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Keywords:

  • blood cells;
  • hematology analyzer;
  • flow cytometry;
  • nonlinear optics;
  • supercontinuum white light laser;
  • laser

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

Multiple wavelength operation in a flow cytometer is an exciting way for cell analysis based on both fluorescence and optical scattering processing. For example, this multiparametric technique is currently used to differentiate blood cells subpopulations. The choice of excitation wavelengths matching fluorochrome spectra (it is currently the opposite) and the use of a broader range of fluorochromes can be made by taking advantage of a filtered supercontinuum white light source. In this study, we first wished to validate the use of a specific triggered supercontinuum laser in a flow cytometer based on white light scattering and electric sizing on human blood cells. Subsequently, to show the various advantages of this attractive system, using scattering effect, electrical detections, and fluorescence analysis, we realized cells sorting based on DNA/RNA stained by thiazole orange. Discrimination of white blood cells is efficiently demonstrated by using a triggered supercontinuum-based flow cytometer operating in a “one cell-one shot” configuration. The discriminated leukocyte populations are monocytes, lymphocytes, granulocytes, immature granulocytes, and cells having a high RNA content (monoblasts, lymphoblasts, and plasma cells). To the best of our knowledge, these results constitute the first practical demonstration of flow cytometry based on triggered supercontinuum illumination. This study is the starting point of a series of new experiments fully exploiting the spectral features of such a laser source. For example, the large flexibility in the choice of the excitation wavelength allows to use a larger number of fluorochromes and to excite them more efficiently. Moreover, this work opens up new research directions in the biophotonics field, such as the combination of coherent Raman spectroscopy and flow cytometry techniques. © 2012 International Society for Advancement of Cytometry


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

In cytometry devices, the use of multiple wavelength excitations (MWEs) represents a great advantage, especially for optical spectroscopic measurements. A few decades ago, MWE was realized using lamps based on QTH (Quartz Tungsten Halogen) or electric discharge in a gas (xenon- or mercury-type) (1, 2). Unfortunately, many drawbacks inherent to the use of such light sources restrict the scale and range of biological analyses; in particular, the spatial coherence of these lamps is too weak for simultaneously measuring low angle light scattering, and the weak energy of the spectral density strongly limits fluorescence measurements. Subsequently, flow cytometry systems including a pulsed laser, such as a copper vapor laser or a semiconductor diode laser, have been proposed (3, 4). These systems allow instantaneous illumination with higher intensity but with only one monochromatic radiation.

Multiple fluorescence measurements require the combination of several monochromatic lasers. In this context, new generation of cytometers can integrate up to six laser sources and allow the detection of 18 or more fluorescence signals (5–8). However, such MWE configuration requires a complex alignment of the optical system, reduces opto-mechanical stability and increases the cost of the system. The inability to tune the wavelengths to a specific value is also a limiting factor of these devices. Although new fluorochromes, including the FRET technology, enlarge the scope of multiple parameter analysis, limiting factors still exist, even if spectral compensations are used. Despite the recent diversification of monochromatic laser wavelengths (9), usual laser sources do not provide any flexibility to well-match the optimal excitation wavelength of any fluorochrome of interest. In addition, commercial lasers allow to excite only a small part of available fluorescent probes.

To overcome these drawbacks, the supercontinuum white-light laser (10, 11) appears as an outstanding potential alternative (12). Once properly filtered, it can be suitable as an illumination source for any selected fluorochromes (for example Texas Red). Such broadband source can even be used to generate a set of spectral channels, thus allowing to simultaneously excite several fluorochromes. In addition, the replacement of several monochromatic lasers by a single light source may significantly improve the mechanical stability of the system and reduce optical alignment issues. Consequently, in the context of multiparametric flow cytometry, the use of supercontinuum sources is a promising approach both in terms of simplicity and quantity of information.

In this article, we introduce a new supercontinuum laser, specifically dedicated to flow cytometry. The implementation of this laser in a hematological analyzer is suggested with a novel “one cell-one shot” (OCOS) configuration, i.e., each biological cell is illuminated by a single optical pulse. To reach this aim, the laser emission is triggered according to the passing of the cells through a preliminary electrical measurement (13). We use a compact triggered supercontinuum laser (TSL), characterized by a low repetition rate (<2 kHz) and a low average power (<6 mW). This approach is fundamentally different from the usual one, which consists in several pulsed illuminations of biological cells (i.e., a lot of optical pulses for each cell) by means of a MHz/multi-watt supercontinuum source. It resolves the problem of handling high optical power in a flow cytometer (12) and is clearly advantageous in terms of size and cost (10–20 keuros).

