Project

The Focinator

Goal: Free and user-friendly foci counting!
https://focinator.com/

Methods: Microscope, Fluorescence, Image Analysis, DNA Damage, DNA Repair, Focinator

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Project log

Sebastian Oeck
added an update
Dear users,
The Focinator v2-40beta is available right now!
  • We fixed an issue with "Remove Scale". The beta will only produce results in pixels
  • We are working on the implementation of a “Remove Scale” option for e.g. area measurements
Please download here https://focinator.com.
You will find a manual how to install and how to run the script on our page.
Thanks for your helpful comments and suggestions!!
Thank you for using the Focinator!
 
Sebastian Oeck
added an update
Dear users,
The Focinator v2-31 is available right now!
  • New intensity measurements added for total nucleus and foci.
  • Fixed some minor stability problems
Please download here https://www.focinator.com.
You will find a manual how to install and how to run the script on our page.
Please also check out the new Stripenator for fast and reliable analysis of micro-irradiated regions and other intensity-based quantifications https://focinator.com/stripe.
Thank you for using the Focinator!
 
Sebastian Oeck
added a research item
DNA- and histone-related research frequently comprises the quantitative analysis of protein modifications, such as histone phosphorylation. Analysis of accumulation and disappearance of protein foci are used to monitor DNA damage and repair kinetics. If the protein of interest doesn’t accumulate in foci, laser micro-irradiation of single nuclei provides an alternative method to monitor DNA repair proteins and histone dynamics at the DNA damage site. We have developed an automated evaluation tool for standardized, high-throughput analysis of micro-irradiated cells featuring single cell background subtraction and detection across multiple fluorescence channels, allowing for robust statistics.
Sebastian Oeck
added 3 research items
The quantitative analysis of cells and foci plays a key role in various cell biological methods. In the field of radiation biology and molecular radiation oncology, the effect of ionizing radiation, chemotherapy or molecularly targeted drugs on DNA damage induction is detected by the analysis of protein clusters or phosphorylated proteins recruited at DNA damage sites, involving for example γ-H2A.X, 53BP1 or RAD51. Consistency in counting and interpretation of these foci is essential, whereas manual counting is time-consuming and subjected to investigator-related bias. We recently developed “The Focinator” as a reliable tool for automated quantitative and qualitative analysis of nuclei and DNA damage foci. It is 23 times faster than manual counting. The software automatically selects nuclei as regions of interest, evaluates the foci in multiple channels at the same time and acquires fluorescence areas and intensities. In the version 2-23 we aimed to further refine the software and make it more user-friendly. We included an R-script-based mode for automated image opening, file naming and progress monitoring. Consequently, the evaluation of several images does no longer require the attendance of the operator and thus saves more time of the researcher. Moreover, the Focinator v2-23 enables multi-channel analysis of four channels with different stainings and even analysis of protein-protein co-localization is possible due to the extension of the Focinator v2-23 to a three channel foci analysis. With the Focinator v2-23 we generated a free, user-friendly tool for fast high-throughput evaluation of DNA repair foci. Overall the Focinator allows improved foci evaluation with respect to accuracy and reproducibility and now also enables colocalization and cell cycle-related analysis with a high analysis speed.
Sebastian Oeck
added an update
Dear users and new users,
The Focinator v2.23 is available right now!
  • Updated to R-3.5.1
  • Reduced size of Focinator window
  • Fixed some minor stability problems
Please download here www.focinator.com.
You will find a manual how to install and how to run the script on our page.
Thank you for using the Focinator!
 
Sebastian Oeck
added an update
Dear users and new users,
The Focinator v2.22 is available right now!
  • R-3.4.3-based problems were fixed.
  • New design of the report file
  • Channel names are now saved in the report file
  • Confirmation of the noise, cutoff, area, etc. values with “Enter” is no longer necessary
  • Fixed some minor stability problems
Please find the Focinator v2-22 attached to this project update or download here www.focinator.com.
You will find a manual how to install and how to run the script on our page.
Thank you for using the Focinator!
The whole team wishes you a happy new year 2018!!
 
Sebastian Oeck
added an update
Now you can find the Focinator v2 using the new URL www.focinator.com.
 
Sebastian Oeck
added a research item
Gastric cancer (GC) is the second leading cause of cancer-related mortality worldwide. The disease develops from the accumulation of several genetic and epigenetic changes. Among other risk factors, Helicobacter pylori infection is considered the main driving factor of GC development. H. pylori infection increases DNA damage levels and leads to epigenetic dysregulation, which may favor gastric carcinogenesis. An early step in double strand break repair is the recruitment of (ataxia-telangiectasia mutated serine/threonine kinase (ATM) to the damaged site, where it plays a key role in advancing the DNA damage checkpoint process. H. pylori infection has been associated with the introduction of double strand breaks in epithelial cells, triggering damage signaling and repair response involving ATM. Thus, the current study analyzed the effect of H. pylori infection on the DNA damage response sensor, ATM, in gastric epithelial cells and in biopsies from patients with GC. In this work, we identified that H. pylori infection stimulated DNA damage, and therefore induced ATM in a virulence factor-dependent manner. In addition, we found that H. pylori might activate ATM through histone H3 and H4 hyperacetylation and DNA promoter hypomethylation. Our findings reveal a mechanism associating ATM signaling induction with H. pylori infection.
Sebastian Oeck
added an update
The new article about the Focinator v2 in the Radiation Research Journal. July 2017, Volume 188 Issue 1:114-120!
DOI 10.1667/RR14746.1
 
Sebastian Oeck
added an update
Dear users and new users,
The first big update since the Focinator v2 release. The Focinator v2.10 is available right now!
All known R-3.4-based problems were fixed! Additionally, we included: colocalization in both directions and your own channel names as well as noise, cutoff, area, etc. values export to the Report file.
Please find the Focinator v2-10 attached to this project update or download here: www.focinator.oeck.de.
You will find a manual how to install and how to run the script on our page.
Thank you for your understanding and patience!
 
