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The aim of this study is to combine multiple excitation wavelengths in order to improve accuracy of fluorescence characterization of labeled cells. The experimental demonstration is realized with a hematology analyzer based on flow cytometry and a CW laser source emitting two visible wavelengths. A given optical encoding associated to each wavelength allows fluorescence identification coming from specific fluorochromes and avoiding the use of noisy compensation method.
©2011 Optical Society of America
1. Introduction
Flow cytometers are currently used to detect blood diseases [1–3] and to monitor pathology evolution such as residual disease [4]. Technically, a flow cytometer gates stained cells previously labeled with antibodies conjugated to dyes or directly stained with dyes or fluorochromes, and determines their relative percentages. The simultaneous use of several dyes introduces difficulties, as spectral overlaps of their beam excitation and/or fluorescence emission spectra. To overcome these problems, specialists generally use a correction method called “compensation” [5–10]. It consists in the removing a part of collected energy by an amount proportional to the spectral overlap between two fluorescence signals. Unfortunately, the main problem induced by the use of this method is the calculation of compensation factor which is based on the average values of chemical properties of fluorochromes. For a same fluorochrome family, these factors can be drastically different and are directly supplier dependent [6]. A same product coming from different manufacturers can give different sets of compensations, causing a critical problem for the calibration of instruments and giving rise to erroneous measurements which could induce dangerous interpretation of results. In order to identify a given fluorescence among multiple signals or to enhance fluorescence detection, encoding techniques have already been used in several systems [10–16]. Indeed, fluorescence detection can be drastically improved if only one fluorochrome is excited at a given time by a single wavelength [17,18]. Unfortunately this method induces a long recording time when several fluorochromes are used. A new technique for simultaneous recording of multiple fluorochromes has been published in 1993 by Aslund et al. [13]. It consists in intensity-modulating the illumination wavelengths at different frequencies and sending them simultaneously on samples. A lock-in detection technique permits to separate the fluorescence contributions arising from each wavelength.
In this paper, we present results on simultaneous measurements of wavelength resolved scattering and fluorescences in a hematology analyzer. An encoding optical technique of incident light beams allows to decrease or to eliminate problems of multiparametric flow cytometry such as fluorescence “compensation”. In flow cytometry, this technique brings new insights in multiple fluorescence issue for multiparametric monitoring of a single particle. Our measurements are performed by using fluorescent polymer microspheres.
2. Experimental set-up and method
Experiments are realized with a hematology analyzer integrating principle of flow cytometry based on hydrodynamic focusing. In addition to flow cytometry technique, this apparatus allows blood cell population counting and identification by using scattering and fluorescence measurements. Particle flow circulating at the speed of 5 m/s is coupled with a specific optical layout presented on Fig. 1 . The optical laser source (COBOLT Dual Calypso) produces two CW lines at 491 nm and 532 nm. It is composed of two different gain medium (Nd:YAG, NdYVO4) pumped by a single CW diode at 808 nm. Sum frequency generation between wavelengths at 914 nm and 1064 nm and second harmonic generation of the 1064 nm radiation allow the generation of the two visible wavelengths by means of a PPKTP crystal with two grating periods. The linearly polarized output laser beams are TEM00 with a narrow spectral bandwidth (<0.01pm) and a low noise (<0.3% rms). An acousto-optic modulator (AOTF nC.TN, company AA Opto Electronic) independently selects and encodes the two radiations. This special acousto-optic tunable filter can control simultaneously up to 8 distinct lines with narrow resolution (1-2 nm) and high extinction ratio. The laser beams are spatially shaped through an optical system composed of two spherical and cylindrical lenses to adjust the elliptical beam to flow chamber dimensions. A standard microfluidic device (HORIBA ABX Pentra 60 instrument, Montpellier, FR) is used to sample and to mix green and red fluorescent microspheres. Particle flow is analyzed during 15 seconds.
Fig. 1 Optical system of the hematology analyzer. The particle flow circulates out of the plane, in our direction.
