1.

Introduction

The task of a biochip reader is to measure the intensity of light emitted from fluorescence-labeled biochemical species in the spots of a biochip that have been printed on a chemically surface-treated substrate.^{1, 2} A number of techniques have been developed during the last several years for this purpose. ^{1, 2, 3, 4, 5, 6, 7, 8, 9} These experimental approaches differ with respect to excitation mechanism (direct irradiation or via evanescent waves; single-spot, multispot, or full-area excitation) and detection technique (type of sensor; temporally, spatially, and/or spectrally resolved signals).

To obtain a high biochip throughput, full-area flood illumination of the fluorescence samples is often employed. Equipped with a CCD-based detection unit, diagnostic systems using this approach are rapid and cost-efficient.^{3, 4} Parallel readout of the biochip enables screening of many fluorescence samples simultaneously in a short time. Also, these systems are versatile with respect to the printing format of the biochips, i.e., they do not require a well-defined pitch of the spots of the microarray. On the other hand, a comparatively high level of background fluorescence significantly reduces their sensitivity.

Alternatively, highly sensitive fluorescence detection can be accomplished by excitation with a focused laser beam.⁵ The fluorescence light from the spots is then detected by a photomultiplier tube (PMT), where the background fluorescence is reduced by a confocally mounted pinhole in front of the PMT. Here, an increased SNR enables an improved detection limit of the fluorescence light. Combined with fluorescence excitation by means of evanescent waves^{6, 7} (sometimes referred to as the total internal reflection fluorescence or TIRF), this method is capable of detecting even single fluorescent molecules.

However, to read out a complete microarray using the laser excitation approach, a mechanical scanning system is required because only one spot can be excited and read out at a time. Due to this sequential readout, the throughput of these systems is low.

Mehta ⁸ and Blom ⁹ demonstrated parallel fluorescence detection through multispot excitation using a laser-illuminated diffractive optical element. In this way, two essential advantages were realized: highly parallel excitation and an excitation only at the position of the spots of the biochip. Highly parallel multispot excitation increases the throughput of samples; excitation only at the position of the spots reduces background fluorescence from unbound marker molecules in the analyte surrounding the spots on the microarray, which means an increased excitation efficiency and improved SNR. However, when using static diffractive optical elements for holographic excitation, matching the diffraction pattern to the spot pattern requires additional imaging optics and mechanical translation stages. Therefore, the adaptation of the excitation unit to new spot patterns requires modification in the hardware of the setup.

Due to the availability of dynamic diffractive optical elements (D-DOEs), this limitation can now be overcome, such that arbitrarily pitched spot arrays can be excited without need for any further optical components. We present here a biochip reader that provides a means for dynamic holographic excitation in combination with a CCD-based hyperspectral detection unit for simultaneous fluorescence analysis at several wavelengths. The combination of these new technologies makes it possible to realize a high-throughput biochip reader with a high sensitivity and great versatility.

2.

Holographic Excitation

The basis of holographic excitation is a diffractive optical element (DOE), which is able to generate an arbitrary spatial phase modulation of an incoming lightwave. The appropriate phase modulation can generate a well-defined, 3-D modulation of the light intensity after reflection from or transmission through¹⁰ the DOE.

The calculation of the required phase modulation for the DOE to achieve a desired arbitrary intensity profile is based on the reversibility of the light paths, i.e., light passes through an optical system backward in the same way as it does forward. Tracing back the light paths from the desired intensity distribution on the image plane (i.e., at the biochip) to the phase-shifting DOE enables the determination of the required phase shift to yield this intensity distribution.^{11, 12}

For adjustment of the pitch of the spot intensity pattern without replacement or mechanical movement of components, reflective liquid crystal displays [for example, a Hitatchi liquid crystal on silicon (LCoS) chip with $1280\times 1024\text{pixels}$ ] can be used as a D-DOE. The liquid crystal has an electric-field-dependent refractive index such that the refractive index of each cell of the LCoS display can be individually modified. The resulting spatial distribution of the refractive index causes a spatial phase modulation of the lightwave reflected from the LCoS display. Thus, for adaptation of the biochip reader to a new fluorescence spot pitch, only the calculation of the new phase modulation pattern of the D-DOE is required. The phase modulation pattern of the LCoS displays can be refreshed dynamically at a rate of $100\mathrm{Hz}$ , thereby enabling a linear movement or rotation or pitch adjustment of the intensity pattern at that rate.

3.

Instrument Design

3.1.

