Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene
2016
Mapping neural circuits that often span distant areas of the brain requires imaging of the entire brain at cellular resolution. However, light scattering in the brain tissue limits the depth of optical imaging. Therefore, until recently, whole-brain imaging was mostly achieved by the slow and labor-intensive method of sectioning and reconstruction1. Recent advances have provided an alternative approach. Optical clearing is a procedure that renders the brain transparent to light by removing the main scattering source – refractive index (RI) differences within the tissue. Since the potential of chemical clearing for imaging of intact brains was first demonstrated2, several other clearing methods yielding better transparency and lower fluorescence quenching have been developed3,4,5,6. However, although numerous improvements and modifications have been reported since then7,8,9,10,11, whole brain clearing still remains challenging, especially for adult, well-myelinated tissue9,12,13.
Another important advancement in brain imaging was applying light-sheet fluorescence microscopy (LSFM) to cleared brains2,14,15,16. By scanning the sample volume plane-by-plane instead of point-by-point, LSFM allows fast imaging of large specimens with sufficient resolution for quantitative neuroanatomy17,18.
The brain clearing procedures have originally been developed and optimized for mouse brain. Processing rat brains is more challenging, not only because of their size, but also higher degree of myelination. Despite the wide use of this species in neuroscience and its superiority in some experiments, including behavioral and physiological investigations, whole-brain clearing and subsequent imaging of an adult rat brain has not been, to our knowledge, reported, although scalability of clearing of periadolscent to adult rat brain has been described19.
In this work we present whole brain imaging of adult rat brain that has been cleared using the FluoClearBABB method, based on dehydration in increasing concentrations of tert-butanol10. For this, transgenic rat expressing GFP under Thy1 promoter, created by us, is used. FluoClearBABB is a simple, inexpensive clearing protocol yielding robust samples that can be easily imaged and stored in the clearing solution. We found that, of the protocols that we tested, that is PACT (passive CLARITY technique), CUBIC and FluoClearBABB, the latter is superior in preserving GFP fluorescence and at the same time providing sufficient tissue transparency for whole-rat brain lightsheet microscopy imaging.
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
31
References
64
Citations
NaN
KQI