Wesley C. Chang, Christopher G. Keller and David W. Sretavan- Isolation of neuronal substructures...

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Journal of Neuroscience Methods 152 (2006) 83–90

Isolation of neuronal substructures and precise neuralmicrodissection using a nanocutting device

Wesley C. Chang a,∗, Christopher G. Keller b, David W. Sretavan a

a Departments of Ophthalmology and Physiology, Program in Neuroscience, Bioengineering Graduate Program,

University of California, 10 Koret Way, K110, Box 0730, San Francisco, CA 94143, USAb MEMS Precision Instruments, Richmond, CA, USA

Received 27 July 2005; accepted 18 August 2005

Abstract

We describe a set of microfabricated nanocutting devices with a cutting edge of less than 20 nm radius of curvature that enables high precision

microdissection and subcellular isolation of neuronal structures. With these devices, it is possible to isolate functional substructures from neurons

in culture such as segments of axons and dendrites, dendritic spines and Nodes of Ranvier. By fine-tuning the mechanical compliance of these

devices, they can also act as alternatives to costly laser capture microdissection workstations for harvesting specific neuronal populations from tissue

sections for analysis. The small size of the device (1 mm2× 100m) allows convenient insertion into researcher specific experimental set-ups. Its

ease of use and possibility for batch fabrication makes this a highly effective and versatile tool for tissue microdissection and the microanalysis of

neuronal function.

© 2005 Elsevier B.V. All rights reserved.

Keywords: Nanoknife; Neuron; Transection; Axonal injury; Dendritic spines; Nodes of Ranvier; Growth cones; Micro-electro-mechanical systems (MEMS); Laser

capture microdissection

1. Introduction

In recent years, significant progress has been made in uncov-

ering the genetic and cellular basis of a wide range of processes

in development, plasticity, behavior and diseases of the nervous

system. Given the highly compartmentalized nature of neurons

and the complexity of neuronal circuitry, a deeper quantitative

understanding will require the convenient and precise isolation

of functionally important neuronal subunits for microanalysis.

Experimental work with CNS and PNS entities such as dendritic

spines and Nodes of Ranvier would be enhanced by the avail-

ability of a set of small, easy-to-use laboratory tools that provide

reliable microscale isolation of specific neuronal substructures

for physiological, imaging and molecular studies. Our under-

standing of neuronal physiology in health and disease will also

benefit from economicaland readily available meansof neuronal

dissection from tissue sections.

Studies of axonal and dendritic function currently rely on

glass micropipettes to severe processes from cultured neu-

∗ Corresponding author. Tel.: +1 415 476 4135.

E-mail addresses: [email protected], [email protected]

(W.C. Chang).

rons or tissue explants in vitro (Araki et al., 2004; Dotti andBanker, 1987; Eberwine et al., 2001; Ju et al., 2004). While

this method can cut many axon segments simultaneously in

bulk for biochemical studies, cutting by micropipettes involves

mechanical shearing and stretching that may lead to unin-

tended cellular and molecular changes. More importantly, this

method of shearing cannot be used to isolate axon and den-

dritic segments smaller than tens of microns in length or be

used within a densely populated and complex axonal or den-

dritic field. Laser ablation systems offer more precise control

(Emmert-Buck et al., 1996; Schutze et al., 1998) but are costly

and require large dedicated workstations that are often diffi-

cult to integrate into researcher-specific experimental set-ups.

Laser cutting is also usually achieved through the localized

heating and vaporization of biological tissues and thus stud-

ies examining real-time molecular function in small subcel-

lular compartments using this approach may require careful

interpretation.

Here we describe a set of small, versatile microfabricated

nanocutting devices that have high precision capabilities such

as the isolation of single dendritic spines and Nodes of Ranvier.

In addition, these devices can effectively dissect entire neuron-

specific layers within tissue sections and thus serve as low

0165-0270/$ – see front matter © 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneumeth.2005.08.020



84 W.C. Chang et al. / Journal of Neuroscience Methods 152 (2006) 83–90

Fig. 1. Design, fabrication and mechanical properties of the nanocutting device. (A) Scanning electron microscope (SEM) image of serpentine flexure suspension

frame fabricated from single crystal polysilicon. Scale = 200m. (B) SEM image of silicon-nitride nanoknife. The nanoknife is an elongated pyramidal-shaped

structure with the top edge serving as the knife or cutting edge. The dimensions of the nanoknife are dictated by the lithographic masks used during the fabrication

process and can be modified as needed. The regions marked by the asterisks represent artifacts from the fabrication process and do not interfere with device function.

