Confocal microscopy

in neurobiology

Gábor Kiszler PhD, 3DHISTECH Ltd.

• Organization of the nervous system

• Investigation of the sensory system

• Ultra modern microscope systems

• New histological technologies

• Pannoramic Confocal system

- Advantages

- Results

Presentation overview

Introduction

The nervous system

– the neural tissue

• Regulates and controls body functions

• Thinking

• Behaviour

• Makes us HUMAN

Introduction

Neurobiology - Neuroscience

Cell body Axon process Axon terminal output

Nucleus

Dendriteinput

Astrocyte Olygodendrocyte

Myelin sheathelectrical insulation

Impulse conduction

Introduction

Neurobiology - Neuroscience Synaptic gap

Introduction

Comparative neuroanatomy labor:

• Conventional light microscopy: Confocal, DIC

• Electron microscopy: TEM, SEM, IHC

• Immunohistochemistry

• Immunocytochemistry

• Molecular biology

• Cytometry

• Invertebrate model animals

An example

Investigation of the nervous system of Earthworm

• Primitive organization from the early stages of the

evolution

• Huge neural elements

- giant axons

- nerve cords

Easy examination

An example

The sensory system of the earthworm

- Primary sensory cells and sense organs- Basic functions:kemo-, mechano-, photoreceptors

Aim: 3D organization, distribution, exact localization

Early scientific results:Electonmicroscopy- ultrastructure

Convencional microscopy - morphology, IHC

An example

Methods:

GABA IHC - inhibitor

Neural tracing technique

- Mechanical

- Electrical

Confocal microscopical examination

- Optical sectioning

- 3D reconstruction

An example

The conventional microscope result

• Cross section of the animal

• 3D reconstruction

An example

The fluorescence microscope result

• Whole mount samples

An example

The confocal solution: Real 3D tissue imaging

The solution

Current questions in microscopy

1. What happened in the last five years in the field of light microscopy?

2. How does it change the thinking about microscopical imaging?

3. What new requests can emerge in the near future?

1.Resolution

The threshold of the optical resolution is limited by the wavelength of the light

Ernst Abbe (1873) – Diffraction limit: 0,2µm

New technologies: Confocal microscopyTwo photon microscopySuper resolution microscopy

„Microscopy becomes nanoscopy”Nobel committee

The confocal microscope

• Signal to noise ratio

• Optical slicing 3D imaging Normal

Confocal

The confocal microscope system

• Laser illumination

• Filter and mirror box

• Laser amplifier

• Optical microscope

• Image recording system

• ...

Two photon imaging

• High resolution

• In vivo imaging

• Ca-imaging

Two photon imaging system

• Laser sources

• Laser amplifier

• Optical microscope

• Cell tracing system

• Electrophysiological setup

• Image recording system

• Special software modules

• ...

Super resolution

Eric Betzig

Stefan Hell

William Moerner

E-coli bacterium with normal fl and STED

• switchable fluorescent protein

jellyfish

• Near-field Scanning OpticalMicroscopy

• Stimulated Emission Depletion

Nobel Prize in Chemistry 2014

Superresolution

• Laser sources

• Laser amplifier

• Optical microscope

• Special real-time

recording and

visualization system

Developmental biology

Whole animal recording

Real time ontogenesis

The model animals:

• Zebrafish embrio: Danio rerio

• Fruit fly: Drosophila melanogaster

• Roundworm: Caenorhabditis elegans

The lightsheet microscopy

• High resolution 3D

• In vivo imaging Embryogenesis

• Long term imaging

• Size limitation

Zeiss 2014

The lightsheet microscopy

• Laser sources

• Two separate light paths

• Multiview imaging

• Large sample investigation

• Optical sectioning effect with

parallel image acquisition

• Extremely fast 3D imaging

Microscopes: summary

• 3D fluorescence imaging is in the main focus

• The resolution is important:

