ABSTRACT
Tissue clearing techniques are useful for large-scale three-dimensional fluorescence imaging of thick tissues. However, high-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we present a water-based optical clearing and mounting media, SeeDB2, which is designed for high numerical aperture (NA) objective lenses with oil or glycerol immersion. Using quick and simple soaking procedures, the refractive indices of samples can be matched either to that of immersion oil (1.52) or glycerol (1.46), thus minimizing light scattering and spherical aberrations. Fine morphology and various fluorescent proteins are highly preserved during the clearing and imaging process. Our method is useful for the three-dimensional fluorescence imaging of neuronal circuitry at synaptic resolution using confocal and super-resolution microscopy. SeeDB2 is also useful as a mounting media for the super-resolution imaging of fluorescent proteins.
ABSTRACT
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ABSTRACT
Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations. During the clearing process, fine morphology and fluorescent proteins were highly preserved. SeeDB2 enabled super-resolution microscopy of various tissue samples up to a depth of >100 µm, an order of magnitude deeper than previously possible under standard mounting conditions. Using this approach, we demonstrate accumulation of inhibitory synapses on spine heads in NMDA-receptor-deficient neurons. In the fly medulla, we found unexpected heterogeneity in axon bouton orientations among Mi1 neurons, a part of the motion detection circuitry. Thus, volumetric super-resolution microscopy of cleared tissues is a powerful strategy in connectomic studies at synaptic levels.
Subject(s)
Microscopy, Fluorescence , Neurons/physiology , Animals , Brain/anatomy & histology , Brain/metabolism , Brain Mapping , Iohexol/chemistry , Mice , Receptors, N-Methyl-D-Aspartate/deficiency , Receptors, N-Methyl-D-Aspartate/genetics , Refractometry , Saponins/chemistry , Synapses/chemistry , Synapses/metabolismABSTRACT
Large-scale three-dimensional fluorescence imaging is essential for comprehensive and quantitative understanding of neuronal circuitry. We describe a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days leaving many types of fluorescent dyes unquenched, including fluorescent proteins and lipophilic neuronal tracers. This method maintains a constant sample volume during the clearing procedure, an important factor to keep cellular morphology intact. After optical clearing with SeeDB, we can reach a depth of >1000 µm with confocal microscopy. When combined with two-photon microscopy, SeeDB allows us to image fixed mouse brains at millimeter-scale level.
Subject(s)
Brain Chemistry , Brain/embryology , Fructose/analysis , Glycerol/analogs & derivatives , Microscopy, Fluorescence, Multiphoton/methods , Solutions/analysis , Animals , Brain Chemistry/physiology , Fixatives/analysis , Glycerol/analysis , Mice , Mice, TransgenicABSTRACT
We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.