Scale = 20m. (C)SEMimageof a crosssectionof theknife showing theradiusof curvature at thecuttingedge. Thesilicon-nitridedeposited bylowpressurechemical

vapor deposition into the etched silicon mold produced a sharp cutting edge of 20 nm in radius of curvature. Scale= 100nm. (D) SEM image of the nanocutting

device consisting of a nanoknife assembled within a suspension flexure. Scale = 200m. (E) Schematic diagrams showing (in cross-sections) the fabrication steps

for the nanoknife. See text for details. (F) Schematic diagrams showing (in cross-sections) the fabrication steps for the suspension flexures. See text for details. (G)

Results of finite element modeling analysis show that the torsional deflection in the “elbows” of the serpentine flexures as well as flexure length govern the overall

stiffness of the nanocutting device during the primary cut stroke. The color-coding indicates the magnitude of local principle strain in the structure, with scale ranging

from 0 (blue) to a maximum strain of 7 × 10−6 (red) (note: strain is dimensionless, L / L) for a 1N load. (The vertical displacement indicated by the arrow has

been accentuated for clarity.) The number of turns in the serpentine flexure can be increased or decreased to produce devices with different compliances that are each

best suited for isolation of specific types of tissues (see also Table 1). (For interpretation of the references to color in this figure legend, the reader is referred to the

web version of the article.)

cost alternatives to conventional laser capture microdissection

systems. The cutting device is created by attaching a nanoknife

to a silicon microsuspensionthat provides compliant mechanical

support. Its small overall size allows convenient insertion into

existing experimental workstations. In this article, we present

the design and fabrication of these nanocutting devices, describe

their tunable mechanical properties and demonstrate their abil-

ity for simple yet highly precise microdissection in a number of

systems of interest to the neuroscience community.

1.1. Device description

The device is an assembly of two components produced from

silicon-based microfabrication: a micromechanical suspension

(Fig. 1A) and a nanoknife (Fig. 1B). The mechanical suspension

is a planar, contiguous silicon structure consisting of a 1 mm2

frame and a pair of serpentine flexures that permit multiaxial

motion for the centrally placed nanoknife. The knife itself is

an optically transparent elongated pyramidal structure, made of

silicon-nitride, with the top edge of the structure serving as an

ultrasharp cutting edge. The radius of curvature of the cutting

edge is approximately 20 nm (Fig. 1C) as determined by scan-

ning electron microscopy (SEM) and is thus on the order of the

diameter of a single microtubule or the width of a synaptic cleft

(Kandel et al., 2000; Scott et al., 2003). The cutting edge can

be fabricated in lengths from one to several hundred microns

depending on intended use. When assembled, the nanoknife

occupies a central position within the supporting suspension

frame, with the pyramidal apex, which forms the knife-edge,

protruding out perpendicularly from the plane of the device

(Fig.1D). Duringuse, theassembled device is simplymounted at

one end of a 10cm longshaft, which in turn isheld and precisely

positioned by a standard micromanipulator. The micromanipu-

lator also delivers the cutting stroke. No additional equipment is

Table 1

Compliance of serpentine flexure with varied number of turns

No. of turns Compliance (N/ m) Tissues

0 >15.4 Tissue microdissection

1 15.4 Tissue microdissection

2 7.7 Axons (myelinated)

Nodes of Ranvier

3 5.1 Axons (unmyelinated)

Dendrites

4 3.9 Dendritic spines

5 3.1



W.C. Chang et al. / Journal of Neuroscience Methods 152 (2006) 83–90 85

Fig. 2. Examples of axons and dendrites isolated using the nanocutting device. (A) Axon field in culture prior to a cutting sequence. Arrow points to an embryonic

mouse retinal ganglion cell axon. The dark rectangular-shaped structure represents the outline of the nanoknife held above the plane of the axon. (B) The nanoknife

has been lowered into position. The axon is visible through the transparent knife. Scale A, B = 100m. (C) The knife is lowered an additional amount to executecutting. (D) The cut (arrow) in the axon is visible after removal of the nanoknife. Scale C, D = 20m. (E, F) Photos of an additional axon before and after cutting

