IMAGING THE NERVOUS SYSTEM 1

Histology and Microscopy

 OVERVIEW

Microscopes
 Light  Histological Methods
 Fluorescent  Tissue preparation
 Confocal  H and E
 EM (Scamming and TM)  Nissl
 Golgi
 Osmic acid
 Immunohistochemistry
LEARNING OUTCOMES

By the end of this lecture you will:

 Know the difference between types of microscopes and some basic

applications
 Have a basic understanding of tissue preparation for microscopy
 Be able to identify different commonly used stains, and understand what
they label and some basic applications.
 Be able to identify neurones, glia and myelin in relevant stains.
WHY LOOK AT THE NERVOUS SYSTEM?
 LIGHT MICROSCOPE

Bright Field Microscopy

 Uses visible light

 Two lenses
 Ocular/eye piece (10X magnification)
 Objectives (4x, 10x and 40X)
(Times one by the other to get total
magnification)
 FLUORESCENT MICROSCOPY
 The sample is the light source
 Fluorescent material emits energy (visible
light) when excited by high energy light
of a specific wave length
 Excitation light is shone onto the sample.
 A filter only lets the wave length that will
make fluorescent marker of interest emit
light through.
 This excites electrons in the sample,
which release a photon (light) when they
relax back to the lower energy level.
 A second filter only lets emitted light
through (not the excitation wave length)

http://slideplayer.com/slide/777953/
CONFOCAL MICROSCOPE

• Focused on one plane

• Removes background out of focus blur
• Allows 3D reconstruction

https://microscopysolutions.ca/
http://slideplayer.com/slide/777953/ 2012/05/31/any_way_you_slice_it/
 ELECTRON MICROSCOPE

 Use electron beam instead of light

waves
 High resolution
 Scanning and transmission

 Scanning – 3D image of surface

(bounces electrons off of the surface)
 Transmission – internal structure of https://uk.pinterest.com/pin/
481603753871604083/
specimens, passes beam through the
object

http://www.slideshare.net/nikki0529/neuroscience-lab-1
 PREPARING TISSUE FOR MICROSCOPY

 Tissue is preserved – Snap Frozen or fixed

 Tissue cut into thin sections
 Can be frozen or embedded in wax or resin to
do this
 Tissue is then mounted on to a glass slide (for
light microscopy)
 Staining
 Coverslip

 Can also image fixed or living cells /

specimens
https://www.youtube.com/watch?v=t9r2J2Yeooo
 STAINING

 Different staining methods allow

different cell types / proteins to be
visualised.

http://www.bioenno.com/catalogue/counterstain-kit
 HEMATOXYLIN AND EOSIN
(LIGHT MICROSCOPY)

Hematoxylin
Dark blue / violet. Positively charged/basic, binds to negatively
charged/acidic DNA and RNA in the nucleus, and RNA in the rough
endoplasmic reticulum (Lots in neurones, Nissl bodies).

Eosin
Pink. Acidic /Negative. Binds to positively charged amino acid side
chains (mostly found in the cytoplasm)

Stains all cells, determine different cell types dependent on

morphology

http://www.lab.anhb.uwa.edu.au/mb140/corepages/nervous/nervous.htm#CNS
http://www.lab.anhb.uwa.edu.au/mb140/corepages/nervous/nervous.htm#CNS
 NISSL STAIN
(ANILINE, THIONINE, OR CRESYL VIOLET)
(LIGHT MICROSCOPY)
http://www.slideshare.net/nikki0529/neuroscience-lab-1

 Basic dye that binds to negatively

charged nucleic acids (RNA and DNA)
 Neurons have a lot of rough
endoplasmic reticulum (as have to
make a lot of protein to support the
large cell membrane).
 Dye binds to RNA in rER and to DNA.
Making granular appearance of cell
body (Nissl substance).
 Stains all cells, so have to distinguish
cell based on morphology / amount of
Nissl substance, shape…
 Neurones have a pale nucleus,
and a prominent nucleolus. In
the cytoplasm Nissl bodies are
often seen.

