typhoon - nhri

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1 /GE /

May 10, 2010

Typhoon -A Versatile Imaging System

產品專員李宗樹奇異亞洲醫療設備股份有限公司

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May 10, 2010

Typhoon OverviewApplications- Radioisotopic (Macroarrays)- Chemiluminescence (Westerns)- Fluorescence (Protein, DNA, Multi-Color)

Technical Summary

Advantages

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May 10, 2010

Typhoon Applications

Chemiluminescent

Radioactive

by label/detection

Fluorescent

Colorimetric

Non-radioactive

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Typhoon Applicationsby image analysis

2D

GFP

Microarray

Microplateanalysis

Differentialdisplay

1D gel/blot

BiotechBiotech

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May 10, 2010

Typhoon storage phosphor imaging

Storage phosphor detection of radioactivity

> 3H, 125I, 14C, 35S, 33P, 32P, 18F, 99mTc, etc.

The proven storage phosphor capabilities

> Limit of Detection (LOD): 2 dpm/mm2 for 14C> Linear Range: 5 orders with <7.5% std. dev.> Uniformity: + 5% over entire scan area

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May 10, 2010

Storage Phosphor - How it worksBaFBr: Eu+2phosphor at ground state

BaFBr - : Eu+3phosphor absorbs x-rays, a, b or g

BaFBr: Eu+2 *excited F-centre absorbs red laser light to enter unstable state

BaFBr: Eu+2phosphor returns to ground stateEmitting Europium luminescence

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Different from film exposureFilm- Not quantitative- Only one copy of the data, not

digital - Low dynamic range

Screen- Quantitative- Save time: 1/5 to 1/10

exposure than film- High dynamic range• Use Image Eraser to erase

Image• DO NOT use detergent on it

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ChemiluminescenceDirect imaging and quantification without exposures to films or screens

Chemiluminescence - Westerns •ECL•ECL Plus•SuperSignal West Femto•SuperSignal West Dura•CDPstar

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Typhoonchemiluminescence modehigh sensitivity

Hyperfilm ECL (PDSI)1 minute exposure

Typhoon ApplicationsECL Plus westerns

Fluorescent Westerns

•Enzyme-amplified>ECF (Enhanced Chemifluorescence)>DDAO Phosphate

•Direct>Cy3>Cy5


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May 10, 2010

Gel separation

Gel transferring

Blocking

1st Ab binding

2nd HRP Ab binding

ECL develop

Film or CCD detect

Gel separation

Gel transferring

Blocking

1st Ab binding

2nd ECL Plex Ab binding

*Fluorescent Scan

ECL PlexGeneral WB

532/526532/526SPSP

Tubulin detected inrat brain homogenate

Western ApplicationsECF

DDAO Phosphate633/670633/670BP30BP30

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532/580532/580BP30BP30

Tubulin detected inrat brain homogenate

Western ApplicationsCy3

Cy5633/670633/670BP30BP30

Western Applications

Cy3 & Cy5 Dual Target Western

Cy3 (green) - actinCy5 (red) - tubulin

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DNA Gel Stains

•Ethidium bromide•Vistra Green•SYBR® Green I•SYBR® Gold


(532/610(532/610DF30)DF30)

DNA Mass LadderDNA Mass Ladder

DNA Gel Stain ApplicationsEtBr

SYBR Green I(532/526(532/526SP)SP)

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May 10, 2010

SYBR Green I•agarose •384(96x4)PCRs/scan•front end to high throughput capillary sequencing

•Courtesy ART - Sunnyvale

DNA Gel Stain Applications

Differential Display Analysis

lung

liver

lung

liver

lung

liver

Rat Liver and Lung cDNA labeled Cy5 and electrophoresed on a 6% denaturing polyacrylamide gel.