Here, the TSL-based OCOS configuration is applied to the multiparametric analysis of human blood cells, including electrical sizing, orthogonal white light scattering, and fluorescence measurements. To the best of our knowledge, this is the first practical demonstration of using a TSL in a flow cytometer.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

Experiment Overview

The general principle of the developed OCOS method is schematically depicted in Figure 1. First, the passing of a biological cell in the electrical window is detected through impedance measurement (Step 1). The produced electrical signal is then reshaped as a rectangular pulse (Step 2) to be used as electrical trigger. The emission of a laser pulse coming from the TSL is then obtained with a controllable time delay tdriver (Step 3). Finally, the laser pulse is effectively emitted after a given time (tlaser), and the biological cell is illuminated by this single laser pulse when passing through the optical window (step 4). Electrical and optical windows are separated by 210 μm.

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Figure 1. Principle of the flow cytometer in OCOS configuration.

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Flow Sample and Specimen Description

Only healthy human peripheral bloods were used during the experiments. We punctured 4 mL blood sample in K3ETDA tubes (VACUETTE®). Then, 30 μL were mixed with 2 mL lytic reagent. Two different lytic reagents were tested to differentiate and to count white blood cells. In a first experiment, a reagent based on saponin (research-used Horiba ABX reagent) was used to demonstrate optical scattering and electrical sizing measurements. In a second experiment, to the same cell prep, thiazole orange (TO) dye was added (14) to stain the nucleic acid of leucocytes (RNA and DNA) and thus to operate fluorescence measurements. A total of 24 μL of each dilution was carried to the flow chamber using hydrodynamic focusing. The flow rate was 5 m/s and the data collection was realized during 15 s.

Illumination Source

The illumination source, i.e., the supercontinuum laser [see picture in Fig. 2 (left)], is described hereafter. This laser system was built collectively by the authors and is now available as a commercial product from Leukos Company under “STM-4” reference. To operate the flow cytometer in OCOS configuration, the laser has to offer the functionality of external triggering by the electrical signal coming from the impedance measurement. Such TSL has been developed by combining specifically designed pump source and photonic crystal fiber (PCF). The pump source is a passively Q-switched Nd:YAG microchip laser operating at 1064 nm and delivering 6 μJ/450 ps pulses at a repetition rate ranging from 0 to 2 kHz. The 6-m-long PCF is based on a highly nonlinear air-silica microstructure with 2.5 μm average hole diameter and 4 μm hole-to-hole spacing. The pump energy is predominantly launched onto the fundamental guided mode of the structure (LP01 mode), having its zero-dispersion point located closely below the 1064 nm wavelength. A prominent spectral broadening is obtained on both sides of the pump by means of third-order nonlinear processes such as modulation instability, soliton propagation/interaction, four-wave mixing (FWM), and cross-phase modulation (XPM). The output supercontinuum ranges over more than two octaves from the blue/violet to the deep near infrared wavelengths (400–2000 nm), for any value of the repetition rate. At 2 kHz, the total visible/infrared average power is ∼6 mW, and the spectral power density is primarily between 6 and 10 μW/nm in the visible range, as plotted in Figure 2 (right).

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Figure 2. Picture of the compact TSL (left) and visible emission spectrum measured at 2 kHz repetition rate (right).

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Laser Safety: Regarding laser safety, the TSL is classified as a Class IIIb laser product, against Class IV for ultrafast supercontinuum sources (12). In spite of the reduced average power, eyes protection is required.

Flow Cytometry

HORIBA Medical Pentra 60 instrument has been set-up and modified to integrate the specific supercontinuum laser source. This automatic hematology analyzer is based on flow cytometry principles using hydrodynamic focusing (15). The flow cell is coupled to a specific optical system as depicted in Figure 3. The flow of cells circulates in the perpendicular plane of the drawing. Before passing through the laser beam, biological cells pass through a microhole which is immersed in a liquid with a defined conductivity. Impedance modification between electrodes, placed on each side of the microaperture, reveals the presence of a cell and delivers information on its volume. The signal produced at the electrical gate drives the TSL using a specific electronic controller performing the following functions:

  • electrical shaping of the trigger and restricting of the repetition rate (2 kHz maximum) to be compliant to supercontinuum requirements,

  • acting as a delay line to adjust the time windowing between electrical and optical gates. This peculiar electronic function permits accurate measurements and suppresses mechanical adjustment between the sensing regions, which is long and fastidious.