Glia are smaller, and you can not

usually see much of their
cytoplasm. It is weakly stained.

It is difficult to distinguish
different types of glia with this
stain.

But astrocytes tend to oval

nucleus and oligodendrocytes
have a round nucleus
http://www.lab.anhb.uwa.edu.au/mb140/corepages/nervous/nervous.htm#CNS
 GOLGI STAIN
CAMILLO GOLGI 7 JULY 1843 – 21 JANUARY 1926
(LIGHT MICROSCOPY)

 Impregnates tissue with potassium dichromate and

silver nitrate
 Some cells are filled with microcrystals of silver
chromate
 Only stains a few cells randomly
 The cells it does stain, are filled completely. So
dendrites, axons, spines are clearly visible.
 As not all neurones are stained, it allows clear
visualisation of the structure of the neuron.
 Not specific to neurones, can stain glia

http://www.lab.anhb.uwa.edu.au/mb140/corepages/nervous/nervous.htm#CNS
 OSMIC ACID
(LIGHT MICROSCOPY)

 Osmic acid stains lipids (therefore

myelin) black
 It also fixes tissue, often used for EM.

 There are a number of other myelin

stains:
 Weigert stains
 Luxol fast blue
 Solochrome cyanine R
 Cross section of neocortex stained by three different methods; the six cortical layers are indicated.
Golgi stain Nissl stain Weigert stain

Molecular layer

External Granular layer

External Pyramidal layer

Internal Granular layer Outer band of Baillarger

External Pyramidal layer

Inner band of Baillarger

Multiform layer

From Broadmann, K: VergleichendeLokalisation lehre

der Grosshirnrinde in ihren prinzipien dorgestellt auf
Grund des Zellenbaues, Leipzig, 1909, JA Barth
 IMMUNOHISTOCHEMISTRY
(LIGHT OR FLUORESCENT MICROSCOPY)

Allows specific proteins to be stained

Can distinguish between different cell types based on
the antigens / proteins they express
Can look for gross changes in protein expression
Can look for the location of proteins on / within cell

http://www.lab.anhb.uwa.edu.au/mb140/corepages/nervous/nervous.htm#CNS
IMMUNOHISTOCHEMISTRY

Direct method:
 An antibody against the antigen of
interest is produced, by introducing the
antigen of interest into an animal, and
collecting the antibodies produced.
 The purified antibody is labelled with a
fluorescent tag (fluorescent
microscopy), or an enzyme complex that
will produce a dye precipitate in the
precise of its substrate (light
microscopy).
 When the antibody solution is added to
the tissue of interest, the antibody binds
to the antigen.
IMMUNOHISTOCHEMISTRY

Indirect method:
 The primary antibody will bind to the
antigen.
 The secondary antibody binds to the
primary antibody.
 The secondary antibody is labelled
with a fluorescent tag, or an enzyme
complex .
 This amplifies the signal, as many
secondary antibodies can bind to 1
primary antibody.
 EXAMPLES
NeuN – against a protein express in neuronal nuclei. Glial fibrillary acidic protein (GFAP) is an intermediate filament
Does not stain the cytoplasm Helps astrocytes to maintain their shape

Stains MOST neuronal cell types

 EXAMPLES
IBa1 is specifically expressed in macrophages /
microglia. It is up regulated during the activation of
these cells.
TH (Tyrosine Hydroxylase) enzyme which catalyses the
conversion of the L-tyrosine to L-DOPA. Expressed in
neurones that produce catecholamines: Dopamine, NA
and A.
http://www.abcam.com/tyrosine-hydroxylase-antibody-ab112.html
SUMMARY
 Seen the difference between types of microscopes
 Light
 Fluorescent
 Confocal
 Electron microscope
 Basic tissue preparation for microscopy
 Common stains and their uses
 H and E
 Nissl
 Golgi
 Osmic acid
 Immunohistochemistry