INSTITUTE OF PROTEIN BIOCHEMISTRY, CNR Luini.pdf · - The accessory equipment comprises centrifuges, ovens, shakers and steromicroscopes for specimen preparation - Eppendorf microinjection

Correlative microscopy

ALBERTO LUINI

INSTITUTE OF PROTEIN BIOCHEMISTRY, CNR

ROMAN POLISHCHUCK

TIGEM

NAPLES

The Castellino Campus comprises a number of Institutes of the National Research Council (CNR), among

which the main ones are the IBP and the Institute of Genetics and Biophysics (IGB). Other Castellino

Institutes operate in the fields of photonics (IMM), informatics and computational modeling, and imaging

(IAC, ICAR). The Campus also hosts a large Telethon research Institute, the Telethon Institute for

Genetics and Medicine (TIGEM).

The IBP, IGB and TIGEM each possess an imaging facility. The IMM, IAC and ICAR develop technology

in the field of photonics and computational image analysis, and collaborate with the imaging facilities. In

addition, the IBP hosts the Telethon service Facility for advanced electron microscopy, tomography and

correlative microscopy. All of these facilities are managed in a coordinated manner that is regulated by

formal agreements among the Institutes, and are open to all of the members of the Castellino Campus.

The Telethon facility at the IBP has extensive experience in access, as it has offered service and assistance

to dozens of Telethon laboratories for a number of years. In many cases the support provided by facility

allowed Telethon-funded scientist to publish their data in top journals (see selected publications below).

he criteria and practice of access developed by the Telethon EM facility at IBP will be used to run the

EuroBioImaging proof of concept study (PCS).

THE IMAGING FACILITY AT THE CASTELLINO CAMPUS

The instrumentation available at the integrated Castellino facilities include

Light microscopy

- Confocal microscope Zeiss 710

- Confocal microscope Leica SP5

- Confocal microscope Leica SP2

- Fully motorized Leica DMI6000 light microscope equipped with incubation system for live cell imaging

- 3 Leica DMI6000 light microscopes equipped with image acquisition and analysis systems

- 2 Zeiss Axiophot microscopes equipped with image acquisition and analysis systems

Electron Microscopy

- FEI TEcnai G2 Spirit BioTWIN for EM tomography

- FEI TEcnai G2 Spirit BioTWIN for EM tomography (accessible in the CNR Institute ICTB)

- JEOL JEM-1011 electron microscope

- Leica Ultracut UCT ultramicrotome

- Leica EM FC7 ultramicrotome

- Leica EM TP automated tissue processor

Other equipment

- The accessory equipment comprises centrifuges, ovens, shakers and steromicroscopes for specimen preparation

- Eppendorf microinjection station

- 5 off line PC stations for EM and light microscopy image analysis

PERSONNEL EXPANSION

Short list of the diseases studied by TeEMCoF

Juvenile nephropathy

Ocular albinism

Juvenile hemochromatosis

Prion protein disease

Lysosomal storage diseases

Muscular dystrophy

Diabetes

Neurodystrophy

Optic atrothy

Roma

Napoli

Bari

Chieti

Siena

Padova

Milano

Perugia

Over 30 projects in

2005-2010

SERVICE

The Telethon Electron Microscopy Core Facility (TeEMCoF)

About 40 papers published in

2005-2010 comprising top journals

(Cell, Nature, etc.)

The Main Goal

To help Telethon funded studies of

genetic disease with electron

microscopy

Services

CLEM

EM tomography

Immuno-EM

Routine EM

Morphometry

Equipment use

Training

Advanced light microscopy is instrumental to

reach the ultimate goal of biological imaging,

to visualise single biomolecules and their

functions and interactions within the context of

live biological systems. The specific nodes will

provide the following key technologies:

Euro-BioImaging.

European Biomedical Imaging Infrastructure- from Molecule to Patient. A project on the European Roadmap for Research Infrastructures under coordination of EMBL and EIBIR

ADVANCED LIGHT MICROSCOPY NODES

Superresolution light microscopy

This node will provide access to

methods that improve the spatial

(and also temporal) resolution of

light microscopy imaging with an

emphasis on technologies

applicable for biological

applications and in live

specimens. Key technologies will

include stimulated emission

depletion (STED),

photoactivation localization

microscopy (PALM) as well as

the use of structured

illumination.