(arrow). Scale = 20m. (G) A pair of axons that was simultaneously cut using a nanoknife with a 200m long cutting edge (arrows). Scale = 20m. (H) DIC image

of an E16 hippocampal neuron from a GFP transgenic mouse with extensive dendritic arborization after eight days in culture. The boxed region is shown in higher

magnification in I–K. Scale = 20m. (I) Fluorescence image of a portion of the dendritic arbor. (J) DIC image of dendritic arbor after cutting. One dendrite within

the complex field has been isolated (arrow). (K) Fluorescent image of the image shown in J. Scale I–K= 10m. (L) Examples of cuts in unmyelinated (arrow) and

myelinated (arrowhead) sciatic nerve axon from an adult mouse. (M) Cut in adult mouse myelinated sciatic nerve axon. Scale L, M = 10m. (N) A pair of adult

sciatic nerve axons from a GFP transgenic mouse. (O) Same axons as in N. The axon on the right has been cut and retains bright GFP fluorescence. The top half of

the cut axon was removed for greater visibility. Scale N, O = 10m.

necessaryand thesmallthin planargeometryof thedevice allows

it to be easily inserted, used and then removed from commonly

used laboratory set-ups for electrophysiological, cell culture, or

imaging studies.

1.2. Microfabrication

Both device components are fabricated using fabrica-

tion techniques originally developed for the manufacture of




silicon-based microelectronics. The nanoknife and the suspen-

sion frame are fabricated separately. The nanoknife is fabricated

by first creating a mold in singlecrystal silicon by theanisotropic

etching of single crystal silicon using a lithographically pat-

terned window (Fig. 1E). This highly directional etching occurs

nearly along crystal planes and produces a pyramidal trench

with a molecularly thin bottom edge. This faceted trench serves

as the mold for the nanoknife. The length of its bottom edge

corresponds to the length of the knife-edge and can be altered

by changing the size (length minus width) of the original etch

window. A 1m thick film of silicon-nitride is then conformally

deposited by low pressure chemical vapor deposition (LPCVD)

into the silicon mold, precisely replicating the mold geometries,

including the sharp edge. The silicon mold is removed by etch-

ing away the backside of the single crystal silicon substrate to

expose the thin,opticallytransparent butmechanically stiff pyra-

midal silicon-nitride film that serves as the “outer skin” of the

nanoknife.

The supporting frame and suspension is fabricated as a

contiguous piece in single crystal silicon (Fig. 1F). The two-dimensional footprint of the structure, including the shape of

the frame and flexures, is patterned via photolithography onto

the surface of the silicon substrate. This pattern is then etched

into the substrate, and the etched component is released. The

shape, size and configuration of the frame and suspension can

be explicitly patterned and thus its mechanical properties can be

explicitly tuned and device footprints substantially smaller than

1 mm2 can be fabricated.

1.3. Compliance tuning

Since a major design goal is to produce a singleset of versatiledevices capable of severing and isolating subcellular compart-

ments as well as neuronal microdissection in tissue sections, it

is necessary to match the range of cutting forces delivered by

the nanocutting devices to the mechanical properties of the tar-

get tissues to ensure effective cutting while limiting wear on the

nanoknife and prolong device longevity. In its current configura-

tion, compliance of the cutting device is governed mainly by the

mechanical properties of the serpentine flexures. During the cut-

ting stroke, the primary motion of the nanoknife is perpendicular

to themajorplaneof thedevice,and device stiffness in this axis is

dictated by the torsional deflection in the “elbows”of the serpen-

tine flexures (Fig. 1G). Finite element modeling of suspensions

with different numbers of switchbacks in the serpentine flexure

was used to calculate increases in device stiffness with progres-

sively fewer switchbacks (Table 1). From experimentation, we

determined that nanocutting devices with high compliance are

optimal for cutting small structures such as single unmyelinated

axons, while devices with lower compliance (higher stiffness)

are required for precise cellular microdissection from fixed tis-

sue sections.