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Figure 3. Optical system of hematology analyzer. The cell flow circulates in the perpendicular plane of this drawing. For experiment 1, operated under white-light supercontinuum illumination: side scattering is measured by detector 1; strictly no filter is used. For experiment 2, operated under blue supercontinuum illumination: dichroic filter is FF506-Di02 (Semrock), bandpass filter 1 is FF01-530/43 (Semrock), and TO fluorescence is measured by detector 1; bandpass filter 2 is FF01-482/35 (Semrock), side scattering is measured by detector 2.

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Thus, the controller operates the laser illumination when a blood cell crosses through the optical window. Thus, this precise driving of the TSL can be effective with OCOS configuration. The optical gate is created using a specific beam shaper (16). It is based on a 50 cm piece of multimode step index optical fiber having a rectangular cross section (RCS) 150 × 70 μm2 core with 0.22 numerical aperture (NA). The output end of the PCF (NA < 0.13 in the visible range) is placed nearly 50 μm front to the RCS fiber. The mismatch between NAs reduces the optical coupling efficiency down to 60%. The PCF end position is adjusted by means of micrometric translation stages so as to obtain the almost full illumination of the RCS fiber core. Therefore, a high number of transverse guided modes are excited in the RCS fiber, which acts as a spatial homogenizer producing a flat top illumination with a RCS. By this way, a polychromatic secondary optical source is available, with adequate brightness and beam profile. Afterwards, the light at the RCS fiber output end is imaged into the flow of cells. This optical illuminator, which chromatic and spherical aberrations have been corrected, produces a well-defined 97.7 × 45.6 μm2 image intercepting the flow of cells. Cells are moving along the largest dimension of the optical gate to get a higher tolerance to laser time jitter as explained later.

A first experiment deals with full supercontinuum spectrum illumination without any spectral filtering in the setup. The orthogonal light scattered by the blood cells is collected with a high NA objective (NA = 0.8), for which sphero-chromatism have been minimized at 530 and 650 nm. To remove the stray light due to multiple reflection and refraction in the optical system, the orthogonal light is spatially filtered through a 500-μm-diameter pinhole. The cleaned light beam is directed toward an optical detector (detector 1 in Fig. 3) using a thin relay lens with 10 mm effective focal length.

A second experiment is dedicated to the measurement of scattering and fluorescence using blue illumination obtained using supercontinuum optical filtering. In this case, a bandpass filter (FF01-482/35, Semrock) is incorporated inside the illuminator where the light beam is collimated. A second detector (detector 2 in Fig. 3) is added to the setup and three filters: bandpass filter 1 (FF01-530/43), a dichroic filter (FF506-Di02), and bandpass filter 2 (FF01-482/35), all purchased from Semrock. TO fluorescence and orthogonal light scattering are measured by detectors 1 and 2. Absorption and emission spectra of TO dye are represented in Figure 4.

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Figure 4. Absorption (line curve) and emission (dashed curve) spectra of Thiazole Orange (TO). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Detectors 1 and 2 are photomultiplier tubes (H9307, Hamamatsu Photonics).The PMT voltages are 590 V (SSC detector) and 600 V (fluorescence detector) for experiment with Thiazole Orange. The PMT signals are amplified using home made electronic amplifiers providing ×10 gain amplification with 100 kHz (−3 dB) bandwidth. SSC and fluorescence pulses are ∼1μs duration with 800 mV average amplitude. We used several steps to digitize and to store subsequent analysis: (1) analog processing and DSP (Digital Signal Processor) allow digitizing, (2) data are stored on the workstation as raw data, each pulse event being characterized by the pulse time arrival, the pulse amplitude, and the pulse width, and (3) data are processed to form in fcs format files. The acquisition time is 10 s and the average number of events collected is 7000.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

Laser Pulse-to-Pulse Temporal and Power Stability

For flow cytometry applications, TSL requires the setting of electro-optical parameters: spectrum extension, beam quality, spectral power density, total average power, long-term power drift, pulse-to-pulse power stability, and pulse-to-pulse temporal stability. The latter depends on two contributions, the pulse creation delay (PCD) and the temporal jitter (TJ):

  • - the PCD is the average time between electrical trigger and pulse light emission,

  • - the TJ expresses a random time due to statistical optical effects within the laser cavity.

