Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 μm) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.
Part 1: Microprism Optics and Microscope Setup
Part 2: Non-survival Animal Surgery and Microprism Insertion
Part 3: Representative Results
Figure 1: Images of the right-angle microprism. Prisms used in this experiment are one-millimeter, BK7 glass prisms with an enhanced silver coating on the hypotenuse to allow internal reflection of excitation and emission light.
Figure 2: Image of layer V YFP pyramidal neurons. Wide field-of-view imaging with the microprism reveals a large population of pyramidal cell bodies ~800 – 900 μm deep from the cortical surface. In addition, apical dendrites extending up towards layer I can also be resolved. Scale bar = 200 μm.
Figure 3: A higher numerical aperture objective used in conjunction with the microprism provides spatial resolution sufficient to resolve dendritic spines on the apical dendrites on layer V pyramidal neurons. Scale bar = 10 μm.
Figure 4: An image obtained using the microprism and a tail-vein injection of fluorescent dye. The image shows vertical, large caliber vessels extending from deep brain regions and smaller capillaries branching off through the cortical volume. Scale bar = 200 μm.
Figure 5: Using a higher numerical aperture objective, the network of capillaries in the neocortex can be imaged with greater detail. Scale bar = 50 μm.
Figure 6: Microprisms also allows for imaging red blood cells (RBCs) flowing through vessels. RBCs do not absorb the fluorescent dye and therefore are seen as dark stripes across the vessel. By line-scanning across the vessel, one can obtain measurements of RBC flux and velocity. Scale bar = 10 μm (horizontal bar in top and bottom image) and 250 ms (vertical bar in bottom image).
Using a microprism in an imaging experiment is relatively straightforward and offers many advantages for in vivo studies on the neocortex. Representative examples using this technique include images taken from transgenic mice expressing yellow fluorescent protein in layer V cortical neurons. Using microprisms one can see a collection of large pyramidal cell bodies in layer V, nearly 1 mm below the cortical surface. Also seen are the apical dendrites that extend through all the superficial layers before diverging into tufts (Figure 2). The mice used for these experiments are transgenic animals designed to express YFP only in layer V neurons (YFP-H2). Using these YFP-H mice and a higher numerical aperture objective with a glass correction collar it is also possible to image the dendrites with greater detail and resolve dendritic spines (Figure 3).
One can also label the cortical blood vessels with a fluorescent dye such as fluorescein-dextran using established tail-vein injection techniques. Typically, for adult mice a 100 ul bolus injection of fluorescein-dextran at 20 mg/ml in physiological saline is appropriate. The best results come from injecting the dye after the microprism has been inserted into the neocortex (after step 2.16). Using this technique one can see the larger caliber vessels from deep layers extending towards the pia and branching off to form the network of microcapillaries (Figure 4). This intricate network of microcapillaries in the neocortex is best appreciated using a high numerical aperture objective (Figure 5).
In addition, it is possible to measure red blood cell velocity and flux from deep neocortical capillaries by line-scanning along the length of a vessel (Figure 6, red line in top image). Since red blood cells do not take up the fluorescent dye, they will appear as dark streaks. Images that result from a line-scan have a spatial dimension in one axes and a temporal dimension in the other axes (Figure 6, bottom image). From these images, one can calculate the flux and velocity of red blood cells through the capillary. In the case of figure 6, the RBC flux was measured to be 36 RBCs/second with a velocity of 0.45 mm/second. This is in agreement with results from the literature3.
The authors have nothing to disclose.
We thank Anthony J. Koleske, PhD for providing the YFP mice.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
With enhanced silver reflective coating-(R77) | OptoSigma Corp. (Santa Ana, CA) | 055-0010 | With enhanced silver reflective coating-(R77) | |
Fluorescein-dextran | Sigma-Aldrich | FD70 | 70 kDa | |
0.28 NA, 4x air objective | Olympus | XLFLUOR 4x/340 | ||
0.60 NA, 40x air objective | Olympus | LUCPLFLN | With 0-2 mm coverglass correction |