Case Studies
Spectral Imaging of Biochips
Dr W-D Schmidt and colleagues of Gesellshaft fϋr Medizin, Bio und
Umwelt technologien, Jena, Germany, are using an iXon EMCCD camera and Imspector
imaging spectrograph to acquire spectral line scans of biochips, followed by component
analysis by spectral unmixing.
This results in a clear spectral distribution of the fluorescence of each biospot,
as well as enabling detection and correction for contribution of unwanted "auto-fluorescence"
background. Spectral imaging detection can be useful for biochip reader applications
to avoid problems of false-positive fluorescence detection, and has the potential
to enhance the accuracy and speed of scanning.
The experimental set-up consists of an Andor iXonEM DV887 back-illuminated in combination
with ImSpector from Spectral Imaging Ltd. The laser excitation sources for fluorescence
dyes Cy3 and Cy5 were realized both by a pulsed green diode laser (532nm; 100nW),
a red diode laser (635nm; 200mW), and a nitrogen pumped pulsed dye laser (1ns; 5
ÌJ).
An appropriate filer set was arranged in the optical pathway. The spectral imaging
system has a spatial resolution of 20 Ìm and spectral resolution of 5 nm. The bioslide
under investigation is moved orthogonally by a micro-translation stage. The whole
measuring arranagement is housed in a closed black-box.
Spectral image of a row of Cy3-labeled biospots and spectrum from one spot.
This measuring system generates a spectral image for each labeled row of biospots.
The fluorescence intensity versus wavelength in the horizontal direction is shown
in figure 8. Also shown is a fluorescence spectrum derived from one of the spots.
For analysis of a biochip, the micro-translation stage moves the slide to the position
of the next row of biospots or interspace, and further spectral images are taken.