Excitation Unit

Figure 1 shows the optical setup for parallel holographic excitation. Two lasers illuminate two reflective liquid crystal displays (LCDs). The first, a $405\text{-}\mathrm{nm}$ laser diode, has a maximum power of $25\mathrm{mW}$ (Chromalase 405, Blue Sky Research) and the second, a diode-pumped solid-state green laser, $32\mathrm{mW}$ at $532\mathrm{nm}$ (GLMC1-30). The intensity of both sources can be individually adjusted by the laser current. The two laser beams are expanded by two-beam expanders with confocal pinholes $\left(5\mu \mathrm{m}\right)$ to fully illuminate the LCDs, which then modulate the phase of the incident light in a 2-D pattern as already described. The two separate light paths are joined together by mirror M1 and the dichroic mirror. A further mirror (M2) directs the modulated laser light through the biochip to excite the sample spots.

Fig. 1

Experimental setup for holographic excitation using two lasers and two LCDs; fluorescence detection for hyperspectral detection, consisting of filters for blocking the excitation light, a dispersive prism, and a highly sensitive CCD camera, equipped with a high-numerical-aperture objective lens. For the line-by-line scan and alignment of the biochip with respect to the excitation intensity pattern, a motorized $xy$ translation stage and a motorized rotation stage is used in the setup.

Generally, the rectangular aspect ratio of the LCDs leads to an asymmetric rectangular beam profile of the reflected light. To correct this asymmetry, the LCDs are illuminated under an oblique angle of incidence, which is given by the arccosine of the aspect ratio of the LCoS display. Then, the rectangular LCoS display appears quadratic to the incident beam, and the reflected beam has a square truncated Gaussian beam profile. At the same time, reflecting the circular expanded laser beam to a square beam profile is more efficient than reflecting it to a rectangular profile. Oblique illumination of the D-DOE also simplifies the optical setup, because no additional beamsplitters are required. On the other hand, oblique illumination requires a higher effort in the calculation of the holograms. First, the hologram must be stretched in one direction because of the projection onto the inclined plane of the LCD. Otherwise the focal spots would become astigmatic. Second, the varying distance from the LCD to the focal plane (i.e., the position of the biochip) must be taken into account because the LCD plane and the focal plane are not parallel. Based on these considerations, holograms were calculated with different spot distributions and pitches.¹³ Figure 2a shows a calculated hologram for generation of 25 light spots with a pitch of $500\mu \mathrm{m}$ at a wavelength of $405\mathrm{nm}$ . Figure 2b (left and middle image) shows the type of aberrations, such as coma and astigmatism, which occur if the already-mentioned compensation for the oblique illumination of the D-DOE is not sufficiently considered in the hologram calculation. Finally, software-based full compensation of all effects resulting from oblique incidence leads to an optimized intensity profile of the spot [Fig. 2b, right image].

Fig. 2

(a) Hologram of 25 spots with compensation for oblique illumination ( $1280\times 640\text{pixels}$ with a $13.7\text{-}\mu \mathrm{m}$ pixel size) and (b) aberrations due to holograms with insufficient compensation of the oblique illumination of the DOE: left, astigmatism; middle, coma; and right, result of optimally compensated holographic structure.

By focusing the spot intensity pattern generated by the dynamic hologram onto a liquid fluorochrome film (rhodamine B, purchased from Fluka, dissolved in distilled water) sandwiched between a glass slide and a cover slide, the quality of the excitation unit was measured directly in the experimental setup. Hyperspectral detection of the fluorescence light does not provide an image of the spots, but rather an exact determination of the intensity of each spot. Figure 3a shows an image plane cross section of 21 spots generated with a D-DOE at a wavelength $405\mathrm{nm}$ . The contrast between bright and dark zones is 0.93, and the standard deviation of the intensity maximum over the 21 foci is 10%. The truncated Gaussian intensity distribution of the incident light is the reason for the smaller intensity in the last two spots on the left side. Under consideration of the light intensities of each spot, the evaluation software can determine the dye concentration from the measured fluorescence intensity of each fluorescence sample. Figure 3b shows a picture of 10 excitation spots imaged directly onto a CCD sensor in the focal plane, i.e., at the position of the biochip.

Fig. 3

(a) Intensity profile of a 1-D array of 21 focused spots generated with a D-DOE, where the intensity per spot is $10\mu \mathrm{W}$ , the beam diameter in the focal plane is $160\mu \mathrm{m}$ , the pitch is $500\mu \mathrm{m}$ , and the pixel pitch of the D-DOE is $13.7\mu \mathrm{m}$ , (b) and image of 10 focused spots on the CCD sensor.

