2. Helpful hints in the processing of experimental dataContinuous processing and evaluation of experimental data is highly needed2.1. Organization of work, type of data- Avoid this approach: This year I am experimenting and next year is for the evaluation of what I have obtained- Miscellaneous software is in every day useTypes 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..

2.1. Organization of work, type of dataSoftware 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 at the Charles University- Open source software - very good tools, often problems with the compatibility- Online software - mainly in bioinformatics, but there is also other software available (e.g. graphical editors)2. Helpful hints in the processing of experimental data

2.1. Organization of work, type of dataWindows 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 drivesDo 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

Windows Explorer 2.2. Data explorers, correct data handling and saving2. Helpful hints in the processing of experimental data

2.2. Data explorers, correct data handling and saving2. Helpful hints in the processing of experimental data

Total Commander 2.2. Data explorers, correct data handling and saving2. Helpful hints in the processing of experimental data

2. Helpful hints in the processing of experimental dataBulk data processing, their editing and graphical outputMicrosoft Excel - suitable for all basic operations including statisticsSpecialized statistical software - in case of need of more specialized statistical evaluation, e.g. software Statgraphics, NCSS Statgraphics, NCSS2.3. Spreadsheets, statistical software, graph editors

Microsoft Excel whole protocol management, automation of numerical operations2. Helpful hints in the processing of experimental data2.3. Spreadsheets, statistical software, graph editors

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

2. Helpful hints in the processing of experimental data2.3. Spreadsheets, statistical software, graph editors

2.3. Processing of structural and sequence dataNotepad

Avoids excessive formatting

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

2. Helpful hints in the processing of experimental data

Vector NTI (Invitrogen)Geneious2.3. Processing of structural and sequence data2. Helpful hints in the processing of experimental data

ImageJ (NIH)Fijihttp://rsb.info.nih.gov/ij/docs/concepts.html2.4 Image analysis, graphical software, presentation software2. Helpful hints in the processing of experimental data

NIS elements2.4 Image analysis, graphical software, presentation software2. Helpful hints in the processing of experimental data

Graphical programes for grabbing and processing of microscopical imagesLeica

Zeiss

Nikon

Olympus2. Helpful hints in the processing of experimental data2.4 Image analysis, graphical software, presentation software

Kleine-Vehn, Mol Syst Biol 7:540, 20118 bit - 256 levels of grey (28)12 bit - 4096 levels of grey (212)16 bit - 65536 levels of grey (216)Color depth in microscopy2. Helpful hints in the processing of experimental data

PIN3::PIN3:YFP Maximln projekcednkov et al., Development 137, 607-617, 2009From image sequence to the image projection2. Helpful hints in the processing of experimental data

Analysis of colocalization Jelnkov et al., Plant Journal 61, 883-891, 20092. Helpful hints in the processing of experimental data

Jelnkov et al., Plant Journal 61, 883-891, 20092. Helpful hints in the processing of experimental dataAnalysis of colocalization

FRAP in tobacco BY-2 cells expressing AtPIN1-GFPPrebleachPostbleachFRAP 30 minAnalysis of intracellular protein dynamics2. Helpful hints in the processing of experimental data

Vandenbussche et al., 2009, Development 137, 597-6062. Helpful hints in the processing of experimental dataAnalysis of intracellular protein dynamics

Vandenbussche et al., 2009, Development 137, 597-606Analysis of intracellular protein dynamics

Lakov et al., in preparationPIN1-GFPPIN1-GFP, 30 min 20 M OryzalinPIN1-GFP, 30 min 0.5 M LatB> 1 m/s < 0.4 m/s 0.4 - 1 m/s 2. Helpful hints in the processing of experimental dataAnalysis of intracellular protein dynamics

GFP peakRFP peak2. Helpful hints in the processing of experimental dataInteraction between two proteins - FRET analysis in vivo (PIN1-ADL1)Spectral detection using ZEISS META 510 detector, laser 477 nm

Average fluorescence intensity measured over the emission spectrum in the following region of interests:ROI1 - middle cell plateROI2, ROI3 - growing ends of the cell plateROI4, ROI5 - transversal and longitudinal plasma membranes500-520 nm unmixed600-620 nm unmixedROI1 - low FRET

ROI2, ROI3 - high FRET ROI4, ROI5 - no FRET12345Interaction between two proteins - FRET analysis in vivo (PIN1-ADL1)

Irfan View - good choice for every day browsing through image documentation2. Helpful hints in the processing of experimental data2.4 Image analysis, graphical software, presentation software

Corel Photo PaintAdobe Photoshop2. Helpful hints in the processing of experimental data2.4 Image analysis, graphical software, presentation software

Corel Photo Paint i Adobe Photoshop - avoid excessive image adjustment!!2. Helpful hints in the processing of experimental data2.4 Image analysis, graphical software, presentation software

Corel DrawAdobe Illustrator2. Helpful hints in the processing of experimental data2.4 Image analysis, graphical software, presentation software

After treatments with cytoskeletal drugs both PIN1-GFP and AUX1-YFP endosomes rapidly decreased their dynamics. *