Functional imaging of live cells

This node will provide access to

methods that visualise

molecular function in live cells.

Key technologies will include

fluorescence lifetime imaging

(FLIM), fluorescence (cross)

correlation spectroscopy

(FC[C]S), photoactivation and

photobleaching (PA, FRAP),

single molecule imaging, and

novel fluorescent reporters of

biochemical reactions.

Correlative light and

electron microscopy

In this node it will be

possible to combine

dynamic functional assays

in live cells directly with

high resolution 3D

morphology at molecular

resolution by EM (cryo)

tomography. This node

will be intimately linked

to EM activities in the

ESFRI initiative

INSTRUCT.

High throughput microscopy

for systems biology

This node will contribute to

systems biology and rational

drug development by

providing access to automation

and high throughput in

advanced light microscopy

methods including ultra high

content screening of genome

level systematic perturbations

of biological systems such as

RNA interference over-

expression or small molecule

screening.

UNIQUENESS

CORRELATIVE MICROSCOPY

INTEGRATED MICROSCOPY

Attempts to apply different microscopy approaches

to the very same object of interest to integrate

information about the dynamics, fine structure and

composition of that object

VIDEO – EM

VIDEO - IF

VIDEO - IF - EM

A FORM OF SUPERRESOLUTION VIDEO MICROSCOPY

Principle

Characterize dynamics by video microscopy

Switch to electron microscopy

Characterizing a dynamic process in live cells

Choosing a particular structure (stage) of interest in the process being

studied

Taking two pictures of the same structure, one at the LM, and the other

at the EM level.

Integrating the information

Advantages

Magnification

Structure (reference space)

Disadvantages

Fixation

Probes

Retracing

Immunoperoxidase

Immunoperoxidase EM 3-D reconstruction

3-D tomography

Correlative video-electron microscopy

Polishchuk et al, J. Cell Biol. (2000)

A COMPLEX INVOLVED IN CARRIER FORMATION AND FISSION

BY SERIAL BLEACHING AND LONG IMMUNO-FRET

COMBINING VIDEO MICROSCOPY WITH

EM TOMOGRAPHY

CRYO-FIXATION

IMMUNO-GOLD LABELING AND TOMOGRAPHY

DETECTION OF MOLECULAR COMPONENTS AND OF

MOLECULAR COMPLEXES IN OBJECTS OF INTEREST



protein complex

FRET distance (Å) from D to A

D

A

Förster Resonance Energy Transfer

THE IDEAL CONFIGURATION FOR LONG RANGE FRET MULTIPLE ACCEPTORS, LONG FRET DISTANCE

Single donor molecule allows an higher efficiency of energy transfer for a

cumulative effect and eliminates the problem of homotransfer

single

donor

multiple acceptors

Molecular complex

distance (Å) from D to A

D

A

Increasing

FRET efficacy

DONOR-ACCEPTOR distance (Å)

SERIAL LABELING – ACQUISITION – BLEACHING

DIFFICULTY: THE SIZE OF THE COMPLEXES

DETECTION OF MOLECULAR COMPLEXES BY FRET

A COMPLEX INVOLVED IN CARRIER

FORMATION AND FISSION

CORRELATIVE MICROSCOPY INTEGRATES INFORMATION ON :

DYNAMICS

ULTRASTRUCTURE

MOLECULAR COMPOSITION

ASSEMBLY OF MOLECULAR MACHINERY

VSVG-GFP

FAPP-2

CERT

PI4K

VSVG-GFP

Release from 20°C block

Multiple labelling of budding post-Golgi carriers by serial bleaching



SERIAL BLEACHING OF PRIMARY ANTIBODIES

Schubert W, 2006

Protein complexes have different sizes, number of components (often many) and stability over time

a proteasome, a large

molecular complex

clathrin, a transient

protein assembly

A few moments of time-lapse video are enough to resolve an

Issue that years of microscopy on fixed cells have failed

to settle- Hugh R.B. Pelham (Nature. 1997)

Unfortunately, light microscopy cannot achieve a sufficiently

high resolution, so spectacular through it is, GFP technology

has its limits-Hugh R.B. Pelham (Nature. 1997)

The development of correlative microscopy can be somewhat arbitrarily

divided divided into two stages

1) An early stage from 1960 or earlier.