Finally, the delay time tlaser (see Fig. 1) between trigger and cell illumination is comprised between law and high values as:

  • equation image(1)

The PCD and the TJ were measured for the supercontinuum laser described previously as a function of the repetition rate and are plotted in Figure 5 (the TJ being depicted by error bars). The PCD ranges from 28.2 to 31.2 μs as the repetition rate varies from 10 Hz to 2 kHz. The TJ varies slightly from 2.5 to 2.6 μs. According to these results, the maximum variation of tlaser is 5.5 μs (32.5 − 27) for a repetition rate ranging from 10 Hz to 2 kHz.

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Figure 5. Evolution of tlaser parameter as a function of the laser repetition rate. tlaser is defined as the time between TSL driving and cell illumination. The error bars depict TJ (temporal jitter). Three repeats for each frequency are realized with 15 min of exposure time.

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The pulse-to-pulse power stability of the laser source is another key feature in a flow cytometer. Here, it has been measured in two cases, i.e., without optical filtering of the supercontinuum and by using a blue filter (FF01-482/35, Semrock), the repetition rate being fixed to a specific value or randomly varied between 100 and 2000 Hz (Table 1). It can be observed that the maximum power variation is 6.8% for unfiltered TSL versus 11.2% with blue filtering. It has been recognized that intensity noise comes from optical nonlinear processes within the microstructured fiber (17). Such amplitude noise could be reduced by using a dedicated detector to measure the peak power of each laser pulse and by applying a correction factor during software postprocessing. Nevertheless, as demonstrated in the following experiments, this optical noise is acceptable to accurately measure scattering and fluorescence from stained leucocytes.

Table 1. Pulse-to-pulse power variation of the TSL as a function of the repetition rate.
 Repetition rate (Hz)100500100015002000Random
  1. Measurements were made in two cases, i.e., without filtering the supercontinuum and by using a blue filter (FF01-482/35, Semrock). The repetition rate is fixed to a specific value or randomly varied between 100 and 2000 Hz.

Pulse-to-pulse power variation (%)Supercontinuum without filter2.33.52.72.95.96.8
Supercontinuum with blue filter6.34.65.44.94.811.2

Side Scattering Under White-Light Supercontinuum Illumination

To obtain an effective OCOS demonstration, time windowing was optimized according to many others parameters among which are tdriver, tlaser, electrical and optical gate size/distance, cell velocity and its variation. The optimized system was first applied to the analysis of white blood cells through scattering measurements under white-light supercontinuum illumination.

Side (orthogonal) scattering coming from each cell after its interaction with the TSL light beam was detected and recorded. The results obtained are illustrated by the biparametric representation of Figure 6, where each dot represents one cell. The electrical signal allows to count cells and to determine their volume. The side scattering signal is a function of the cell lobularity and granularity (1). The discrimination of monocytes (5.9%), lymphocytes (32.4%), and granulocytes (60.6%) is then demonstrated, proving the effectiveness of our compact supercontinuum sources for hematological diagnostics in OCOS configuration. To the best of our knowledge, this is the first demonstration of flow cytometry based on triggered supercontinuum illumination.

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Figure 6. Biparametric analysis of human peripheral white blood cells using a TSL in OCOS configuration, operated under white-light supercontinuum illumination. Each dot stands for one cell. The electrical signal intensity (x-axis) is a function of the cell volume. The orthogonal scattering intensity (y-axis) is a function of the cell lobularity and granularity.

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Side Scattering and TO Fluorescence Under Blue Supercontinuum Illumination

To point out the attractiveness of our system, the TSL light beam was filtered in the blue range, allowing TO excitation. TO fluorescence, measured in the green range of wavelength, is correlated with the amount of nucleic acids (RNA and DNA). The multiparametric analysis of leukocytes was, therefore, conducted combining electrical, side scattering, and TO fluorescence signals. In Figure 7a, the side scattering intensity is plotted as a function of the electrical intensity. As previously, this cytogram displays the discrimination of monocytes (9.4%), lymphocytes (28.1%), and granulocytes (58.4%). Figure 7b exhibits the fluorescence versus scattering diagram. Immature granulocytes (0.16%) and cells with high RNA content (0.16%), which are monoblasts, lymphoblasts, and plasma cells, are visualized.

