Continuous processing and evaluation of experimental data is highly needed

2.1. Organization of work, type of data

2. Helpful hints in the processing of experimental data

- Avoid this approach: „This year I am experimenting and next year is for the evaluation of what I have obtained“

- Spectrum of software must be managed

Type od data:

- Numerical data - raw data or data converted to the numerical form, may be obtained with certain tool (ruler) or equipment (spectrophotometer)

- Graphical data - very important set of results, their quantification is highly needed

- Structural data - analytical data, outcomes from sequencing machines, etc..

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Software used in modern laboratory:

- Software controlling machines - sometimes very complex (PCR cycler), the most important is the form of the output (table, text)

- Specialized software - very important in the field of image analysis and processing of sequence data

- General software - text and spreadsheet editors, graphical software, presentation software, mainly products of Microsoft

- Open source software - very good tools, often problems with the compatibility

- online software – mainly in bioinformatics

2. Helpful hints in the processing of experimental data

2.1. Organization of work, type of data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Windows Explorer or similar software (Windows Commander)

Protocols named according to date - the folder in format YYMMDD (081030). Must correspond to date in the laboratory book.

Never modify folder with raw data - copying to the folder with other files accompanying the experiment.

Back up data - external HDD or DVD or CD, online hard drives

Do not delay backuping!!!

Experiment must be always finished with complete documentation, do not start others without finishing the previous.

2. Helpful hints in the processing of experimental data

2.1. Organization of work, type of data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Windows Explorer

2. Helpful hints in the processing of experimental data

2.2. Data explorers, correct data handling and saving

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

2. Helpful hints in the processing of experimental data

2.2. Data explorers, correct data handling and saving

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Windows Commander

2. Helpful hints in the processing of experimental data

2.2. Data explorers, correct data handling and saving

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Bulk data processing, their editing and graphical output

Microsoft Excel – suitable for all basic jobs including statistics

Specialized statistical software - in case of need of more parameters, software Statgraphics, NCSS

2. Helpful hints in the processing of experimental data

2.3. Spreadsheets, statistical software, graph editors

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

2. Praktické postupy zpracování experimentálních dat

Microsoft Excel – whole protocol management, automation of numerical operations

2.3. Spreadsheets, statistical software, graph editors

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

2.3. Spreadsheets, statistical software, graph editors

Sigma Plot – line/scatter plots, bar charts, histograms

2. Helpful hints in the processing of experimental data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Notepad

- Avoiding excessive formatting

Plenty of online and stand alone applications for the processing of sequences

2. Helpful hints in the processing of experimental data

2.3. Processing of structural and sequence data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

2.3. Processing of structural and sequence data

Vector NTI(Invitrogen)

2. Helpful hints in the processing of experimental data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

ImageJ (NIH)

2. Helpful hints in the processing of experimental data

2.3. Image analysis, graphical software, presentation software

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

NIS elements(Lucia)

2. Helpful hints in the processing of experimental data

2.3. Image analysis, graphical software, presentation software

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

NIS elements (Lucia)

2. Helpful hints in the processing of experimental data

2.3. Image analysis, graphical software, presentation software

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Leica LSM

2. Helpful hints in the processing of experimental data

2.3. Image analysis, graphical software, presentation software

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Zeiss LSMbrowser

2. Helpful hints in the processing of experimental data

2.3. Image analysis, graphical software, presentation software

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

From the gallery of optical slices to the maximal projection

PIN3::PIN3:YFPMaximální projekce

2. Helpful hints in the processing of experimental data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Žádníková et al., Development 137, 607-617, 2009

Kleine-Vehn, unpublished

2. Helpful hints in the processing of experimental data

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8 bit – 256 grey scale (28)

12 bit – 4096 grey scale (212)

16 bit – 65536 grey scale (216)

FRAP in cells of BY-2 expressing AtPIN1-GFP

Prebleach Postbleach FRAP 30 min

Dynamic distribution of PIN1 auxin carrier in the plasma membrane of tobacco cells.

2. Helpful hints in the processing of experimental data

2.3. Image analysis, graphical software, presentation software

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

2. Helpful hints in the processing of experimental data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Vandenbussche et al., 2009, Development 137, 597-606

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Vandenbussche et al., 2009, Development 137, 597-606

(a) (c)FM 4-64 PIN1-GFP Merged

Colocalization analysis

2. Helpful hints in the processing of experimental data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Jelínková et al., Plant Journal 61, 883-891, 2009

0

50

100

150

200

250

0 50 100 150 200 250

Intensity of PIN1::PIN1:GFP(green channel)

Absolute intensity

Inte

nsi

ty o

f F

M 4

-64

(re

d c

han

nel

)

(e)

2. Helpful hints in the processing of experimental data

Colocalization analysis

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Jelínková et al., Plant Journal 61, 883-891, 2009

Spectral detection using ZEISS META 510 detector, laser 477 nm

GFP peak

RFP peak

Interaction between two proteins - FRET analysis in vivo (PIN1-ADL1)

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480 500 520 540 560 580 600 620 640 660 680 700Em ission w avelength (nm )

585

1085

1585

Intensity

R O I 1 R O I 2 R O I 3 R O I 4 R O I 5

Average fluorescence intensity measured over the emission spectrum in the following region of interests:

ROI1 - middle cell plate

ROI2, ROI3 - growing ends of the cell plate

ROI4, ROI5 - transversal and longitudinal plasma membranes

500-520 nm unmixed

600-620 nm unmixed

ROI1- low FRET

ROI2, ROI3

- high FRET

ROI4, ROI5 - no FRET

1

2

3

45

Interaction between two proteins - FRET analysis in vivo (PIN1-ADL1)

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Irfan View – ideal for every day browsing through image documentation

2. Helpful hints in the processing of experimental data

2.3. Image analysis, graphical software, presentation software

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Corel Photo PaintAdobe Photoshop

2. Helpful hints in the processing of experimental data

2.3. Image analysis, graphical software, presentation software

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

Corel Photo Paint or Corel Photoshop – avoid excessive image adjustment!

2.3. Image analysis, graphical software, presentation software

2. Helpful hints in the processing of experimental data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek

2.3. Image analysis, graphical software, presentation software

Corel DrawAdobe Illustrator

2. Helpful hints in the processing of experimental data

B130P16E: Practical basics of scientific work Department Experimental Plant Biology FSCU http:/lhr.ueb.cas.cz/petrasek