CoLocalizer Pro papers keep coming...

Dr. Osvaldo Rey and colleagues from the University of California at Los Angeles published a very interesting paper about the role of calcium-sensing receptor (CaR) in the generation of transient Ca2+ oscillations. Dr. Rey used CoLocalizer Pro software to elegantly quantify the colocalization of CaR and transient receptor potential gene, TRPC1, according to Manders' overlap coefficient. They employed CoLocalizer Pro to quantify colocalization of CaR and TRPC1 and presented corresponding scatter grams. The paper is a good read and leaves a very positive impression. The paper appeared in the Journal of Biological Chemistry (281:38730-38737 (2006), one of the best journals in the medico-biological field.

To do or not to do

A number of researchers contacted CoLocalization Research Software Support lately and said they tried either CoLocalizer Pro or Express versions of colocalziation analysis software and the results of colocalization coefficients were not what they expected. They wondered why is this so and what should they do to obtain more suitable calculations. After asking them few questions about the way they prepared samples and how they used confocal microscopes, it became clear that their images were simply unusable for quantitation purposes. For these images, even calculations which seem correct will no doubts be erroneous. We put together some simple guidelines for researchers using confocal microscopy and doing quantitative colocalziation analysis so that they can avoid mistakes in future. Here they are:

Sample preparation:
- Make sure that the used antibodies are specific and do not cross-react.
- Choose fluorophores with well-separated excitation and emission spectra.
- Use the same mounting medium for the samples you will be comparing.
- Try to avoid using anti-fading reagents, as they can increase background fluorescence.

Microscope set-up:
- Use plan apochromatic lenses to reduce chromatic shift.
- Use optimized emission filters to maximize emission collection while avoiding bleed-through effect.
- Use the same objective lens for the samples you will be comparing.
- Make sure that the size of microscope pinhole is properly set.

Image acquisition and handling:
- Acquire images sequentially to minimize bleed-through effect.
- Do not acquire too bright and too contrast images, as it will result in their saturation.
- Do not manipulate images in graphics-editing programs, as it may ruin usability of your images for quantitation.
- Do not resave files in any other format than TIFF. It may cause loss of original image data needed to perform calculations.

If you have not followed one of these guidelines, just don`t use your images for calculating colocalization. You will not get reliable results.

CoLocalizer Pro 2 released!

We upgraded CoLocalizer Pro to version 2. New version of CoLocalizer Pro is a significant improvement over the previous one. CoLocalizer Pro 2 adds one of the most requested features: the ability to reveal colocalized and selected pixels. The upgrade is free to all registered users! It can be downloaded here: http://homepage.mac.com/colocalizerpro/purchasesupport.html

Colocalization studies performed using multidimensional microscopic robot technology

October issue of Nature Biotechnology published an article about the latest trend in studying the high-throughput protein colocalization studies: multidimensional microscopic robot technology. The technology consists of cycles of fluorescence tagging, imaging and bleaching in situ. It is said that the technology combines three advances: a) a fluorescence technique capable of mapping hundreads of different proteins in one tissue section or sample, b) a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors, c) and a system for imaging the distribution of these protein clusters in a so called toponome map. It is claimed that this approach is able to reveal rules of hierarchical protein network organization, in which state-specific lead proteins control protein network topology and function.

The method is relied on a so called multi-epitope-ligand cartography (MELC), that can map the location of several proteins in the one samlel of cells or tissue using sequential rounds of fluorescent detection in situ. Importantly, it appears that MELC can be used to show when and where proteins colocalize in cellular or tissue compartments, which proteins are excluded, how these associations are altered by pathology and how proteins are functionally interrelated.

MELC appears to be a significant step forward in colocalization analysis, because it enables obtaining considerably more reliable images for latter use with colocalization analysis software. It would be interesting to see whether these images will still require background correction, a critical step performed by the current software.

Study colocalized antigens? Make sure you`ve got CoLocalizer...!

A whole lot of new researchers doing confocal microscopy will now learn about amazing capabilities of CoLocalizer Pro and CoLocalizer Express! Microscopy and Analysis, the premier international journal for users of microscopical, analytical and imaging equipment with 46,000 subscribers and 120,000 readers worldwide, in its special issue dedicated to "Light Microscopy in the Life Sciences" published our review article about quantitative colocalzition analysis. The article describes the applicability of our colocalization analysis software for studying dynamics of cellular responses: Microscopy and Analysis 2006, September. For the convenience of users, we included table with characteristics of coefficients used to estimate colocalization. We describe the range of their values and give tips on how they should be applied.

