doi:10.1016/j.bios.2004.06.050
Copyright © 2004 Elsevier B.V. All rights reserved.
Short communication
Confocal reader for biochip screening and fluorescence microscopy
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Thomas Ruckstuhl
, 
, Andreas Walser, Dorinel Verdes and Stefan Seeger
Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland
Received 14 April 2002;
revised 30 June 2004;
accepted 30 June 2004.
Available online 17 August 2004.
Abstract
We developed a fluorescence reader for the sensitive detection of surface-generated fluorescence. The system is applicable for high resolution imaging as well as for the readout of large biochips. The surface of a microscope coverslip is scanned with a laser beam focused to a waist diameter of 500 nm (FWHM) by means of a single aspheric lens. Scanning large areas with a focused beam usually evokes the need of automatic control elements to adjust the laser spot to the designated position at the surface. Due to the special design of the reader, the focus keeps at the plane of the surface even when scanning large areas, obviating the requirement of any real time control. Thus the instrument is straightforward and inexpensive. Nevertheless it features a high sensitivity and high optical resolution. The versatility of the instrument is demonstrated by imaging cells and reading out a DNA-chip. The excellent sensitivity is shown by detecting single fluorescently labeled antibodies.
Keywords: Fluorescence scanner; Biochip reader; Aspheric lens
Fig. 1. Scheme of the confocal fluorescence reader.
Fig. 2. (a) Displacement of expander lens in order to focus on coverslips of varying thicknesses. The solid line give the calculated position, the circles gives the measured lens position for four different coverslips. (b) Dependence of expander lens position on illumination of the asphere (beam waist diameter).
Fig. 3. CEFs calculated for three different coverslips, 140 μm, 150 μm and 160 μm thick. The dashed line denotes the interface, the scale bar corresponds to 1 μm, with the optical axis in z-direction.
Fig. 4. Simulated focal intensity. The dashed line denotes the water/glass interface, with the glass below.
Fig. 5. (a) Confocal images of fluorescence beads on four surface sections with a lateral separation distance of 5 mm. The focus was adjusted to the interface before scanning the first segment and left unmodified afterwards. (b) The image on the left gives the calculated point-spread function of the instrument, the solid curve shows the cross-section. The image on the right gives the superposition of eight beads, the circles show the cross-section.
Fig. 6. (a) Image of a 3.5 mm × 5 mm DNA-chip. (b) Plot of fluorescence intensity against Cy5-DNA concentration. The circles give the average fluorescence intensity of the spot columns of the DNA-chip. The error bars show the intensity deviation within one column.
Fig. 7. Fluorescence signal obtained from fixed surface position of the (a) blank glass/water interface and (b) with a Cy5-antibody concentration of 10−12 M.
Fig. 8. High resolution fluorescence image of stained cells.
Corresponding author. Tel.: +41 1 635 4443; fax: +41 1 635 6813.
Abstract
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