Summary

نهج السريع إلى التصوير الإسفار عالية الدقة في الدماغ شرائح شبه سميكة

Published: July 26, 2011
doi:

Summary

نحن هنا وصفا لطريقة سريعة وبسيطة لصورة fluorescently المسمى الخلايا في الدماغ شبه شرائح سميكة. عن طريق تحديد ، تشريح ، وإزالة أنسجة الدماغ بصريا وصفنا كيف يمكن استخدام معيار التصوير epifluorescent مبائر أو لتصور الخلايا الفردية والشبكات العصبية داخل الأنسجة العصبية سليمة.

Abstract

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.

Protocol

1. الدماغ في تثبيت الوضع الطبيعي * تحضير حقنة 10 مل (28 إبرة قياس) مليئة الفوسفات مخزنة المالحة (PBS). * تحضير حقنة 10 مل (28 إبرة قياس) مليئة بارافورمالدهيد 4 ٪ (PFA) في برنامج تلفزيوني. الاحتياطي إلى ترخيص إضافي 5-10 PFA / لتثبيت برنا?…

Discussion

نظرا للتطبيق على نطاق واسع من استخدام البروتينات الفلورية لاستهداف الخلايا العصبية فرعية للتحقيق عبر المجهر الضوئي ، والحاجة إلى سرعة الشاشة والصورة وتحليل الشبكات العصبية داخل أنسجة الدماغ سليمة أصبحت لا تقدر بثمن.

التقدم الت…

Disclosures

The authors have nothing to disclose.

Acknowledgements

وقد تم تمويل هذا العمل عن طريق الدعم من خلال مؤسسة ماكنير ، NARSAD ، ومنح NINDS R00NS064171 – 03.

Materials

Name of the item Company Catalogue number Comments (optional)
bone scissors F.S.T. 16044-10 -or equivalent
dissection scissors F.S.T. 14084-08 -or equivalent
type I-B agarose Sigma A0576  
Compresstome Precisionary Instruments VF-200 -other vibratomes are compatible
double sided adhesive Grace Bio-Labs SA-S-1L  
Superfrost Plus slides VWR 48311-703  
Cover glass VWR 48383-139  
glycerol EMD Chemicals Inc. GX0185-6 -or equivalent

References

  1. Pfister, H., Lichtman, J., Reid, C. . The Connectome Project. , (2009).
  2. Briggman, K. L., Denk, W. Towards neural circuit reconstruction with volume electron microscopy techniques. Curr Opin Neurobiol. 16, 562-570 (2006).
  3. Micheva, K. D., Smith, S. J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron. 55, 25-36 (2007).
  4. Arenkiel, B. R., Ehlers, . Molecular genetic and imaging technologies for circuit based neuroanatomy. Nature. 461, 900-907 (2009).
  5. Capecchi, M. R. Altering the genome by homologous recombination. Science. 244, 1288-1292 (1989).
  6. Luo, L., Callaway, E. M., Svoboda, K. Genetic dissection of neural circuits. Neuron. 57, 634-660 (2008).
  7. Shimomura, O., Johnson, F., Saiga, Y. Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell Comp Physiol. 59, 223-239 (1962).
  8. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Prasher, D. Gene fluorescent protein as a marker for gene expression. Science. 263, 802-805 (1994).
  9. Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science. 312, 217-224 (2006).
  10. Micheva, K. D., Smith, S. J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron. 55, 25-36 (2007).
  11. Svoboda, K., Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron. 50, 823-839 (2006).

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Cite This Article
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).

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