Particle counting is realized by using impedance measurement placed close to the electric flow chamber. The particles propagate through a micro-hole immersed in a liquid with known conductivity. The impedance modification between electrodes placed on each side of the micro-aperture reveals the presence of a particle. The impedance value also allows obtaining information on particle volumes [19].
Optical measurements are performed after the electrical gating. In our set-up, the optical detection is composed of three channels. The first one is used to measure the forward light scattering (FSC) obtained in the vicinity of the beam axis. This scattered light is detected by a photodiode (S1223 Hamamatsu) placed behind a bar stop. The FSC is more sensitive to cell size than to other cellular characteristics. The axial light forms two half-moon of 1-3° apparent diameter coming from the scattering region. Fluorescence emissions at the red and green wavelengths coming from lit microspheres (respectively Duke Scientific Corporation R0300 and G0300) are selected through a Semrock dichroïc filter (FF01-513/17 and FF01-582/75). Excitation and emission spectra of these microspheres are shown on Fig. 2 . Signal channel 1 (S1) is composed of a Semrock filter FF01-513/17, a Semrock dichroïc filter FF562-Di02, a focusing lens and a photomultiplier tube (Hamamatsu) to selectively record green fluorescence. Signal channel 2 (S2) is composed of a Semrock filter FF01-582/75, a focal lens and a photomultiplier (Hamamatsu) to measure red signal close to 610 nm. It is important to note that spectral emission of the two microspheres are overlapping in the 560-650 nm spectral window, thus increasing signal to noise ratio and decreasing accuracy of characterizations obtained from channel 2.
Fig. 2 Absorption and emission spectra of green fluorescent microspheres. Absorption and emission spectra of red fluorescent microspheres. Excitation wavelengths of laser (λ = 491 nm and λ = 532 nm). Bandpass filters for two fluorescent detections (rectangles).
In our system, a programmable optical synthesizer based on an acousto-optic modulator allows the selection and the encoding of each incident beam. A given sinusoidal intensity modulation at 200 kHz and 300 kHz is associated to λ1 = 491 nm and λ2 = 532 nm respectively. In these conditions the incident multicolor beam expression is given by Eq. (1) and Eq. (2), sm1 and sm2 are the first and the second terms of Eq. (2) respectively.
I10 and I20 are the maximum intensities of optical signals; M1 and M2 represent the modulation amplitudes and w 1 and w 2 are the modulation frequencies.The optical tunable filter is able to induce different modulations on optical signals with variable efficiency. This parameter has been measured for the two input wavelengths at 491 nm and 532 nm and is illustrated on Fig. 3 . A modulation efficiency higher than 40% can be experimentally achieved for both wavelengths but only for modulation frequency between 0 and 300 kHz.
Fig. 3 Optical modulation efficiency of the tunable acousto-optic filter versus frequency for laser beams at 491 nm and 532 nm.
3. Results
The incident and encoded radiations (Fig. 4(a) ) interact with green and red fluorescent microspheres propagating in microfluidic exposition chamber at a speed of 5m/s. Because of the microsphere size and speed, the interaction time with the CW multi-wavelength laser beam gives rise to output pulsed signal with a quasi-Gaussian shape and a duration of ~50µs (Fig. 4(b)). The modulated output radiations (sm, Eq. (3) and Eq. (4)) are shown on Fig. 4(b) and Fig. 4(c).
