continuous processing and evaluation of experimental data is highly needed
DESCRIPTION
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D. 2. Helpful hints in the processing of experimental data. 2.1. Organization of work, type o f data. - PowerPoint PPT PresentationTRANSCRIPT
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 the approach „I am experimenting this year and next year is for the evaluation“
- 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 http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
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
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.1. Organization of work, type of data
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.
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.1. Organization of work, type of data
Windows Explorer
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.2. Data explorers, correct data handling and saving
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.2. Data explorers, correct data handling and saving
Windows Commander
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.2. Data explorers, correct data handling and saving
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
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Spreadsheets, statistical software, graph editors
2. Praktické postupy zpracování experimentálních dat
Microsoft Excel – whole protocol management, automation of numerical operations
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2.3. Spreadsheets, statistical software, graph editors
2.3. Spreadsheets, statistical software, graph editors
Sigma Plot – line/scatter plots, bar charts, histograms
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
Notepad
- Avoiding excessive formatting
Plenty of online and stand alone applications for the processing of sequences
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Processing of structural and sequence data
2.3. Processing of structural and sequence data
Vector NTI(Invitrogen)
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
ImageJ (NIH)
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
NIS elements(Lucia)
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
NIS elements (Lucia)
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
Leica LSM
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
Zeiss LSMbrowser
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
MD 2009, Malenovice, Beskydy
From the gallery of optical slices to the maximal projection
PIN3::PIN3:YFPMaximální projekce
2. Helpful hints in the processing of experimental data
Kleine-Vehn, unpublished
8 bit - 256 grey scale
12 bit - 4096 grey scale
16 bit - grey scale
2. Helpful hints in the processing of experimental data
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.
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
Vandenbussche et al., 2009, Development, accepted
2. Helpful hints in the processing of experimental data
MD 2009, Malenovice, Beskydy
Vandenbussche et al., 2009, Development, accepted
(a) (c)FM 4-64 PIN1-GFP Merged
Jelínková et al., prov. accepted, Plant Journal, 2009
Colocalization analysis
2. Helpful hints in the processing of experimental data
0
50
100
150
200
250
0 50 100 150 200 250
Intensity of PIN1::PIN1:GFP(green channel)
Absolute intensity
Inte
nsity
of F
M 4
-64
(red
chan
nel)
(e)
Jelínková et al., prov. accepted, Plant Journal, 2009
2. Helpful hints in the processing of experimental data
Colocalization analysis
Spectral detection using ZEISS META 510 detector, laser 477 nm
GFP peak
RFP peak
Interaction between two proteins - FRET analysis in vivo (PIN1-ADL1)
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)
Irfan View – ideal for every day browsing through image documentation
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
Corel Photo PaintAdobe Photoshop
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
Corel Photo Paint or Corel Photoshop – avoid excessive image adjustment!
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2.3. Image analysis, graphical software, presentation software
2. Helpful hints in the processing of experimental data
2.3. Image analysis, graphical software, presentation software
Corel DrawAdobe Illustrator
B130P16E: Practical basics of scientific work http:/lhr.ueb.cas.cz/petrasek Department of Plant Physiology FS CU RNDr. Jan Petrášek, Ph.D.
2. Helpful hints in the processing of experimental data