With the advent of confocal microscopes and 3D/4D image acquisition capabilities, image processing and analysis can be overwhelming. There is a variety of microscopes in the market and almost all have acquisition software that saves images in a different format. This can result in the daunting task of finding something that will support the file format as well as provide the proper analysis tools. Bitplane AG has created Imaris®, a program that supports images acquired from almost all microscope manufacturers. Some of the formats include Tiff series, BMP series, Leica, Olympus, and Zeiss datasets.
The possibilities are endless with regards to the capabilities of Imaris®. Four-color immunofluorescence on cell culture/tissue samples is in high demand, and having Zeiss LSM 510 and Nikon PCM-2000 Laser Confocal Scanning Microscopes provides for very complex datasets. Imaris® not only supports the file formats of these microscopes but it enables the user to quickly resample or crop large datasets directly from the original file. This provides for a refined dataset and ultimately a more precise evaluation of protein expression patterns.
Upon loading a dataset in Imaris®, it is easy to process the images for a spectacular three-dimensional rendering. Imaris® offers smoothing capabilities with Gaussian and Median filters, which eliminate excessive pixilation. Thresholding and Background Subtraction can also be used to refine the dataset. Colors can be changed and made transparent with ease for optimal visualization. The combination of all these tools, which take a matter of minutes to use, will result in a dataset that can provide amazing pictures. The Snapshot tool allows one to image various views of samples, including x, y, and z views as well as gallery and 3D rotational views. The Keyframe Animator Module of Imaris® generates incredible movies.
In addition to the valuable images generated with Imaris®, a variety of statistical data can also be acquired. Some of the most popular applications at our facility include distance, area, volume, voxel intensity, and co-localization statistics. Objects can also be identified based on intensity and objects of different sizes can be distinguished with the Spot feature. Not only is one able to flip and rotate the z-stack in real time, but tools such as the Clipping Plane allow the user to cut a cell in half for internal review. This is most useful for those interested in protein localization. All statistics are conveniently exported to Excel® for further analysis.
I have used Imaris® quite extensively over the past two years in helping scientists to create amazing 3D renderings of their proteins of interest. Statistical values, such as co-localization, are also often measured. In particular, the Contour Surface feature of Imaris® has become quite popular. The Cavalier Method to determine volume has been widely used. However, with extensive sample sizes, this method proves to be overwhelming. Imaris® makes it effortless to ‘outline’ your region of interest across slices of a z-stack and obtain volume measurements. In a particular study, this method was used to compare the volume of a structure in the brain of a normal vs. diseased model. The sample size was close to 50 for each group, and with Imaris® the volume statistics were obtained in less than a few days.
The only downside to the product is that it will only work on PC platforms, namely Windows® 2000 and XP. It is also very important that the computer which will run Imaris® has a top-of-the-line graphics card. Computer memory is also essential, as extremely large datasets can take a lot of time to process in Imaris®. Bitplane AG provides all the necessary information for optimal system requirements. The higher-end suggestions will allow the user to analyze tasks at a much faster rate, which can be especially convenient for larger datasets.
Aside from great product performance, Bitplane AG also provides a wealth of knowledge on their website. They offer web seminars on a regular basis for their users as well. This is most convenient, as Imaris® is constantly upgraded to include one or two new features every few months.
Surita Banwait
Morphology & Imaging Core
Research Associate II
Buck Institute for Age Research