Wednesday, January 7, 2009

microarray spot morphology


Seen here are scans of a 100 micron diameter microarray spot scanned at 500 nanometer resolution (top) 10 micron resolution (bottom). It is amazing to see the difference! The 500 nanometer scan (0.5 micron) was done with the Innoscan 900A microarray scanner. What do we learn from this image? Our microarray surface chemistry creates very well defined spots and no bleeding or gradient of signal exists at the edge of the spot. This is good news for spot finding and quantification systems. Regardless of the resolution as long as there are enough pixels to quantify the data will be reliable. The basic rule of thumb is 10 pixels are required. 30 micron spots should be scanned at at least 3 micron resolution...100 micron size spot scanned at 10 micron and so on... The Innoscan 900 scanners have adjustable resolution to scan different spots sizes ranging from 5 microns and up (given the 10 pixel rule for reliable quantification). This flexibility is important since when the resolution of the scan doubles, the computer image file quadruples. Keeping images as small as possible help save on valuable computer resources.

Another nice piece of information is
that we always thought that the smaller the tip of our micro spotting pin, the more round our spots were made on the microarray (we make 15 different tip sizes to accommodate the resolution of different detection instruments). Our smallest tip is 50 microns, and largest 375 microns...with the end of the tip being a small square (see images below). Since we have a capillary delivery mechanism, we have always known that the buffer system and surface chemistry can have an effect on spot morphology. More wettable samples and hydrophilic surfaces will create more round spots than more viscous and hydrophobic surfaces. In general the larger the tip of the micro spotting pin, the easier it is to make squarish looking spots...however, what we learn from being able to scan a very high resolution, that the printing mechanism of all our pins is the same regardless of the tip size. The spots above were made with 946MP3 micro spotting pins. See figure for a image of the device, the different loading volumes of the tips and a image of the end of the tip:


It is really fun to see in a very high resolution scan the real spot morphology of a 100 micron size spot on a high quality microarray surface chemistry. Contact me at todd@arrayit.com for more information on scanners, microarray slides and microarray manufacturing.