Fluorescence Reader module

For PCR-based systems, detection of amplified products is surely the most important thing to fulfill requirements of an entire modular “sample-in-answer-out” µTAS device, while optical detection methods still dominate over others due to sensitivity purposes. However, for complexity reasons in most µTAS the optical detection is commonly accomplished using a microscope located off-chip. Due to the modular and “open” character of the LOC comprising a perfect accessibility to chips installed in the CytoCycler PCR device, a fluorescence detection unit “Fluorescence Reader” could be integrated easily as not being part of any fabrication process. The Fluorescence Reader consisted of a commercially available CCD camera and control equipment. A similar simple, portable and modular fluorescence detection system for lab-on-a-chip applications was developed by Novak L et al. (2007).

The Fluorescence Reader module of the lab-on-a-chip system typically consisted of a) a light source for emitting light at a suitable wavelength range (blue LED λmax = 470±2 nm with collimating optics (inhibiting power losses over the length of the optical path), b) an ET482/35 excitation and ET536/40 emission filter set (λmax ex = 482 nm, λmax em = 536 nm), c) a CCD camera as detector for signal processing (capturing emitted light), d) external electronics like a LED power supply control box and a trigger signal break-out box and e) software for image data analysis. The optical path of excitation light and emission light was designed in a 45° arrangement for the optical separation of excitation and emission channels (figure 12). A LED was chosen as light source as fluorescence systems based on light emitting diodes (LEDs) became popular in the last few years for their low cost, due to their long lifetime and that LED’s light output can be modulated (Dasgupta PK et al., 2003). Additionally, traditionally used light sources like mercury lamps and lasers, were too bulky and expensive for combination with the LOC devices. Due to collimation, the stray light of the LED was minimized, in order to reduce the signal-background relation. The optical system including LED and filter set was adapted to the fluorescence requirements of SYBR Green I providing a typical standard fluorescence detection system. SYBR Green I is a fluorescence dye intercalating into double-stranded DNA molecules, absorbing blue light at an absorption maximum of 498 nm and emitting green light at an emission maximum of 521 nm (figure 11). Accordingly, integrated filter sets included an excitation filter with a transmission of λmax = 482 nm (spread 36 nm = 464-500 nm) and an emission filter with a transmission of λmax = 536 nm (spread 40 nm = 516-556 nm).

Figure 11. SYBR Green I spectra. Excitation and emission curves of the DNA intercalating fluorescence dye

SYBR Green I are shown (from Fluorescence Dye and Filter Database at www.micro-shop.zeiss.com). The dye comprised an emission maximum of 521 nm when enlightened with excitation light at a maximum of 498 nm.

The Fluorescence Reader was positioned stationary in the middle of the LOC slide, between microscope and BioSpot (figure 12). For excitation the LED as light source was placed at an angle of 45° shining to the sample positioned on reaction center B on the LOC chip surface. LED collimated light was filtered by an exciter ET482/35, exciting the SYBR Green I dye to produce fluorescing light. Fluorescent light was detected passing through an emission filter ET536/40, followed by the collection of light by a CCD camera (figure 12).

Figure 12. Design of the Fluorescence Reader including CCD camera detector, LED light source and filter sets. The Fluorescence Reader was integrated on the LOC slide, located between microscope and BioSpot. A fluorescent sample was positioned to reaction center B of a LOC chip installed in the CytoCycler. For detection of fluorescent sample signals, an angled arrangement of excitation and emission devices including appropriate filters was chosen. Thus the optical part could be split into two paths: excitation light was directed in an angular way to a fluorescent sample (blue light path), while emitted fluorescing light was detected and captured direct vertically by the CCD camera positioned above (green light path). CAD (SolidWorks 2006, Solid Works Corp.) image was kindly provided by G. Lieckfeld.

The basic setup of the Fluorescence Reader, including LED light source, filter set, LED power control box, trigger signal break-out box and CMOS (Complementary Metal Oxide Semiconductor) sensor as fluorescence signal detector was developed in the context of a diploma thesis (“Fluoreszenzreader zur Detektion von Biomolekülen auf einem ‘Lab-on-a- chip’/Fluorescence reader for detecting biomolecules on a lab-on-a-chip device”, submitted by Taner Sari, April 2008). This work dealed with the coupling of an optical Fluorescence Reader to a “lab-on-a-chip”, whereas DNA molecules (enriched with fluorescent marker) were optically excited and quantitatively detected via a detection unit. The setup was optimized to the actual state in the context of an internship program (“RT-PCR automation for lab-on-a-chip using LabVIEW”, submitted by Muhammad Atyab Imtaar in January 2009), where an appropriate LabVIEW-based detection software was written (“Grand_NIVision_Intensity_Consec_Subtract_Loopback_NewCamera.VI”) for automatic picture taking and the CMOS chip detection unit was exchanged by a CCD camera for sensitivity and resolution purposes. Additionally, this software was adapted for manual picture taking purposes (“Norbert.VI”).

For performing fluorescence detection, the chip-holder of the hardware heating-device CytoCycler needed to be centered to the Fluorescence Reader (figure 12). The temperature control box of the CytoCycler provided the connecting to the software control, but also was programmed to give trigger signals to the trigger signal break-out box of the Fluorescence Reader. Trigger signals were produced for indicating the end of a PCR cycle. There were three trigger signal output-plugs at the backside of the temperature control device named 1, 2 and 3, representing the three periodically-repeated temperature steps of a PCR protocol. Outlet 1 gave a signal after the denaturation step, outlet 2 after the annealing step and outlet 3 after the extension step. Which of these outlets was connected to the Fluorescence Reader depended on the kind of PCR performed. In 2-step PCR, when annealing and extension were combined in one step, outlet 2 was the choice, in 3-step PCR outlet 3 needed be connected. The trigger signal break-out box captured the trigger signal from the temperature control box and activated the image capture and image processing process controlled by the particular LabVIEW-based software. The LED was turned on to illuminate the sample for fluorescence and a picture of the sample was captured by the CCD camera arrangement (figure 13).

Figure 13. Illumination of a fluorescent sample during a PCR cycle. The inserts show the software captured

images at the specific temperatures of a PCR cycle. The optical units for excitation of a fluorescent sample and detection of emitted fluorescence signal were placed above reaction center B of the LOC chip in a 45° design. For excitation of the fluorescing dye in the sample through a blue LED, the optical excitation path was focused through the oil into the aqueous reaction mix solution. A) When the sample droplet was enlightened at 94°C during the denaturation step, no fluorescence was emitted from the fluorescent dye inside the oil-covered sample droplet. This was due to the denatured DNA strands, being single-stranded and thus eliminating binding of the fluorescent dye SYBR Green I. Thus, software captured images showed no fluorescence signals. B) When the sample droplet was enlightened at 60°C during the annealing and extension step of a 2-step PCR performance, green fluorescence signals were emitted from the SYBR Green I dyed sample and captured by the optical detection unit. This was due to the double-stranded DNA molecules at the end of this temperature step, enabling the incorporation of the fluorescent dye.

Taken pictures were stored in a separate folder and fluorescence intensities produced at the end of each cycle were plotted graphically by the software. At the end, an excel file was generated summarizing all the collected and measured intensities. There were two kinds of pictures generated, “original” ones as well as “processed” ones. “Original” pictures represented the real image, while “processed” pictures represented subtracted fluorescence intensities. The previous image was subtracted from current image, so just the fluorescence increase was displayed.