Resolving Low-Expression Cell Surface Antigens by Time-Gated Orthogonal Scanning Automated Microscopy

We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of ∼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning automated microscopy (OSAM) technique to rapidly process a large population of target-only cells on microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events

Absorption (continuous line) and fluorescence (dotted line) properties of the isolated PEII complexes

<p><b>Copyright information:</b></p><p>Taken from "Diversity and evolution of phycobilisomes in marine spp.: a comparative genomics study"</p><p>http://genomebiology.com/2007/8/12/R259</p><p>Genome Biology 2007;8(12):R259-R259.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2246261.</p><p></p> PEII-A (as in sp. WH7803); PEII-B (as in sp. RCC307); PEII-C (as in spp. CC9605 and WH8102). Type IV chromatic adapters have a PEII-B under white or green light and a PEII-C under blue light [34]

Proposed models of PBS structure for the different pigment types and subtypes

<p><b>Copyright information:</b></p><p>Taken from "Diversity and evolution of phycobilisomes in marine spp.: a comparative genomics study"</p><p>http://genomebiology.com/2007/8/12/R259</p><p>Genome Biology 2007;8(12):R259-R259.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2246261.</p><p></p> PBS cores are generally composed of three cylinders, but in some chromatic adapters possessing an extended L, it is likely composed of two additional half cylinders (see, for example, [58]). In pigment type 1, rods are composed of C-PC only; in pigment type 2, rods are composed of either C-PC, or R-PCIII and a PEI-like phycobiliprotein; in pigment type 3, rods comprise R-PC and two PE types (PEI and PEII). Cells of the latter pigment type bind PEB and PUB at a low (3a), medium (3b), high (3c) or variable (3d or type IV chromatic adapter) ratio. Colored stars indicate the pigment type of each strain (see Figure 1 for color code)

Absorption (continuous line) and fluorescence (dotted line) properties of isolated PBP complexes

<p><b>Copyright information:</b></p><p>Taken from "Diversity and evolution of phycobilisomes in marine spp.: a comparative genomics study"</p><p>http://genomebiology.com/2007/8/12/R259</p><p>Genome Biology 2007;8(12):R259-R259.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2246261.</p><p></p> C-PC (as in spp. RS9917, WH5701 and WH8018); PEI-A* (as in spp. WH8018 and WH7805); PEI-A (as in spp. WH7803, Almo03 and RS9912); PEI-B (as in spp. WH8102, CC9605 and Oli31)

Comparison of PBS rod gene regions of the different pigment types of marine

<p><b>Copyright information:</b></p><p>Taken from "Diversity and evolution of phycobilisomes in marine spp.: a comparative genomics study"</p><p>http://genomebiology.com/2007/8/12/R259</p><p>Genome Biology 2007;8(12):R259-R259.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2246261.</p><p></p> Rectangles above and below the lines have a length proportional to the size of ORFs and correspond to the forward and the reverse strand, respectively. In several genomes, the sense of the rod region was inversed so that the regions all appear in the same direction. For the group formed by the chromatic adapters and RCC307, a few variations can be found with regard to the region shown here, which corresponds to BL107. First, the lyase-encoding gene(s) located near the 3'-end can either be a operon or , a -like fusion gene (see text). Second, the gene organization at the 5'-end can vary: is located elsewhere in the genome of RCC307 and the gene following is either the lyase gene in RS9916 and RCC307, in BL107 and CC9902, or none of these in CC9311. Colored stars indicate the pigment type of each strain (see Figure 1 for color code)