Sebastian Oeck
added an update
We will upload the Focinator update to v2.07 this week. It will solve the version conflicts with R-3.4 and various other related problems.
Thank you for your patience.
 
Sebastian Oeck
added an update
R-3.4.0 released!
Dear users and new users,
the Focinator v2.06 will not run with R-3.4.0. We are working on a stable version for R.3.4.0!
If you don't want to wait, please download R-3.3.3 here: https://cran.rstudio.com/bin/windows/base/old/3.3.3/
Thank you for your understanding and patience!
 
Sebastian Oeck
added an update
Currently, there is a problem occurring during runs with large image numbers. For some images, depending on the computer performance, the ROI window is counted instead of the first foci channel. This is visible through mean and max intensity values of 255 as the ROI channel is converted to 8-bit before the ROIs are marked.
 
Sebastian Oeck
added an update
Patch notes
  • v2.06 || New function "Don't panic" for difficult images with high background/many cells --> extra 8-bit conversion of ROI channel
  • v2.05 || Increased stability, all windows close command after last foci channel
  • v2.04 || Increased stability:: v2.03Increased stability, correction of colocalization function
  • v2.02 || Export of mean and max intensity values for all three foci channels
  • v2.01 || New R-script-based GUI, 4-channel evaluation now possible
  • v1.50 || R-Script based automatic mode
 
Sebastian Oeck
added a project goal
Free and user-friendly foci counting!
 
Sebastian Oeck
added 4 research items
The quantitative analysis of cells and foci plays an important role in various cell biology methods. In the fields of radiation biology and molecular radiation oncology, the effect of ionizing radiation, chemotherapy or molecularly targeted drugs on DNA damage induction is performed by the analysis of protein clusters or phosphorylated proteins recruited to foci at DNA damage sites, involving for example γ-H2.AX, 53BP1 or RAD51. Consistency in counting and interpretation of foci remains challenging. Manual counting is time-consuming and subjected to investigator-related bias. Currently available software is often expensive or consists of instructions for semi-manual analysis or incomplete algorithms. We recently developed “The Focinator” as a reliable tool for automated quantitative and qualitative analysis of nuclei and DNA damage foci. It is 23 times faster than manual foci counting and proved to be highly accessed (Oeck et al. 2015). The software automatically selects nuclei as regions of interest (ROIs), evaluates the foci in multiple channels at the same time and acquires fluorescence areas and intensities. Here, we aimed to further refine the software and make it even more user-friendly by adding further features. In the Focinator 2.0 we included an R-script-based mode for automated image opening, file naming, progress monitoring and an error report. As a consequence the evaluation does no longer require the attendance of the operator and thus saves more time of the researcher. Moreover the Focinator 2.0 enables multi-channel analysis of four channels with different stainings. Furthermore, analysis of protein-protein co-localization is possible. Due to the extension of the Focinator 2.0 to a four channel analysis, it is now possible to compare up to three foci channels. Furthermore, we established a cell-cycle based foci evaluation by using a cell cycle phase marker like Cyclin B1 in one channel. With the Focinator 2.0 we generated a free, user-friendly tool for fast high-throughput evaluation of DNA repair foci. Overall the Focinator allows improved foci evaluation with respect to accuracy and reproducibility and now also enables colocalization and cell cyclerelated analysis with a high analysis speed.
Background The quantitative analysis of foci plays an important role in many cell biological methods such as counting of colonies or cells, organelles or vesicles, or the number of protein complexes. In radiation biology and molecular radiation oncology, DNA damage and DNA repair kinetics upon ionizing radiation (IR) are evaluated by counting protein clusters or accumulations of phosphorylated proteins recruited to DNA damage sites. Consistency in counting and interpretation of foci remains challenging. Many current software solutions describe instructions for time-consuming and error-prone manual analysis, provide incomplete algorithms for analysis or are expensive. Therefore, we aimed to develop a tool for costless, automated, quantitative and qualitative analysis of foci. Methods For this purpose we integrated a user-friendly interface into ImageJ and selected parameters to allow automated selection of regions of interest (ROIs) depending on their size and circularity. We added different export options and a batch analysis. The use of the Focinator was tested by analyzing γ-H2.AX foci in murine prostate adenocarcinoma cells (TRAMP-C1) at different time points after IR with 0.5 to 3 Gray (Gy). Additionally, measurements were performed by users with different backgrounds and experience. Results The Focinator turned out to be an easily adjustable tool for automation of foci counting. It significantly reduced the analysis time of radiation-induced DNA-damage foci. Furthermore, different user groups were able to achieve a similar counting velocity. Importantly, there was no difference in nuclei detection between the Focinator and ImageJ alone. Conclusions The Focinator is a costless, user-friendly tool for fast high-throughput evaluation of DNA repair foci. The macro allows improved foci evaluation regarding accuracy, reproducibility and analysis speed compared to manual analysis. As innovative option, the macro offers a combination of multichannel evaluation including colocalization analysis and the possibility to run all analyses in a batch mode.