1.4. Device performance: isolation of axons and dendrites

We found the nanocutting devices to be highly effective and

easy to usefor isolatingboth axonaland dendriticprocesses from

neurons in vitro. Axons from retinal ganglion cells (Fig. 2A–G)

and dendrites of hippocampal neurons (Fig. 2H–K) were imme-

diately severed upon contact with the nanoknife. There was

minimal lateral movement and shearing of the cellular pro-

cesses, resulting in clean and sharp ends. Given the small size

of the nanoknife and its optical transparency, it was possible to

reach into a complex dendritic field under microscope guid-

ance to sever a specific process, leaving adjacent structures

untouched (Fig. 2H–K). Unmyelinated and myelinated axonsfrom acutely isolated adult mouse sciatic nerves (Fig. 2L, M)

and optic nerves (not shown) were also tested. The nanocutting

device produced uniform and repeatable cutting of both types of

axons, serving as a very precise microscale surgical scalpel. The

high tensile strength of silicon-nitride (Ciarlo, 2002), together

with a suspension design that limited the forces impinging on

the nanoknife-edge, produced a robust nanocutting device. The

knives can be coated with 3% bovine serum albumin or perflu-

oropolyether (Ausimont Fomblin) to minimize tissue sticking.

(The perfluoropolyether lubricant forms a molecularly thin layer

to cover the knife surface but does not dissolve in aqueous solu-

tions.) We have used a single device repeatedly in more thantwo hundred cutting cycles without any observable degradation

in performance. The nanocutting devices do not require special

handling or storage and we have successfully used devices that

have been stored in air at room temperature for up to two years.

Silicon and silicon-nitride based devices can also be autoclaved

for use in sterile conditions. Furthermore, if necessary, the knife,

along with its entire support structure, are strong enough to with-

stand gentle wiping and are resistant to a variety of cleaning

solvents.

Axons and dendrites obtained from GFP transgenic mice

were cut and tested for membrane resealing. Cells are known to

possess an intrinsic calcium-dependent self-repair mechanism

and areas of membrane damage are repaired via vesicle exocy-

tosis that is similar to neurotransmitter vesicles release (McNeil

Fig. 3. Isolation of neuronal subcompartments and tissue microdissection of neuronal subpopulations. (A) Brightfield image showing myelinated sciatic nerve axons

from an adult mouse. The arrow points to a Node of Ranvier of interest. (B) Brightfield image of same axons as in A. The Node of Ranvier has been isolated using the

nanocutting device. The 5m long isolated segment consists of the nodal region and a portion of the paranodal region. (C) Immunolocalization of sodium channels

in the Node of Ranvier (arrow) shown in A. (D) Fluorescence image of B showing sodium channels within the isolated Node of Ranvier. Scale A–D = 20m. (E)

Brightfield image showing a dendritic spine of interest (arrowhead) on a rat hippocampal neuron after eight days in culture. (F) The same dendritic spine after

removal of its distal half. The remaining proximal stump is indicated by the arrow. Scale E, F = 10m. (G) Brightfield image showing dendritic spines (arrowheads)

on a hippocampal dendrite. (H) Both dendritic spines have been cut leaving the distal spine regions as isolated compartments (arrows). Scale G, H = 10m. (I) Ten

micron thick cryostat section of the retina from an adult mouse. The cryostat section is attached to a plastic membrane for microdissection. Arrow indicates location

of retinal ganglion cell layer. (J) The same retinal cryostat section shown in I after microdissection using the nanocutting device. (K) The dissected tissue containing

the retinal ganglion cell layer (arrow). Scale I–K = 100m.




and Steinhardt, 2003; Steinhardt et al., 1994; Zeng et al., 1995).

Resealing has also been described for invertebrate axons, PC12

cell axons and developing DRG axons in culture injured by

glass micropipettes (Detrait et al., 2000; Godell et al., 1997;

Howard et al., 1999). We have tested the nanocutting devices on

adult tissues obtained from transgenic animals that express sol-

uble cytoplasmic GFP under the -actin promoter (Okabe et al.,

1997). The results showed that adult mammalian CNS and PNS

axons as well as CNS dendrites also have the capacity for self-

repair and readily retained GFP after cutting. (Axon resealing

shown in Fig. 2N, O). In a recent study of local protein synthesis

in isolated dendritic segments (Ju et al., 2004), it was reported

that only 25% of dendrites severed using glass micropipettes

remained sufficiently structurally intact for experimental use.

We have found that greater than 95% of hippocampal dendritic

segments isolated by the nanoknife remained structurally intact

with no blebbing or diminution of GFP fluorescence. The highly

consistent sharp cuts produced by the nanocutting device may

make this instrument a good choice for studies of physiological

processes in isolated axonal and dendritic compartments.

