 dimensional modelling of early human brain development using optical projection tomography. BMC Neurosci. 2004;5:27
  2. Sharpe J, Ahlgren U, Perry P et al: Optical Projection Tomography as a Tool for 3D Microscopy and Gene Expression Studies. Science  19 Apr 2002: Vol. 296, Issue 5567, pp. 541-545
  3. Walls JR, Coultas L, Rossant J, Henkelman RM. Three-dimensional analysis of vascular development in the mouse embryo. PLoS One. 2008;3:2853
  4. Fisher ME, Clelland AK, Bain A, Baldock RA, Murphy P, Downie H, et al. Integrating technologies for comparing 3D gene expression domains in the developing chick limb. Dev Biol. 2008;317:13–23.
  5. Koshiba-Takeuchi K, Mori AD, Kaynak BL, Cebra-Thomas J, Sukonnik T, Georges RO, et al. Reptilian heart development and the molecular basis of cardiac chamber evolution. Nature. 2009;461:95–98.
  6. Bryson-Richardson RJ, Berger S, Schilling TF, Hall TE, Cole NJ, Gibson AJ, et al. FishNet: an online database of zebrafish anatomy. BMC Biol. 2007;5:34.
  7. McGurk L, Morrison H, Keegan LP, Sharpe J, O'Connell MA. Three-dimensional imaging of Drosophila melanogaster. PLoS One. 2007;2:834
  8. Lee K, Avondo J, Morrison H, Blot L, Stark M, Sharpe J, et al. Visualizing plant development and gene expression in three dimensions using optical projection tomography. Plant Cell. 2006;18:2145–2156.
  9. Hajihosseini MK, De Langhe S, Lana-Elola E, Morrison H, Sparshott N, Kelly R, et al. Localization and fate of Fgf10-expressing cells in the adult mouse brain implicate Fgf10 in control of neurogenesis. Mol Cell Neurosci. 2008;37:857–868
  10. Gupta, S,  Utoft, R, Hasseldam, H, Schmidt-Christensen, A, Hannibal Dahlbeck, T,  Hansen, L,  Fransén-Pettersson, N, Gupta, N A, Rozell, B, Andersson, Å and Holmberg, D :Global and 3D spatial assessment of neuroinflammation in rodent models of multiple sclerosis. PLoS One  2013, 8(10): e76330.
  11. Alanentalo T, Asayesh A, Morrison H, Lorén CE, Holmberg D, Sharpe J, et al. Tomographic molecular imaging and 3D quantification within adult mouse organs. Nat Methods. 2007;4:31–33
  12. Davies JA, Armstrong J. The anatomy of organogenesis: novel solutions to old problems. Prog Histochem Cytochem. 2006;40:165–176
  13. De Langhe SP, Carraro G, Warburton D, Hajihosseini MK, Bellusci S. Levels of mesenchymal FGFR2 signaling modulate smooth muscle progenitor cell commitment in the lung. Dev Biol. 2006;299:52–62.

 

For further information and access to the machine please contact: Autoimmunity group (Prof. Dan Holmberg), CRC 91:10 (lab 026), dan.holmberg@med.lu.se 

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