Advanced Microscopy and Quantitative Imaging

le.ac.uk

Advanced Microscopy and Quantitative Imaging

Dr. Kees Straatman


CBS Centre for Core Biotechnology Services

EM

PNACL

Prof Andrew Tobin (Director)

AIF

Ila Patel (Manager)

AIF

BBASH

Geneta

Workshops

Level 3

Facilities

Protex

Biomarkers

MRI


CBS Centre for Core Biotechnology Services

Prof Andrew Tobin

(Director)

Ila Patel (Manager)

Calendar booking

Student use of systems

AIF

Academic lead:

Prof Andrew Fry

MMC:

Dr Kees Straatman (chair)

Prof Andrew Fry

Dr Nicola Royle

Prof Nick Hartell

Prof David Twell


CBS Centre for Core Biotechnology Services

HWB

3 Nikon TE

1 Nikon Eclipse Ti-E

Leica SP5 CLSM

Typhoon scanner

BD FACSCanto II

RKCSB

Nikon C1Si CLSM

AIF

Adrian Building

Olympus FV1000 CLSM

Olympus CellR/ScanR

Cyto system

MSB

Zeiss MP7

Nikon TIRF

Olympus LV200


CBS Centre for Core Biotechnology Services

HWB

Technical support:

Tara Hardy

Bipin Patel

RKCSB

Technical support:

Shashi Rana

AIF

Adrian Building

MSB

Technical support:

Matt Barker


Focus on

� 2D/3D imaging

� Live cell imaging

� Multi position imaging

� F-techniques

� High content imaging/Quantitative imaging

� Image analysis and processing

� Data storage

� Training and teaching

Info on the AIF website


Available microscope systems:

Systems Slides Live cell

imaging/

autofocus

FRAP/

FLIP

TCS SP5 CLSM X X/- X X

FV1000 CLSM X X/- X X

C1Si CLSM X

Zeiss MP7 LSM

X/-

3 Nikon TE300 X (X/-)

eclipse Ti X X/-

Cyto system X

X

X X

X/- X X

FRET Microinjection

X

X

X X

cell^R/scan^R X X/+ X X

TIRF X X/+

Olympus LV200 X/-

X

X X

Screening Spectral

imaging

X

X

X

X

X

X

X

Karyo

typing

X X X


Available microscope systems:

� Resolution for all these systems is diffraction limited.

XY = (0.61 * λ)/NA

XZ = 2λ /(NA) 2

(Rayleigh criterion)

With being refractive index of the

mounting/immersion media


1. Confocal laser scanning

microscope

Scan pixel by pixel

sample

Z-series

3D-reconstruction

Laser Bleaching experiments (FRAP, FLIP)

Photoactivation (PA-GFP)


1. Confocal laser scanning

microscope

The CLSM is widely used in numerous disciplines for:

� High resolution optical sectioning

� Z-series with 3D reconstruction

� Live cell imaging

� Bleaching experiments

But is relatively slow


2. Multi-photon laser scanning

microscope

� Penetration depth CLSM ~ 30-50 µm

� Use of pulsed infrared laser increases penetration

depth to up to 1000 µm

� 20x NA=1 dipping objective

� No pinhole

CLSM

MP-LSM


3. Total internal reflection

microscope

n= refractive index


3. Total internal reflection

microscope

To study:

� Focal adhesion dynamics

� Membrane development/fusion

� Membrane receptor binding

� Uptake experiments

� Actin/microtubule dynamics

BUT:

System can also be used for normal FITC/TRITC/DAPI

imaging.


4. Bioluminescent imaging system

� Bioluminescence: emission of light by a living

organism as the result of a chemical reaction during

which chemical energy is converted to light energy.


4. Bioluminescent imaging system

Advantage:

Imaging at cell level; analyse cell hetrogenicity or

subcellular localization

Imaging for hours or days

BF Biolumin Merge Olympus

Nucleus

ER


4. Bioluminescent imaging system

Possible assays

� Promoter assay of clock genes for long period

� Promoter assay among heterogeneous cell populations

� ATP release assay

� Gene activity in morphogenesis and metamorphosis

� BRET imaging and imaging split assay are applicable


At present:

User AIF

Sample preparation Imaging systems

Data storage

Image analysis


5. Data server

User

Sample preparation Imaging systems

Image analysis

AIF

Data storage


5. Data server

� Large amount of data

� Microscopes at multiple locations

� Data analysis at different locations

� Easy data access

� Back ups

The web server is maintained by the computer centre.


6. Software

Adrian Building:

Phot0shop

Imaris

Huygens deconvolution

CellR/ScanR

ImageJ

Henry Wellcome Building:

Photoshop

Volocity

NIS- Elements

Leica LAS AF

ImageJ

RKCSB:

NIS-Elements


Deconvolution

Deconvolution


New equipment ‘wish list’

� Super resolution system

� Spinning disk system

� LEDs for TIRF

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