1.5. Nodes of Ranvier and dendritic spines

In order to determine whether the nanocutting device can

operate at an even smaller scale, we tested its performance in

isolating functional substrucures of interest such as Nodes of

Ranvier in peripheral nerve axons and dendritic spines from hip-

pocampal neurons. Individual myelinated axons from the sciatic

nerves of adult mice were isolated (Fig. 3A) and the sodium

channels that mark the Nodes of Ranvier were visualized by

antibody staining (Fig. 3C). Individual Nodes of Ranvier could

be isolated using the nanocutting device resulting in a smallsegment containing sodium channels together with a portion

of the paranodal region on either side (Fig. 3B, D). The abil-

ity to specifically isolate an individual compartment responsible

for saltatory action potential conduction may further our under-

standing of channel protein trafficking, molecular mechanisms

of myelination and the pathology of demyelinating diseases.

Dendritic spines contain the postsynaptic apparatus of most

excitatory CNS synapses and are thought to be the site of synap-

tic plasticity (Hering and Sheng, 2001; Kasai et al., 2003; Tsay

and Yuste, 2004). The dendritic spines of hippocampal neu-

rons maintained in culture for eight days were cut using the

nanocutting device. It was relatively easy to specifically target a

particular dendritic spine for isolation. Dendritic spines can besevered in half, leaving a proximal stump (Fig. 3E, F), or their

distal ends can be isolated as separate compartments (Fig. 3G,

H). The high degree of precision by which important neuronal

substructures, such as single dendritic spines, could be isolated

may enable new studies of plasticity, learning and memory at a

much finer level of spatial resolution.

1.6. An alternative to laser capture microdissection

Studies of regional specificity in the nervous system have

been greatly aided by the ability to microdissect desired neu-

ronal subpopulations or white matter regions from frozen tissue

sections for molecular and biochemical analysis. Currently, this

type of tissue harvesting is typically performed using dedi-

cated laser capture microdissection systems. Such systems have

the disadvantage of being relatively high cost, large, stand-

alone workstations that are not widely disseminated. By fitting

nanoknives with suitably low compliance suspensions, nanocut-

ting devices can serve as small, low cost instruments that enable

the precise dissection of subregions of interest from frozen cryo-

stat sections, comparable to what is achieved using a dedicated

commercial laser capture microdissection system. Fig. 3I–K

shows the dissection of the retinal ganglion cell layer (arrow)

from a 10m thick cryostat tissue section from an adult mouse

retina. The tissue section was transferred from the cryostat onto

a commercially available microscope slide covered with plastic

membrane (Leica). Microdissection was performed on a stan-

dard inverted laboratory microscope simply by using a nanocut-

ting device mounted on a micromanipulator and with the cutting

path manually traced by movement of the microscope stage. The

dissected piece (Fig. 3K) consisted of a 25m wide strip of

retinal tissue containing the retinal ganglion cell layer (arrow)along with a section of the plastic membrane. Although only

rectangular-shaped pieces have been isolated from tissue sec-

tions thus far, the use of knives with shorter knife edges (1 m)

should make it possible to trace and cut out any arbitrary shaped

region from tissue sections. The ability of simple nanocutting

devices to perform tissue microdissection potentially make them

a widely disseminated, low cost alternative to conventional laser

capture microdissection systems.

1.7. Studies of de novo growth cone formation

Given its ability for repeatable and precise cutting actionwithout the need for destructive tissue ablation, the nanocut-

ting device can be a useful investigative tool for studies of

axonal responses to injury and growth cone regeneration. The

phenomenon of new growth cone formation from a cut axon

stump has previously been studied in vitro in invertebrates and

DRG neurons, where glass micropipettes were the instrument of

choice (Godell et al., 1997; Howard et al., 1999; Ziv and Spira,

1998). Following injury to the mammalian nervous system in

vivo, injured axons in the PNS, and to a more limited extent

axons in the CNS, will form a new growth cone, exhibit other

forms of axon terminal remodeling, and attempt to re-extend

(Allcutt et al., 1984; Cajal, 1928; Howard et al., 1999; Kulbatski

et al., 2004; Selles-Navarro et al., 2001; Zeng et al., 1995 ). Themolecular mechanisms that control how mammalian axons or

dendrites recover from injury to issue a new growth cone are of

substantial interest for understanding the cell biology of neuro-

trauma and process regeneration. These molecular mechanisms

have been difficult to address without a precise and repeatable

method that can selectively injure individual axons or dendrites

for study. Fig. 4 shows results from an experiment in which a

nanocutting device was used to sever a specific dendrite within

the complex dendritic arbor of a hippocampal neuron in culture.