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PicoGreenDNA Gel Stain Applications

532/526SP

PicoGreen - DNA Solution QuantitationLOD = 10 ng/mlLinearity = 200-fold

R2 = 0.9972

0

200

400

600

800

1000

0 500 1000

DNA (ng/ml)

Fluo

resc

ence

Lambda DNA

Protein Gel Stains

•SYPRO Orange•SYPRO Red•SYPRO Ruby (2-D gels)•DeepPurple


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Protein Stain Applications

532/610BP30

SYPRO Red

SYPRO Red Linearity

R2 = 0.9982

0.E+005.E+051.E+062.E+062.E+063.E+063.E+064.E+064.E+06

0 500 1000 1500

[BSA] (ng per band)

Fluo

resc

ence

BSA Protein

Protein Stain Applications

532/610BP30

SYPRO Ruby2-DSYPRO Ruby 1-D

Carrot Seed ProteinProtein Mass Ladder

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Fluorescent Covalent DNA LabelsPolyacrylamide Gels

•Fluorescein/FAM•HEX, TAMRA, ROX•Cy3, Cy5


Covalent DNA Labels Applications

•Agarose gel•+3 mm plane•Genomic

mapping•3-color:

>FAM (blue)>HEX (green)>ROX (red)

Mapping

(Courtesy Laurie Gordon, LLNL, Livermore, CA, USA)

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•Mnt repressor protein•180bp end-labeled operator DNA fragment•Sandwich gel•4-color: FAM (green), HEX (red), TAMRA (blue) and ROX (yellow)• FluorSep processed(Courtesy Chris Man, WashU, St Louis, MO, USA)

Gel Shift Assay

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May 10, 2010

Microarray

•Corning slide spotted with cDNAfrom APB ScoreCard control and cloned cDNA from Incyte Genomics

•Hybridized with 25pmoles of Cy3 and Cy5 labelled mRNA:

•Liver - Cy5

•Skeletal Muscle - Cy3

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May 10, 2010

Challenges for 2D electrophoresis

n Sensitivity (CBB)

n Linear Dynamic Range (CBB、Silver)

n Reproducibility (Gel-to-gel variation)

n Accuracy (Statistics needed)

n Consumption (Gels, time, samples)

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Presentation of the problem

6 gels made from the same sample, run in parallel (SYPRO Ruby)

Nice guy

Bad guy

Jörgen Östling, AstraZeneca R&D Mölndal, Sweden. June 2004

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Advantage of fluorescent detection:increased dynamic range

3-D plots of same spot areas on gel

2-D DIGE

Silver staining

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Measuring induced biological change

Differences due to disease state, drug treatment, life cycle stage

Induced biological change - what we want to measure

System variation

(gel-to-gel variation)

Experimental factorsDifferences in IEF/SDS-PAGE conditionsGel distortionsuser-to-user variation

Data analysisuser-specific editing and interpretation

Inherent biological variation

Intrinsic differences that occur within populationseg animal-to-animal, plant-to-plant or culture-to-culture, subjected to identical conditions

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Difference gel electrophoresis (DIGE)

Internal standard and 2 samples per gel

2-D DIGE using an internal standard

Cy5Cy3Cy2Standard Sample 1 Sample 2

Gel A

Cy2Standard

Cy3Sample 3

Cy5Sample 4

Gel B

Cy2Standard

Cy3Sample 5

Cy5Sample 6

Gel C

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2-D DIGE: internal standardizationSample 2 Sample 1

Gel A

Sample 3 Sample 4

Gel B

Standard (Cy2)

Standard (CyTM2)

Virtual elimination of gel variation: induced biological change with statistical accuracy capable of revealing differences in abundance of less than 10% between samples

Without internal standard

With internal standard

(Cy3)

(Cy5)

(Cy5)

(Cy3)

Normalisation of spots between gels

Not normalised to standard Normalised to standard1.79-fold with greater than 99.9% t-test confidence.

