1Department of Molecular & Human Genetics, Baylor College of Medicine (BCM), 2Precisionary Instruments Inc., 3Departments of Molecular & Human Genetics and Neuroscience, Baylor College of Medicine (BCM), 4Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital
This article is a part ofJoVE Neuroscience. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Selever, J., Kong, J., Arenkiel, B. R. A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices. J. Vis. Exp. (53), e2807, doi:10.3791/2807 (2011).
A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.
1. In situ brain fixation
*Prepare a 10 ml syringe (28 gauge needle) filled with phosphate buffered saline (PBS).
*Prepare a 10 ml syringe (28 gauge needle) filled with 4% paraformaldehyde (PFA) in PBS. Reserve an additional 5-10 ml of PFA/PBS for post fixation.
2. Dissection and brain extraction
3. Agarose embedding
4. Sectioning brain slice with the Compresstome
5. Optical Clearing
6. Slice mounting for imaging
7. Representative Results:
Processing, imaging, and analyzing fluorescently labeled brain tissue have become indispensible to the study of neurobiology. Many of these investigations require sophisticated genetic manipulations to obtain reporter expression in targeted neuronal subsets, followed by both low- and high-resolution image analyses. Often experimental and technical limitations make it impossible to obtain these types of data from the same animal, or in a rapid manner. Preparation of optically-cleared, semi-thick brain sections for fluorescent imaging aids in this challenge. An example of an intact thick brain slice labeled with a genetically modified rabies virus engineered to express EGFP, and imaged using epifluorescent and confocal microscopy is shown in Figure 4 and Figure 5 respectively. For epifluorescent imaging we used a Leica M205 FA, and for confocal image acquisition we used a Zeiss LSM 510. Due to the relative simplicity of the attached protocol, this method is capable of yielding useful image data from tissue expressing fluorescent reporters with a turnaround time of less than one day, and is compatible with both low- and high-resolution light microscopy.
If the investigator chooses to incorporate additional postmortem labeling methods, immunohistochemical staining, or make thinner sections, the protocol lengthens accordingly. However, the method described above represents a simple and relatively high-throughput method for screening patterns of vital reporter expression in intact brain.
Figure 1. Flow chart diagraming the fixation, dissection, slicing, clearing, and mounting procedure of semi-thick brain slices.
Figure 2. Diagram of sequential cutting steps to extract the intact mouse brain.
Figure 3. Steps to mount and slice brain tissue using the Compresstome. A) Placement of brain tissue onto cutting plunger using superglue. B) Drawing down the mounted brain tissue into the plunger for agarose embedding. C) Solidifying an agarose brain plug using a chilled compression block. D) Insertion of agarose brain plug and plunger into Compresstome cutting chamber. E) Alignment of razorblade to the plunger device. F) Cutting and collection of brain slices into Compresstome buffer chamber.
Figure 4. Light microscopy images of a thick slice from glycerol-cleared brain tissue through the frontal cortex expressing enhanced green fluorescent protein (EGFP). A) Coronal slice through a mouse brain (200 um thick) labeled with a viral vector expressing EGFP and imaged at low resolution using an epifluorescent stereoscope. Scale bar, 2 mm.
Figure 5. High magnification image of fluorescently labeled layer 5/6 cortical neurons in a cleared thick brain slice. A) High resolution confocal image of a maximum projection Z-stack (150 um thick) through the region highlighted in (Figure 4). Scale bar, 25 um.
Given the widespread application of using fluorescent proteins to target neuronal subsets for investigation via light microscopy, the need to rapidly screen, image, and analyze neural networks within intact brain tissue has become invaluable.
Technical advances in the development of user-friendly viral vectors, in vivo electroporation techniques, and genetically modified mouse strains now provides a seemingly unlimited source of labeled cell types to investigate. However, image analysis of intact brain tissue still remains a bottleneck to experimentation. The method described here has proven to be extremely valuable for our routine analysis of brain tissue expressing fluorescent reporters. It significantly truncates the time needed to prepare and analyze tissue from brain sections, is compatible with both low-power and high-resolution fluorescence imaging, can be used in combination with immunocytochemistry, is translatable to other experimental animal models, and importantly, is broadly applicable to routine laboratory environments that may not have access to multiphoton imaging equipment. It is important to note however, that optical clearing with glycerol and other aqueous and organic solutions has practical limitations. Here we describe the use of glycerol to aid clearing of brain slices up to 200 um thick. Although this method works quite well to improve the imaging resolution in tissues up to 200 um, we do not see an improvement beyond this. Other organic solvents are capable of clearing tissue further, however there is always a trade off with the loss (or extraction) of fluorescent signal.
Possible variations of the method described above may include perfusion using perstaltic pumps, clearing with other aqueous or organic phase solvents that are compatible with FP fluorescence, automated slicing and serial imaging, or pairing with multi-photon micropscopy to allow imaging in even thicker brain slices.
Jian-Qiang Kong is an employee of Precisionary Instruments, Inc., that manufactures a product used in this article.
This work was funded by support through the McNair Foundation, NARSAD, and NINDS grant R00NS064171-03.
|bone scissors||F.S.T.||16044-10||-or equivalent|
|dissection scissors||F.S.T.||14084-08||-or equivalent|
|type I-B agarose||Sigma-Aldrich||A0576|
|Compresstome||Precisionary Instruments||VF-200||-other vibratomes are compatible|
|double sided adhesive||Grace Bio-Lab Inc.||SA-S-1L|
|Superfrost Plus slides||VWR international||48311-703|
|Cover glass||VWR international||48383-139|
|glycerol||EMD Millipore||GX0185-6||-or equivalent|