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