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1 color 2D vs. DIGE -Number of gels

2-D DIGE/internal std/DeCyderTM 1 colour 2-D

8 samples

2 samples per gel = 4 gels

x 3 biological replicates

0 gel replicates

= 12 gels total

= 6 runs by SE600

or 2 runs by DALTsix

= 6 days (3 days/run)

8 samples

1 sample per gel = 8 gels

x 3 biological replicates

x 3 gel replicates

= 72 gels total

= 36 runs by SE600

or 12 runs by DALTsix

= 36 days (3 days/run)

Benefits of Ettan™ DIGE

Reduce number of gels

Reduce hands on time

Reduce amount of sample required

Ensure accurate quantification of differences in protein abundance through the use of an internal standard

Increase the number of real hits*)

*) Friedman et al. Proteomics 2004; 4(3): 783-811

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DIGE paper reference

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May 10, 2010

Typhoon Overview

Applications> Radioisotopic (Macroarrays)> Chemiluminescence (Westerns)> Fluorescence (Protein, DNA, Multi-Color)

Technical SummaryAdvantages

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May 10, 2010

Typhoon – Versatility• Sample Requirement:

• Fluorescence (633nm, 532nm, 488nm)• Radioactive• Chemiluminescence samples

• Gels and sandwich gels• Blotting membranes• Macroarray and microarray chips• Micro-plates• Tissue sections (not in situ)• Radio-TLC & X-Ray diffraction

• Large format: 43 x 35 cm maximum

• Scans mounted & un-mounted screens• Most screens even from other suppliers

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May 10, 2010

Typhoon - Sensitivity• Optical Design:

• Confocal optics• 25, 50, 100, 200, 500, 1000 µm pixels• 5 log dynamic range• Twin PMT’s, one is cooled• Objective changer 0 & +3mm

• Emission and excitation:• 6 std emission filters, total 13 filter seats• 3 std beam splitters 560, 580nm & 630nm• 2 excitations 532nm (green laser) & 633nm (red laser)• Blue laser is optional

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Typhoon excitation & collection

Lens

532nm

633nmShut

ter

Asse

mbl

y

Scan Head

LaserLine Filter

FocusingOptics

Objective LensChanger Assembly

PMT & Filter Changer Assembly

PMT

1PM

T 2

Glass Platen

Gel

B/S

Focu

sing

Lens

Focu

sing

Lens

Mirrors

Lens 1

Colli

mat

ing

Lens

Lens 2

B/S B/S

Emis

sion

Filte

rsConfocal optics

Dual excitation

Dual emission

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May 10, 2010

Sample

Glass platen

Excitation Laser

Objective

Collection Lens

Fiber Optic Cable

Z-Plane MirrorLaser reflection filter

Advantages of Confocal Optics• Rejection of Scatter• Flat Field• Focus Control

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Galvonometer

Laser

Sample Tray

f-Theta Lens

Galvo Mirror

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May 10, 2010

Galvanometer scanning - Distortion problemGalvanometer

Typhoon

Two problems

1. Very obvious flaring of the high activity samples

2. Distortion of wells

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Typhoon Scan Setup• Focal Depth

> “Platen” setting– default for storage phosphor– choice for membranes, thin gels on platen

> “+ 3mm” (3 +/- 0.5mm above platen surface)– thick agarose gels– microplates– polyacrylamide sandwich gels

platen (support)

gel

Typhoon

3mm

free gel

(glass plate)

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May 10, 2010

Emission filters

Filter Fluorophore520DF30 ECL Plus, Cy2

526SP Fluorescein, FAM, SYBR, ECF, PicoGreen555DF20 TET, HEX, R-6G

560LP Multiple fluors (single channel scans)580DF30 Cy3, HEX, TAMRA, SYPRO Orange610DF30 ROX, C-X-R, SYPRO Red/Ruby, EtBr670DF30 Cy5

Emission filter assembly for custom filters is available 63-003399

* 520DF30 is optional filer

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May 10, 2010

Technical Manual: Fluorescence Imaging

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May 10, 2010

Excitation & Emission

575LP Filter

Emission Filter:

- The more is the better

- More specific is more better

Typhoon 580BP30

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May 10, 2010

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May 10, 2010

768 384 192 96 48 24 12 6 3 1.5 0.8 ng

All three laser lines offer same sensitivity for SYPRO Ruby- stained 1D protein gel

Pixel Size: 200µm; PMT: 550V; Focal Depth: Platen

Excitation: 457 nm Emission: 610BP30

Excitation: 532 nmEmission: 610BP30

S/N > 3

Excitation: 488 nmEmission: 610BP30

SYPRO Ruby

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May 10, 2010

Thanks for your attention

typhoon - nhri

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