Imaging large arrays of supported lipid bilayers with a macroscope
8.6.2007
Edward T. Castellana and Paul S. Cremer/Courtesy AVS Biointerphases Journal
A computer aided design rendering of the inside of the EF/TIR macroscope
Fluorescence microscopy has proven to be an invaluable tool for the investigation of supported lipid bilayers inside microfluidic devices and on array-based platforms. When combined with total internal reflection (TIR) illumination, this technique becomes even more powerful, allowing for the investigation of surface specific binding events that occur between membrane bound ligands and aqueous proteins containing complementary receptor sites. Imaging arrays of bilayers with standard fluorescence microscopy has been performed by shrinking the membrane patches to the micron size scale. This technique has been especially valuable for making one-shot equilibrium dissociation constant measurements in which the contents of a linear bilayer array are identical, but the aqueous solutions above them contain various protein concentrations.
While fluorescence microscopy has the advantage of one-shot imaging, it is limited in its field of view. In order to employ a platform with more individually addressable membrane patches, one needs to either shrink the size of the patch or expand the field of view. Excellent methods are now available for creating micron-scale patterns of lipid bilayers. So long as each bilayer in the array is chemically equivalent to the others, patterning can be scaled down to the 1 m level or even below. On the other hand, there are a limited number of methods available for making arrays with variable contents at each address. One method is to employ laminar flow of vesicle solutions side by side inside a microfluidic device. This creates a gradient of bilayer chemistries that can be confined by a patterned surface. This method is, however, limited in the number of unique chemical constituents that can be patterned. Moreover, spatial control over the patterned membrane array is somewhat difficult. Another method for patterning unique lipid membrane chemistries relies on the use of polydimethylsiloxane (PDMS) stamping and backfilling. Again, this method can fabricate arrays with a few unique chemistries, but spatial alignment limits this number for practical rapid prototyping purposes. Several light directed methods have also been developed for the patterning of lipid bilayers. Such techniques, however, have not yet been proven for patterning more than a few components.
Fully controlled spatial addressing of lipid bilayers with unique chemistries at each individual location has been achieved by transferring picoliter- and nanoliter-sized droplets of vesicle solutions to a hydrophilic interface patterned within hydrophobic barriers (i.e., the microcapillary injection method). The array size, however, has been typically limited to 3 3 or 4 4 because of the need to pattern all the contents within the field of view that is compatible with imaging by fluorescence microscopy. Very small boxes (<50 m) become impractical because of the difficulties that arise from pinpoint liquid delivery. In fact, microcapillary injection already becomes tedious to employ for large arrays with individual elements of 250 m on each side. On the other hand, larger arrays could be achieved more practically if the element size were bigger (i.e., 1 1 mm2). Such a strategy would require the design and construction of an epifluorescence/total internal reflection macroscope.
Standard epifluorescence microscopes often come equipped with low magnification objectives, which certainly allow for large fields of view. The problem associated with conventional microscopes, however, is that the numerical aperture (NA), or light gathering capability, rapidly decreases with decreasing magnification. In order for the magnification to be decreased, the working distance (distance between the objective and the sample) must be increased. By increasing the working distance and maintaining a fixed objective diameter, one unfortunately also reduces the solid angle at which light can be collected from a given point on the sample. Low NA/large field of view imaging therefore requires longer exposure times, which decreases the signal-to-noise ratio and increases the amount of photobleaching in the sample. The solution to this problem is to build a system with large diameter objectives.
Epifluorescence macroscopes employing 1 magnification have been previously developed for imaging animal tissues. The basic design uses a tandem-lens imaging system much like those used in x-ray video radiography and up-close photography. Surprisingly, however, such instruments have seen limited employment for imaging protein arrays on chip. To the best of our knowledge, a total internal reflection fluorescence macroscope has not previously been developed. Herein, we demonstrate the ability to fabricate dozens of uniquely addressed phospholipid bilayers and image them simultaneously with an epifluorescence (EF)/TIR fluorescence macroscope. This technology is then used to demonstrate fluorescence quenching as a function of probe density as well as a ligand-receptor binding assay. These experiments clearly demonstrate for the first time the ability to array large numbers of bilayers with unique chemistries at each address and employ them in model membrane studies. This should greatly aid biophysical measurements of lipid membrane/protein interactions.