The goal is generally to look at the same field by both light (imuno-fluorescence)

and electron microscopy, to exploit the advantages of the two techniques:

the broad field of view of light and the resolution of electron microscopy.

2) A recent stage from 2000 onward.

Correlative microscopy as the first kind of GFP-based super-resolution video

microscopy with some disadvantages and a few substantial advantages over other super-

resolution video techniques that are being developed today

1986

Hayat MA. Correlative microscopy in Biology instrumentations and methods.

Academic Press. 1987

1995

TRANSPORT CARRIERS What kind of fine structure do they have?

With what organelles do they interact?

GFP-BASED CORRELATIVE LIGHT-ELECTRON MYCROSCOPY (CLEM):

EXPERIMENTAL PROCEDURE

1. DNA transfection of cells grown on

CELLocate coverslip.

2. GFP-based time-lapse confocal microscopy

and fixation of the cells.

3. Immunoperoxidase or gold labelling and

embedding in resin.

4. Cutting of serial sections and identification

of structure of interest at EM level.

knife

resin block

section

Perfect Loop

Slot grid

Collection of serial sections on the slot grids and their

analysis at the electron microscope

Serial sections

3D reconstruction Analysis of serial sections

Post-Golgi transport carriers in living cell

ER

Golgi

Plasma membrane

VSVG-GFP

Questions to address

• How are post-Golgi carriers

organized during different

stages of their life-cycle?

1. Formation

2. Transition through cytosol

3. Fusion

Golgi

Plasma membrane

1

2

3

Lumen

Cytosol

VSVG-GFP

VSVG is a widely used protein to study membrane transport

VSVG protein is a ts045 mutant strain of vesicular

stomatitis virus

40°C-VSVG is in the ER

20°C-VSVG is in the Golgi (TGN)

32°C-VSVG is moved out of the ER or the Golgi

Correlative light-electron microscopy of VSVG-GFP

labelled post-Golgi membrane carrier

VSVG-GFP

Polishchuk R.S. et al. ( 2000, JCB)

a b

c

d f g

r s

l k

n o p u

t

e

q

h i j

m

Golgi

Plasma membrane

Golgi

Plasma membrane

Formation of post-Golgi carriers

Formation of post-Golgi carriers

VSVG-GFP

VSVG-GFP

Ultrastructure of exit site of post-Golgi carrier

Polishchuk E.V. et al. ( 2003, MBC)

Ultrastructure of post-Golgi carrier formation site



1

2

3

4



1

2

3

4


Visualization of post-Golgi carrier fusion with

the plasma membrane by transmission EM VSVG-GFP VSVG-GFP Anti-VSVG HRP

Anti-VSVG HRP


VSVG-GFP

confocal microscopy Anti-VSVG gold

Visualization of post-Golgi carrier fusion with

plasma membrane by scanning EM


scanning EM

Constitutive transport from the Golgi to the

plasma membrane

Golgi

Plasma membrane


Golgi

Apical surface

Basolateral surface

ER

QUESTIONS TO ADDRESS

• Is ultrastructure of apical and basolateral

carriers different?

• Are apical and basolateral cargoes ever

packed into the same post-Golgi carrier?

•If so, how do they distribute within these

structures?

Transport of cargo proteins from the Golgi to

the apical and basolateral surfaces

Fluorescent proteins with apical and basolateral sorting

signals show polarized distribution in MDCK cells

GPI-GFP

Anti-occludin

XY

XZ

VSVG-GFP

Anti-occludin

XY

XZ

Apical marker Basolateral marker GPI-GFP

VSVG-GFP

Lumen

Cytosol

Lumen

Cytosol

Polishchuk R.S. et al. ( 2004, NCB)

Dynamics of Golgi-to-plasma membrane transport

of apical and basolateral markers in living cells

GPI-YFP

VSVG-CFP

merge


Ultrastructure of post-Golgi carriers containing both apical and

basolateral markers

GPI-YFP

VSVG-CFP

2

1

merge


Ultrastructure of Golgi-to-plasma carriers in

cell expressing apical and basolateral markers

2

2

1

1


Intracellular membrane transport

trans-Golgi

network (TGN)