3.3.

Filters

The fluorescence emission peak is shifted towards longer wavelengths (“Stokes shift”) with respect to the excitation wavelength;¹⁵ this shift is typically about $20\mathrm{nm}$ . To detect weak fluorescence signals, the excitation wavelength must thus not reach the detection unit, such that efficient filters for blocking the excitation light are required.

In the setup of Fig. 1 a special filter combination is used to measure the fluorescence of different sets of fluorochromes. A $435\text{-}\mathrm{nm}$ long-pass filter (Schott Glas; GG435) blocks the shortest excitation laser wavelength $\left(405\mathrm{nm}\right)$ , and a notch-filter (Semrock StopLine™ 532.0) with an optical density higher than ${10}^6$ at $532\mathrm{nm}$ blocks the second excitation laser line at $532\mathrm{nm}$ . With this combination of filters, most fluorochromes in the visual spectrum can be detected. The blue and green fluorochromes can be stimulated with the near-UV light of the blue laser. To stimulate green and red fluorochromes, the laser at $532\mathrm{nm}$ can be used. Figure 4 illustrates the transmission spectra of the filters employed in relation to the two laser lines as well as the emission and excitation spectra of the three fluorochromes, Dy415, Dy550, and Dy631.

Fig. 4

Emission (Em) and excitation (Ex) spectra of the fluorochromes Dy415, Dy550, and Dy631; the laser lines used are 405 and $532\mathrm{nm}$ ; and the transmission spectra of the low-pass (LP) and notch filters.

4.

Quantitative Concentration Measurement

To quantify the performance of the system, a DNA-based test chip was employed. On an amino-silane-coated glass slide (GAPS™ II, Corning), a microarray with different oligonucleotide concentrations was printed. The format of the chip was $25\times 75\mathrm{mm}$ and the distance between the spots $500\mu \mathrm{m}$ ; a standard VHL sense-strand oligonucleotide (SVSO) was used. Concentrations from $1000\text{to}5\mu \mathrm{M}(\mathrm{M}=\mathrm{mol}∕\mathrm{l})$ were printed with a high precision needle printer on the slides. After washing, UV crosslinking and drying, the slides were hybridized for $16\mathrm{h}$ at $42°\mathrm{C}$ with $100\mathrm{mM}$ SVSO-FL-Cy3 fluorescence labeled oligonucleotide solution. The slides were subsequently washed and dried again.

To verify the hybridized oligonucleotide concentrations, the chip was first examined with two commercial biochip readers, an Axon GenePix® 4000 and a GeneScan Biodetect 645/4. The Axon reader, with photomultiplier detection, was able to detect all concentrations $\left(5\text{to}1000\mu \mathrm{M}\right)$ with a resolution of $10\mu \mathrm{m}$ in $240\mathrm{s}$ . The CCD-based reader (GeneScan) detected the concentrations from 1000 down to $50\mu \mathrm{M}$ with an integration time of $90\mathrm{s}$ .

Figure 5 (a, top) shows a picture of the area of the test chip measured with the system developed here. With an illumination power of $15\mu \mathrm{W}$ per spot, the chip was read out line by line using holographic excitation. The exposure time of each line was $1\mathrm{s}$ . The readout of the $4\times 4$ subareas shown in Fig. 5 took $80\mathrm{s}$ in total ( $2\mathrm{s}$ for the whole readout of one line). After integration of the fluorescence signal of each spot the signal shows the correspondence between the measured fluorescence intensity and the oligonucleotide concentration. As seen in Fig. 5c, the system was able to measure oligonucleotide concentrations from 1000 down to $50\mu \mathrm{M}$ with a linear dependence between the dye concentration and the fluorescence intensity. In spite of the hyperspectral evaluation, which decreases the sensitivity of the detection by the distribution of the fluorescent light on more camera pixels, the same sensitivity as the commercial CCD-based microarray reader is attained,¹⁶ showing that implementation of hyperspectral detection in microarray biochip readers may not necessarily adversely affect performance.

Fig. 5

Detection of oligonucleotide concentrations at several concentrations: (a) test chip configuration with $500\text{-}\mu \mathrm{m}$ spot grid, including reference spots (line 1); hybridization test-spots with different match-mismatch constellation (lines 2 and 3) and oligonucleotide concentrations from $1000\text{to}50\mu \mathrm{M}$ (remaining lines); on one chip $4\times 4$ of these subareas are printed; (b) with the system measured image of one subarea of the test chip; and (c) integrated fluorescence signal of the four concentration lines with varying nucleotide concentrations showing the linear dependence of the fluorescence signal on the dye concentration.

