Biomedical Engineering Reference
In-Depth Information
Fig. 2 Schematic drawing of the VideoScan hardware components and the heating/cooling-unit
(HCU). a The main hardware components are a motorized fluorescence microscope, a stage, and
a CCD camera controlled by a personal computer. b Scheme of the HCU for dynamic heating of a
single well of standard PCR modules. Changes in fluorescence intensity can be monitored over a
broad temperature range in up to six ''ligand channels'' for both solution and microbeads
FastFluoScan contains a hardware abstraction layer, so it can deal with hardware
from different vendors. Currently we are using a Olympus IX81 microscope illu-
minated by xenon lamp or LED unit, and a Märzhäuser ScanStage IM 120 9 100.
Our cameras (Kappa DX2H, DX2HC, DX4C-285, PS4C-285) are equipped with a
Sony ICX285AL CCD-chip. Camera cooling is only required to detect extremely
weak signals, e.g., luminescence applications. FastFluoScan has a modular design.
It can plug in a variety of analysis modules for different measurement tasks. Cur-
rently there are modules for microbead measurement, fluorescence reading, and
bacteria detection and counting. Any kind of sample carrier with a transparent,
planar bottom can be used, e.g., 96-, 384-well-plates, microscope slides, or Adva-
lytix
slides. In particular, Advalytix
slides are interesting because they provide 48
''spots'' for small reaction volumes of less than 2 lL even under PCR conditions (see
Sect. 5
).
VideoScan can be operated with a HCU. This unit, using Peltier elements, fits
into the scanning stage (Fig.
2
b). Currently we are using a prototype made by
Berthold Detection System (Germany) for a single module (NucleoLink
TM
, Nunc)
with heating and cooling rates of up to 15 and 10 K/s, respectively, in a tem-
perature range between 15 and 105 C. With a temperature resolution of less than
0.1 C, high-resolution melting curve analysis is feasible (see
Sect. 6
).
2.2 Data Acquisition
Microbeads are located on the transparent bottom of a microplate well. Microbead
immobilization is not mandatory. In each well, simultaneous measurement of
several microbead populations (multi-parametric) is enabled. Microbead measure-
ment is usually done with a 10 9 /0.30 objective, which is able to image microbeads