Golgi

Endosomes

Lysosomes

Secretory

granules

Plasma membrane

ER

Clathrin

AP1

GGAs

MPR 1

2

3

Structure of Golgi-to-endosome carriers

GFP view

- pleiomorphic carriers

- frequently tubules

- tubular or vesicular clusters

EM view

- clathrin coated vesicles

- or vesicular clusters

CLEM as a tool to characterize newly-formed

endosomal transport carriers

Constructs

CD-MPR-GFP-Cation-Dependent Mannose 6-Phosphate Receptor-GFP

GGA1-GFP-Golgi-localized Gamma-ear -containing ARF-binding

protein1-GFP

Clathrin light chain-GFP

GGA-GFP positive carriers in living cells

Polishchuk R.S. et al. ( 2006, Traffic)

CLEM of GGA-GFP positive carriers

GGA-GFP

TRITC-dextran


CLEM of GGA-GFP positive carriers

GGA-GFP

TRITC-dextran


Clathrin-coat domains are always present on

GGA-GFP positive carriers


Three-dimensional organization of GGA-GFP

positive carriers


Variability of shapes through GGA-GFP and

MPR-GFP positive TC populations

vesicle

ovoid

tubule

grape-like


SUMMARY

• TGN-to-endosome TCs exhibit various morphology ranging

from vesicle-like to complex grape-like

• Frequently such TCs only partially covered with clathrin

• GGA adaptors are not restricted to the clathrin-coated domains

A laser-scanning microscopy with a resolution of 80-100 nm along

the xy axes and even the Z axis! (Hell, Nature Biotechnology, 2003)

2Pi angle 2Pi angle becomes a 4Pi angle

100 nm

PSF

nm

I

nm

4Pi-microscopy: Engineering the Point Spread Function (PSF)

Overcoming resolution limits in light microscopy

I

500 nm

200 nm

z

x

z

y

z

x

z

y

Confocal

4Pi

Perinetti G. et al. ( 2009, Traffic)

4Pi-CLEM Technique

DNA transfection EM processing

Serial Sectioning

3D Reconstruction

Comparison

Deconvolution EM serial recording

4Pi-microscopy Electron microscopy

3D Reconstruction

Confocal microscopy

Confocal Recording

Threshold Zero-Crossing

Threshold Zero-Crossing

Comparison

3D Reconstruction

4Pi Recording

Non –Confocal

Trasmission

Non Confocal

Fluorescence TEM

Confocal 4Pi

Mini-stack visualization under the microscopes

Mini-stack identification under the TEM

recording

MS01

MS03 MS02

MS04

MS05

MS01

MS02

MS04

MS05

MS03

Perinetti G. et al. ( 2009, Traffic)

TEM serial recording and Imod labelling

4Pi-EM overlap

Threshold 20% Zero-Crossing

z

y

z

y

y

x

y

x

4Pi-EM overlap

z

y

z

y

y

x

Perspectives

• Combination with advanced light microscopy methods

• Development of the new light-electron microscopy

probes

• Combination with proteomics approaches

Mario Negri Sud Institute and TIGEM

Alexander Mironov

Albeto Luini

Elena Polishchuk

Alexander Mironov Jr.

Giuseppe Perinetti

Alexander Spaar

Acknowledgements

MPI (Goettingen)

Tobias Muller

Alexander Egner

Stefan Hell

NICHD (NIH) Bethesda

Jennifer Lippincott-Schwartz

Juan Bonifacino

The Monte Carlo analysis:

a simulation of the system

single

donor

multiple

acceptors

protein complex

distance (Å) from D to A

D A

video-microscopy to survey

the cellular behaviour upon

the stimulating event

cells are fixed at the time of interest,

then sequential steps of:

immunolabeling with a donor and various acceptors

long range FRET assessments

selective bleaching of acceptors

staining another protein with new acceptors

TIME AND SPACE ONLY SPACE NOR TIME NEITHER SPACE INFORMATIVE

complexes composition should

be determined using standard

biochemical techniques

A typical experiment:

1994

Documents

INSTITUTE OF PROTEIN BIOCHEMISTRY, CNR Luini.pdf · - The accessory equipment comprises centrifuges, ovens, shakers and steromicroscopes for specimen preparation - Eppendorf microinjection