Both CCD-based systems showed a reduced SNR at longer integration times due to a strong increase of the background noise caused by the autofluorescence of the slide and the dark current noise of the CCD. To minimize the autofluorescence of the slide, we used amino-silane-coated glass slides, which showed a much lower autofluorescence compared to TOPAS^® and PMMA plastic slides. The dark current noise of the CCD camera can be reduced by cooling the CCD chip. To detect lower concentrations than those demonstrated here, the use of a cooled CCD camera is reasonable and with this technology the system could lead to sensitivities comparable to a photomultiplier-based system.

5.

Detection of Emission Spectra

Modern biochips are able to assay complex biochemical reactions. To obtain more information from a single spot, several fluorochromes are frequently used simultaneously.¹ This requires a biochip reader capable of a detailed spectral analysis of the fluorescence signal to discriminate different fluorochromes, one motivation for the hyperspectral detection unit presented here.

To evaluate the performance of the microarray scanner, a test system with the fluorochromes Dy631 (red), Dy550 (green), and Dy415 (blue) (Dyomics GmbH) was made. Different mixtures of these dyes were spotted onto a glass slide using a commercial needle printer. As we can see in Fig. 6 , the hyperspectral detection unit can measure the fluorescence intensity of the three different dyes simultaneously. The color picture illustrates the spectral distribution of each spot of the microarray biochip and a qualitative correlation between the concentrations and the intensity of the fluorescence signal can be observed. Figure 7 shows the measured fluorescence intensities versus the wavelength for different dye mixtures; in each case, the spectrum of the individual dyes is also shown for comparison. By comparing the fluorescence spectra of the spots with the known spectra of the fluorochromes employed, evaluation of the spectra may be used to determine the dye concentrations. It is clear in the figure that the fluorescence intensity peaks correspond to the dye concentrations.

Fig. 6

Hyperspectral picture of a multicolored test biochip. The image shows eight spots with different dye mixtures of Dy415 (blue), Dy550 (green), and Dy631 (red). The horizontal axis corresponds to the wavelength. The table on the left illustrates the dye-mixture of each spot.

Fig. 7

Emission spectrum of multicolored spots illuminated with 405 and $532\mathrm{nm}$ . The plots show the measured data and the native spectra of the corresponding fluorochromes: (a) three emission spectra of spots with Dy415 and Dy550 at different mixtures, (b) three emission spectra of spots with Dy415 and Dy631 at different mixtures, and (c) two emission spectra of three color spots at different mixtures. The minimum at $532\mathrm{nm}$ is caused by the notch filter used as a wavelength reference.

In addition, the different fluorescence intensities measured for identical concentrations of different dyes is due to the different quantum efficiencies of the dyes. As we can see in Figs. 7a and 7b, the fluorescence intensity of the red fluorochrome Dy631 is nearly the same as that of the Dy550 (green), even though the excitation with the green laser at $532\mathrm{nm}$ is not optimal for Dy631. The results also show that excitation with the two lasers at 405 and $532\mathrm{nm}$ is sufficient to excite all three fluorochromes at the same time. However, if red dyes are extensively employed, the use of a third red laser source would be preferable.

Figure 7c shows the spectral distribution of two spots with three different dyes. In this case, the fluorescence intensity of the green dye is less than the intensity of the red dye. This dimming of the green fluorescence intensity is caused by the fluorescence resonance energy transfer (FRET) from the green to the red fluorochrome, as described by Förster¹⁷ in 1948.

6.

Conclusion

A new approach for highly parallel hyperspectral fluorescence spectroscopy using holographic excitation of a microarray biochip was presented. A key feature of the instrument is the flexible, multipoint excitation of fluorescence spots using spatial light modulators. This new approach has four notable advantages. First, due to the holographic multipoint illumination, excitation light is focused only onto the spots under investigation; background fluorescence is thereby reduced and the measurement shows a higher contrast when compared to flood illumination. Second, an adaptation to arbitrary spot configurations can be achieved by changing the holograms by software modification only; modifications of the hardware are not required. Third, parallel illumination and readout of 1-D spot arrays increase the throughput compared to single-point scanning techniques. Finally, continuous spectral fluorescence detection enables discrimination of various dyes without modification of the optical setup, providing additional information on the chemical reactions that take place on the biochip and increasing the sensitivity by eliminating significant background fluorescence. The presented system thus represents a fast, universal, and reliable prototype biochip readout unit and enables high throughput complex DNA detection.