Biomedical Engineering Reference
In-Depth Information
2 The VideoScan Platform
Flow cytometry technologies are common for measuring multiplex microbead
assays [
4
-
6
]. The measuring period of microbeads in suspension is limited to the
time it takes for a microbead to pass through the detector, i.e., a few microseconds,
so a detector of very high sensitivity is required, e.g., a photomultiplier [
7
]. Using
flow cytometry it is also extremely difficult to measure the same microbead several
times to acquire reaction kinetics. To overcome these disadvantages we developed
the patented VideoScan technology [
8
,
9
]. VideoScan is based on automatic
capture and analysis of two-dimensional fluorescence images utilizing algorithms
of digital image processing. Similar approaches have been reported previously
[
10
-
13
].
The VideoScan measures and stores the signal of individual microbeads as a
single-event detection principle already described for flow cytometry or micro-
bead-based arrays [
4
]. This technology utilizes miniaturized wells and can be used
in combination with standard technologies like multiplex PCRs and direct
hybridization assays. The arrays are characterized by randomly ordered micro-
beads which carry specific capture probes. Within a microbead mixture, each
microbead is characterized by a specific dye ratio and a given size. Microbeads
which match these parameters are grouped into one microbead population. The
population is assigned to one analyte only, allowing an unambiguous identification
of individual microbeads in multi-analyte assays. This is a very simple yet pow-
erful encoding strategy [
14
]. Other microbead encodings were reported previously
[
2
,
14
,
15
]. In contrast to other technologies, VideoScan decodes the randomly
dispersed microbeads prior to every measurement of the ligand by their intrinsic
color-code and microbead size. This is different to Illumina BeadArray technology
which defines microbead spots prior to the assay by sequential hybridization
[
14
,
16
]. Real-time decoding has multiple benefits. The redundancy per microbead
population is adjustable. There is no requirement for a defined spot size for one
population nor for a defined spot position. Assays are customizable by adaptation
of number and analyte of microbead populations for each experiment. The readout
concept is adaptable to scenarios where endpoint or real-time measurement at
defined but variable temperatures is desirable.
2.1 Hardware and Software Components
VideoScan consists of our software package FastFluoScan developed in-house and
commercial available hardware components (inverse motorized fluorescence
microscope with a minimum of three fluorescence channels, motorized scanning
stage, digital camera with at least 1.3 megapixels, and optionally a heating/cool-
ing-unit (HCU)) (Fig.
2
a).