MacResearch

In 'Showcase' reviews, the reviewer is the developer. No claim of objectivity is made, but it’s a chance for the developer to show off his/her app. Here, Dr. Vadim Zinchuk, M.D., Ph.D., who consults CoLocalization Research Software, talks about CoLocalizer Pro, a program for determination of the degree of colocalization in medico-biological images obtained using confocal microscopes.

Overview

CoLocalizer Pro is a professional scientific software for quantitative colocalization analysis of confocal fluorescence microscopy images.

Colocalization is defined as the presence of two or more types of fluorescence molecules at the same location. Although colocalization is relative, the information on the appearance of distinct molecules at the same location can be of particular significance to researchers. If the question is “Does protein A overlap/colocalize with protein B in a given image, and if so to what degree?”, quantitative colocalization provides the answer.

The software performs analysis of images with colocalization and prepares obtained results for presentation and publication.

Interface

CoLocalizer Pro is a Cocoa application designed specifically for the Macintosh platform. Images to be analyzed are opened in CoLocalzier Pro main window by drag and drop or from image drawer.

The software features are accessible from a fully customizable toolbar:

Compatibility

CoLocalizer Pro works with images in the most popular file formats, such as TIFF, Zeiss LSM 4 TIFF Chunky, Zeiss LSM 4 TIFF RGB Planar, JPEG, PICT, and BMP. It also exports data in these formats and ensures that your colleagues using Windows PC will have no problems viewing your files.

Background correction

The biggest problem in quantitative colocalization analysis is overestimation of colocalization. This can be experienced when using ImageJ software. Overestimation happens because confocal fluorescence images include a certain amount of background noise. If not removed, this noise undermines accuracy of calculations by introducing false-positive results.

In CoLocalizer Pro, the problem of reliability of analysis is solved by using it’s original tools. CoLocalizer Pro introduces procedure called background correction, which removes pixels of background levels from the image. To do so, the software uses implementation of the originally developed algorithms for correcting background for the most common types of confocal images. The image below shows preset for images with average contrast and fluorescence:

In addition, it is also possible to correct background manually by using a selected in the image background region of interest (ROI):

Background correction functionality is implemented in the way to ensure that this crucial step is not accidentally skipped:

Quantification of colocalization

Quantification of colocalization is based on calculations of several coefficients. Colocalization window of CoLocalizer Pro contains current image ROI preview (1), it’s scatter gram (2), pixel information of the ROI (3) a channel pair according to which quantification will be made (4), and the area where coefficients are calculated (5):

Exporting and saving data

CoLocalizer Pro is able to export all data it calculates and save these calculations in various file formats. It`s export window provides the options to choose what and how to export and save. Coefficients calculations can be saved as Microsoft Excel and Text files. Images can be saved in TIFF, JPEG, PICT, and BMP formats. Reports can be saved as PDF and HTML files:

iPhoto integration

CoLocalizer Pro software is fully integrated with iPhoto, so that it is possible to access images stored in iPhoto library directly. When you are using CoLocalizer Pro, you do not even need to open iPhoto to view images stored in it.

In addition, if you have selected in General preferences of iPhoto application to edit photo in CoLocalizer Pro, you will be able to open double-clicked in iPhoto images directly in CoLocalizer Pro ready to be examined:

References

Top-ranked medico-biological journals, such as Blood, Journal of Clinical Investigation (JCI), Proceedings of the National Academy of Sciences (PNAS), Journal of Biological Chemistry (JBC), and many others publish studies performed with the help of CoLocalizer Pro software. Current Protocols of Cell Biology (CPCB), a highly respected source of protocols in cell biological field, has recently published a comprehensive quantitative colocalization analysis protocol based on the use of CoLocalizer Pro software. Importantly, the protocol listed suitability requirements for images needed to perform quantification. CoLocalization Research Software website contains a wealth of knowledge about quantification of images and interpretation of obtained results.

Pricing

Single-User license of CoLocalizer Pro costs USD 449 (USD 399 for Academic users), which is more than fourty times less than other types of commercial software that can calculate coefficients, but lack any background correction functionality. Single-User license of CoLocalizer Express, lite version of Pro with scaled down background correction and image viewing tools, costs USD 129 (USD 99 for Academic users). Volume-discount options are available too. Both applications can be evaluated for a limited time before purchase. Free demo trials can be obtained from CoLocalization Research Software website by request.