Another American Journal of Physiology paper empoyed CoLocalizer Pro software

Dr. Tstutsumi and colleagues from University of California in San Diego reported results of their study about role of 12-lipoxygenase in volatile anesthetic-induced delayed preconditioning in mice. Researchers tested the hypothesis that expression and activity of 12-LO mediate delayed cardiac protection induced by isoflurane in the mouse heart in vivo. Among other tools, CoLocalizer Pro software was used in this study to find out that 12-LO is an important mediator of isoflurane-induced delayed preconditioning. Am J Physiol Heart Circ Physiol 2006, 291:H979-H983.

CoLocalizer Pro in the Journal of Biological Chemistry paper

Dr. Sweaney and colleagues from University of California in San Diego published interesting paper about the mechansim of fibroblast-myofibroblast transformation. The paper appeared in Journal of Biological Chemistry (JBC), one of the best and highly credited medico-biological journals J Biol Chem 2006 281:17173-17179. Fibroblast-myofibroblast transformation is a critical event for enhanced extracellular matrix deposition. It involves formation of an actin stress fiber contractile apparatus that radiates from focal adhesions (FA) in the plasma membrane. The researchers used adult rat cardiac fibroblasts to examine distribution and expression of adenyly cyclase (AC), phospho-cav-1, and FA proteins to define mechanisms that link increases in cAMP to caveolin-1 phosphorylation, actin/FA assembly, and fibroblast-myofibroblast transformation. The authors detected AC in both cav-1 and phospho-cav-1 immunoprecipitates, but FA kinase (FAK), phospho-FAK (FAK Tyr-397), paxillin, and vinculin were detected only in phospho-cav-1 immunoprecipitates. Treatment with the AC activator forskolin or a cAMP analog increased cav-1 phosphorylation but decreased FAK Tyr-397 phosphorylation in a cAMP-dependent protein kinase-dependent manner. These events preceded actin cytoskeletal disruption, an effect that was blocked by small interfering RNA knock-down of cav-1. Inhibition of protein tyrosine phosphatase 1B abrogated cAMP-mediated disruption of actin cytoskeleton, cav-1 phosphorylation, and FAK Tyr-397 dephosphorylation. CoLocalizer Pro software was used to make important conclusions about functional significance of phospho-caveolin-1. According to the authors "the data define a novel organization of signaling molecules that regulate fibroblasts: scaffolding of AC by phospho-cav-1 at FA sites in a caveolae-free microdomain along with components that mediate inhibition of actin/FA assembly and fibroblast-myofibroblast transformation via increases in cAMP".

American Journal of Physiology paper cites CoLocalizer Pro software

Dr. Patel and colleagues from University of California in San Diego reported results of their interesting study about the role of caveolar microdomains and δ-opioid receptors in protection of adult rat cardiac myocytes from ischemic cell death. They tested the hypothesis that that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. The authors used tools of quantitative colocalization to determine colocalization of DOR with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells. CoLocalizer Pro software was used for this task. They also investigated the effect of the treatment with methyl-β-cyclodextrin (MβCD), which binds cholesterol and disrupts caveolae. It was found that MβCD fully attenuated the protective effects of ischemic preconditioning or SNC-121, a selective DOR agonist, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated MCD, which maintained caveolae structure and function. This article suggests a key role for caveolae, perhaps through enrichment of signaling molecules, in contributing to protection of cardiac myocytes from ischemic damage. Am J Physiol Heart Circ Physiol 2006, 291:H344-H350.

Journal of Immunology paper reports data obtained using CoLocalizer Pro

Dr. Cario and colleagues from Division of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany, and Harvard and University of Massachusetts Medical Schools published intersting article about trypsin-sensitive modulation of intestinal epithelial MD-2 as mechanism of lipopolysaccharide tolerance. The paper appeared in Journal of Immunology, one of the best journals in the medico-biological field.

The article characterized the expression and subcellular distribution of intestinal epithelial MD-2 and delineated potential functional interactions with trypsin and then alteration in inflammatory bowel disease (IBD). The authors obtained confocal images using Zeiss LSM510 and then quantified the extent of colocalization in the sections using CoLocalizer Pro software. Pearson’s correlation and the Manders’ overlap coefficients were employed.

It was concluded that endoplasmic reticulum-associated MD-2 expression in IBD may be altered by ileal protease in inflammation, leading to impaired LPS recognition and hyporesponsiveness through MD-2 proteolysis in IEC, thus implying a physiologic mechanism that helps maintain LPS tolerance in the intestine. J Immunol 2006, 176:4258-4266.

Review article in the works

I am working on a review article about quantitative colocalization analysis. We have accumulated significant knowledge about this subject over the last two years and I am convinced that such article is needed for the research community. I will be giving theoretical background of this research technique, summarizing tips on how it should be used properly, and provide practical examples of its applicability.