The cut dendritic process resealed (Fig. 4B) and within minutes

of injury, a new actively motile growth cone regenerated and

grew in place of the original dendritic process (Fig. 4C–E). The




Fig. 4. De novo dendritic growth cone formation in hippocampal neurons. Panels represent images of a portion of the dendritic field of a GFP transgenic mouse

hippocampal neuron after eight days in culture. (A) The arrow points to the site on the dendrite where the cut will be made. (B) Immediately after cut. (C) New

growth cone formation after 8min. (D) Further elaboration of the growth cone at 10min. (E) At 15 min. Scale A–E = 10m.

nanocutting device thus provides an easy to produce and highly

repeatable model of single axon or dendrite injury. The viability

of the parent neuron and the ability of the cut stump to regrow

also demonstrate the suitability of the nanocutting devices for

usein biological studies. Given thesimplicity of device design, it

should be possible to combine a nanocutting device with phar-

macological and molecular perturbation protocols to identifysignaling pathways and cytoskeletal elements required for this

adaptive response to injury.

2. Discussion

In this study, we describe a small, easy to use nanocutting

device that is ideally suited for the precise isolation of neuronal

substructures and neuronal subpopulations from tissue sections.

In addition to useful features such as its optical transparency

and the lack of autofluorescence, its small footprint allows easy

insertion into existing imaging and physiological experimental

workstations, immediately endowing these workstations withadditional utility at the microscale. Although the nanocutting

devices can be used repeatedly and can be sterilized if needed,

its manufacturing process allows large numbers of devices to be

produced simultaneously and these devices can almost be used

as disposable labware.

Thedesign of thedevice is focused on twoparameters that are

important for any cutting tool. One is the sharpness of the cut-

ting edge, and the other is the mechanism that ensures sufficient

cutting forces are delivered for the task at hand without com-

promising the integrity of the knife. These issues are addressed

by taking advantage of the excellent mechanical properties of

silicon-based materials and the ability of photolithographic pro-

cessing to form highly precise features such as a nanoscalecutting edge and the mechanically compliant suspension flex-

ures. Although the current generation of nanocutting devices

described here can serve a wide range of function by merely

being attached to a micromanipulator and using the microma-

nipulator to deliver the cutting stroke, future versions of the

device will incorporate an on-board force generation (actuation)

mechanism to perform cutting. With this improvement, a micro-

manipulator is required only to position the device and is then

removed. The use of an on-board self actuation mechanism pre-

vents external vibration from interfering with cutting and will

fully realize the microscale precision intrinsic to these fabricated

devices.

A reliable andhighly precise methodto isolate specific axonal

and dendritic compartments from a single identified neuron may

find application in a wide range of studies. This includes work

addressing issues of mRNA transport and translation in sin-

gle axons or dendrites (Eberwine et al., 2001; Kanai et al.,

2004; Willemsen et al., 2004), and protein transport and syn-

thesis in axons, dendrites and growth cones (Campbell andHolt, 2001; Hirokawa and Takemura, 2004; Sutton et al., 2004).

Additional research questions include the molecular mecha-

nisms of axonal and dendritic fate determination and factors

that govern the critical period during which axonal and dendritic

fates remain plastic (Bradke and Dotti, 2000; Dotti and Banker,

1987). The nanocutting devices may also find utility in areas of

potential clinical significance. Axonal segments that have been

severed from their cell bodies undergo a degenerative process

called Wallerian degeneration. The biological mechanisms for

this process appears to involve the ubiquitin-proteasome sys-

tem (Ehlers, 2004; Zhai et al., 2003), but the complete details

remain unknown. The ability to specifically trigger Wallerian

degeneration in an identified axon could be combined with other

molecular perturbation strategies to gain a deeper understand-

ing of the mechanisms involved. We believe that this nanocutting

device provides the neurobiological research community with a

low cost tool that exhibits a high level of reliability and precision

that will enhance the microscale analysis of neuronal function.

Acknowledgements

The authors wish to thank members of the Sretavan labora-

tory for their comments and technical assistance, and the UC

Berkeley Microfabrication laboratory for use of their facilities.

This research was conducted through the support of the SandlerFamily Foundation, That Man May See, NIH and the Research

to Prevent Blindness Foundation. W. Chang was supported by

NEI postdoctoral vision training grant awarded to UCSF.

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Wesley C. Chang, Christopher G. Keller and David W. Sretavan- Isolation of neuronal substructures...

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