Imaging the structure and organization of mouse
cerebellum and brain stem with second harmonic
generation microscopy
Xiuli Liu (刘秀丽)
1,2,†
, Daozhu Hua (华道柱)
1,2,†
, Ling Fu (付 玲)
1,2,
*,
and Shaoqun Zeng (曾绍群)
1,2
1
Britton Chance Center for Biomedical Phot onics, School of Engineering Sciences, Wuhan National Laboratory for
Optoelectronics–Huazhong University of Science and Technology, Wuhan 430074, China
2
MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of
Science and Technology, Wuhan 430074, China
*Corresponding author: lfu@mail.hust.edu.cn
Received May 4, 2017; accepted June 16, 2017; posted online July 11, 2017
To visualize the structure and organization of the brain is a fundamental requirement in the research of neuro-
science. Here, combining with two-photon excitation fluorescence microscopy and transgenetic mouse GAD67,
we demonstrate a custom-built second harmonic generation (SHG) microscope to discriminate brain layers and
sub regions in the cerebellum and brain stem slices with cellular resolution. In particular, the cell densities of
neurons in different brain layers are extracted due to the cell soma appearing as dark shadow on an SHG image.
Further, the axon initial segments of the Purkinje cell are easily recognized without labeling, which would be
useful for guiding micropipettes for electrophysiology.
OCIS codes: 170.3880, 180.4315, 190.4180.
doi: 10.3788/COL201715.090003.
The organization of neurobiological tissue containing the
arrangement of cells or the relative amount of cell numbers
in some special layers and brain regions is crucial to our
understanding of the structure and function of the
brain
[1–4]
. Magnetic resonance imaging (MRI) and positron
emission tomography (PET) have been employed into
brain structure and function research, but the relatively
low spatial resolution and tissue contrast hamper them
in providing organization morphology with a resolution
enough to distinguish cells
[5,6]
. Fluorescence microscopy
is powerful in the research of the brain structure and func-
tion with submicron spatial resolution
[7,8]
, while it often
needs labeling fluorescent indicators, such as fluorescent
proteins and chemical dyes, to provide an enough con-
trast. Coherent anti-Stokes Raman scattering (CARS)
microscopy, based on the molecular vibration in the tissue,
can achieve label-free biochemical imaging with high
spatial resolution, but the relative low sensitivity and
poor contrast of detection, due to an unavoidable lower
resonant background
[9,10]
, restrict its application to
brain study.
Second harmonic generation (SHG) microscopy, based
on the nonlinear interaction mechanisms, not only has
the capability of submicron resolution and inherent optical
sectioning, like two-photon excitation fluorescence (TPEF)
microscopy
[11,12]
, but also provides label-free imaging for
some structural proteins, such as collagen, actomyosin,
and tubulin
[13]
. Although the extracellular matrix in brain
tissue is short of collagen, SHG microscopy has succeeded
in imaging a label-free acute hippocampal slice and cul-
tured living neuronal cells, which have an abundance of
rich uniform polarity microtubules in their axon struc-
ture
[14–16]
. Combined some special dyes, SHG microscopy
also serves to record the electrical activity in intact neuro-
nal networks
[17–19]
.However,atpresent,therehasnostudy
to report on the structure and organization of the cerebel-
lum and brain stem using the label-free SHG imaging tech-
nique. Recently, studies show that, besides the major role in
motor function, the cerebellum might contribute to diverse
aspects of behavior
[20,21]
. The brain stem controls the flow of
messages between the brain and the rest of the body, and it
controls basic body functions, such as breathing, swallow-
ing, heart rate, blood pressure, and consciousness
[22]
.There-
fore, we now try to study the cerebellum and brain stem
using SHG microscopy.
In this Letter, by using a custom-built large area SHG
microscope, we first demonstrate it to characterize the mor-
phological features of the intact coronal brain slice of the
cerebellum and brain stem. From the SHG images, different
brain regions and subregions can be easily distinguished
from their morphology, and the detailed structures can
be depicted. Moreover, due to cell soma appearing as dark
shadows on the SHG image, we can easily extract the cell
number in each layer of cerebellum. Interestingly, on the
SHG image the soma and axon initial segment (AIS) struc-
ture of a Purkinje cell (PC) can be easily recognized as a
brush surrounded by the SHG signals, maybe from axons
of basket cells. Our results demonstrate that the custom-
built large area SHG microscopy provides a useful tool
for the research of brain organization and structure.
In this study, the GAD67-GFP adult transgenic female
mice were used. The Animal Experimentation Ethics
COL 15(9), 090003(2017) CHINESE OPTICS LETTERS September 10, 2017
1671-7694/2017/090003(5) 090003-1 © 2017 Chinese Optics Letters
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