I10 and I20 are the maximum intensities of optical signals; M1, M2 represent the modulation amplitudes, w 1 and w 2 are the modulation frequencies; kG, kR and k’R (Eq. (5)) are factors depending on quantum yield of fluorescent beads (ρG and ρR of R0300 and G0300 beads respectively (Duke Scientific Corporation)).The emitted fluorescence signal is detected close to 510 nm and 610 nm by two photomultipliers placed in channels 1 and 2 respectively. The photomultiplier of channel 1 (signal S1) only collects part of the green microsphere fluorescence signal (a.kG.sm1(w 1)) (Fig. 4(b)). Detector 2 (signal S2) collects a great part of the red microsphere fluorescent signal excited by both 491 nm (b.kR.sm2(w 2)) and 561 nm lights (c.k’R.sm1(w 1), and a small part of the green microsphere fluorescence (d.kG.sm1(w 1)) (Fig. 4(c) and Fig. 2) (a, b, c and d factors are depending of bandpass filters). The digital signal processing is realized with a home-made algorithm based on the Labview software allowing computation of each frequency component and identification of fluorescent signals coming from green or red microspheres.Fig. 4 (a) Incident light beams at 491 nm and 532 nm modulated with frequencies w 1(200 kHz) et w 2(300 kHz) respectively. (b) Fluorescence signal at 510 nm from channel 1. (c) Fluorescence signal at 610 nm from channel 2 (experimental data).
Channel 2 signal processing is based on Fourier transforms and on filters. This processing provides fluorescence efficiency of red microsphere and allows its identification (Fig. 4(c)). The result of the numerical calculation showing envelope of the two initial modulated signals at two different frequencies is displayed on Fig. 5 . Useless signal (Fig. 5(b)) represents more than 50% of useful signal (Fig. 5(a)). Usually in flow cytometry, the crosstalk is defined as the percentage to be removed from one channel to another, due to overlap of fluorescence spectrum. Using a single laser excitation, red microsphere fluorescence (FluoRED) depends on signals of detectors 1 and 2 (respectively S1 and S2), and crosstalk factor (ε ~0,04) (Eq. (6). Using this new electro optical processing and filtering, this crosstalk has been drastically reduced.
It is important to note that numerical processing of the data is drastically affected by the modulation frequency shift Δf ( = Δw / 2π) introduced in the two laser wavelengths. Indeed, it exists a crosstalk between the two modulation frequencies because of the modulator response. Thus, a part of the amplitude modulation mainly devoted to the first optical radiation is also printed on the second one. To enlighten this phenomenon we illuminated green fluorescent microspheres only with the two modulated radiations at 491 nm and 532 nm. According to previous equations, modulated signal sm2 should be equal to zero. Unfortunately, a significant signal is detected at signal frequency sm1. This crosstalk efficiency is increasing as the frequency difference Δf between the two modulations is decreasing. For two modulation frequencies of 200kHz and 300kHz (Δf = 100kHz) the parasitic signal is estimated close to 8% with respect to the first one modulation at 200 kHz (Fig. 6(a) ). More than 20% of the noise is then observed for two modulation frequencies at 200kHz and 250kHz (Δf = 50kHz) (Fig. 6(b)).The envelope of signals at the two frequency modulations are presented on the Fig. 6.Fig. 5 Optical signals of fluorescent microspheres extracted from experimental data recorded on channel 2 (S2). (a) Useful optical signal which is red microsphere fluorescent (FluoRED) and (b) useless optical signal.
Fig. 6 Crosstalk between sm1 and sm2 signals with respect to the frequency detuning of the two frequency modulations.(a) w1 = 200kHz, w2 = 300kHz; (b) w1 = 200kHz, w2 = 250kHz.
4. Conclusion
We demonstrated microsphere characterization by using a dual laser source emitting two CW radiations at 491 nm and 532 nm. Measurements are realized with a specific hematology analyzer based on microfluidic technique. Encoding of each incident optical signal allows to discriminating fluorescence signals coming from green and red fluorescent microspheres despite their spectral overlap in the 550 - 650 nm spectral window. This new technique of spectral encoding opens new ways to improve diagnostic accuracy in the hematology field when fluorescence multiplexing is required. Digital signal processing of scattering and fluorescence channels enables to deliver accurate optical information for quantitative and qualitative experiments. This technique can be extended with a broadband source such supercontinuum light source operating in CW regime [20–22].
Acknowledgments
This work was supported by ANRT.
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