- Molecular analysis and live cell imaging

• Huge region of interest:

- Whole animal recording

• Flexible solution with many upgrade possibilities

• These systems are very special

• Produced directly for scientific purposes

• Market opportunities are limited

New histological technique

2. Deep analysis

• Whole tissue recording

• High Z-resolution

• No background

• No crosstalk

• No artefact

Tissue clearing

Tissue clearing

Make the tissue transparent

Optical requests:

• High numerical aperture

Depth of field

• Large field of view

Clarity

Tisssue fixation Molecular network Remove other molecules

Clarity

• A method of making brain tissue transparent

Kwanghun Chung et all.: Structural and molecular interrogation of intact biological systemsNature 497, 332–337 (16 May 2013)

Clarity: See-through brains

3-D tour of intact mouse brain

Revolution in microscopical imaging

The connectome project

Brain cartography: mapping neural connections

„Neurons don’t use cellphones”

Connectivity data: „guides your thinking into how information flow can occur in the brain.”

3. Large investigation area

Human connectome project

• These investigations are mostly focused on electronmicroscopic

level, because the neural connections (synapses) are present on

ultrastructural magnification.

• The electron microsope concentrates only on a very small field

of view.

• These technologies need to have an overview information about

the investigated region to find the correct orientation.

Human connectome project

Summary

• Neural mapping in 3D is the main request in

neurobiological investigations

• The directions of the processes and the detection

of the connections are important

• The background can be eliminated

• Huge region of interest needs to be examined

• Big data needs to be analyzed

Conclusions

What do neuroscientists exaclty need:

• 3D fluorescence imaging

• Deep tissue imaging

• High resolution in Z-direction

• Whole tissue scanning

• Excellent image quality

Conclusions

Our solution:

Pannoramic confocal whole slide scanner microscope

The aim of the development:

• Compact solution: easy to install in the laboratory

• Full automatic: easy to use – spreading the technology

• Closed system: excellent fluorescent imaging

decreased malfunction possibilities

The Pannoramic Confocal system

Main features:

• Water immersion objective with high numerical

aperture excellent depth of field

• Solid state LED light sources

• Fast scanning speed

• High image resolution

• Optical knowledge is not needed for the calibration

• Brighfield and fluorescent scanning

• 3D visualization software is included

The Pannoramic Confocal system

Whole cerebellum digital slide

The Pannoramic Confocal system

Cell line with neural processes

High magnification image

Comparison: The system

Conventional confocal system Pannoramic Confocal

The conventional confocal system

• Space consuming: Two rooms are needed, because

of the several laser sources

• Complex system: Several malfunction possibilities

• Hign customization potential

• Cumbersome calibration process

• Intensive bleaching, because of the laser sources

• Can be used for a wide range of investigations

• High magnification objective

The conventional confocal system

• Compact, closed system

• Minimal beaching: sample can scanned multiple

times

• Huge area scanning possibility

• Closed system approach: not so wide application

field

• Easy to use: produces the final result fast

• The whole scanned sample can be presented in 3D

• Extremely fast scanning speed

The Bleaching

This is the main problem in confocal examinations, because the

confocal recording takes a long time and because of this the

bleaching effect is increased.

That is why we focused on decreasing the bleaching:

• The microscope is in a closed box

• The LED light source protects the sample

• The camera is triggered directly from the light source so the

sample is illuminated only during the exposure time

The result

Conventional confocal system Pannoramic confocal

Anti-Fade Mounting Medium

The most popular

• ProLong® Gold antifade reagent

- from molecular probes

• VECTASHIELD® Mounting Medium

- from VECTOR laboratories

• FluorSave™ Reagent

- from MERCK

Confocal publications

0

1000

2000

3000

4000

5000

6000

Confocal microscopy

1993 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

Why neuroscience?

Conferences in 2014

52%

31%

12%

5%

31.00018.000

7.000

3.200Registered participants

Thank you for your attention