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Figure 7. Multiparametric analysis of human peripheral white blood cells using a TSL in OCOS configuration, operated under blue supercontinuum illumination. Each dot stands for one cell. The number of events collected is 7000 on average. Representation (a): orthogonal scattering intensity versus electrical signal intensity. Representation (b): TO fluorescence intensity versus orthogonal scattering intensity.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

A pulsed PCF-based supercontinuum optical source, synchronized with cell passing through an optical window, was coupled to a flow cytometry device for hematology purpose. The novelty of this system relies in the triggering mode, allowing cell by cell illumination and both qualitative and quantitative scattering and fluorescence measurements.

In comparison with solid-state lasers currently used in multicolor flow cytometers, microchip-based supercontinuum sources provides flexibility for multiple wavelength experiments by offering compactness and by reducing optical alignment constraints. The use of a supercontinuum as a programmable optical source considerably extends the field of multiple fluorescence measurements without the complexity of breakthrough technologies recently published (18). In addition, it is a cost-competitive approach, compared with the use of several monochromatic lasers, however, providing a significant quantity of biological information.

In comparison with supercontinuum technologies based on ultrafast pulsed lasers (10), the microchip laser technology allows a significant reduction of power consumption while producing super bright visible emission. Within the scope of our investigation, power and TJs are considered as the main cause of optical noise, and theoretical understanding and technical progress are needed to bring the technology to an industrial level. Despite these required improvements, the TSL used in our flow analyzer prototype made possible leukocyte differentiation and counting. The full discrimination of monocytes, lymphocytes, granulocytes, immature granulocytes, and high RNA content cells was demonstrated, by means of a multiparametric analysis using impedance, scattering and fluorescence signals.

The total energy received by biological cell is an important parameter to compare TSL technology, mode locked technology, and continuous wave (CW) laser. For TSL technology, the power density is about 10 μW/nm (in the visible range) for 0.45 ns pulse duration and 2 kHz repetition rate. Therefore, each pulse provides the total energy density of 5 nJ/nm. In a mode locked technology, the power density is about 1 mW/nm, whereas the pulse duration is 6 ps and the repetition frequency is 80 MHz (see http://www.fianium.com/pdf/whitelase-sc400.pdf). Therefore, each pulse provides the energy density of 10.5 pJ/nm. As the transit time is 3 μs (corresponding to a flow rate of 10 m/s and a beam size of 30 μm), each biological cell is illuminated with 240 light pulses. Therefore, the total energy density received by a biological cell is 2.5 nJ/nm. With TSL technology, the total energy density is twice higher than the one of mode locked technology (respectively, 5 nJ/nm and 2.5 nJ/nm). The only difference comes from the temporal distribution of the energy transmitted to the cell. In the TSL technology, this energy is provided by a single pulse illumination, whereas it is based on multiple pulse illuminations using mode locked operation. Also, in flow cytometry application, 20 mW CW laser is often used. Based on 3 μs transit time, laser beam provides 60 nJ of energy. This configuration can be reached by using 12 nm wavelength bandwidth using TSL technology, which is very acceptable for fluorescence applications.

In our research work, we did not consider the fluorescence saturation effects which depends on several other parameters such as laser intensity and chemical compounds used for staining cells (19, 20).

We are currently investigating how electro-optical or acousto-optical (AOTF nC.TN, AA Opto Electronic Compagny) devices (21, 22) can be combined with the supercontinuum technology so as to realize a fully programmable optical source for which one or several wavelengths are selected with adjustable energy. Such a programmable laser could be applied to cell diagnosis using several fluorochromes conjugated antibodies (6, 7) with reduced spectral compensation (23), resulting in more accurate fluorescence measurements. The possibility to select multiple optical radiations with specific wavelength and intensity is an important improvement for single cell monitoring, allowing wider flexibility in multicolor flow cytometry applications. It is worth to note that using compact supercontinuum optical sources will open up new research avenues in biophotonic instrumentation, such as the implementation of coherent Raman spectroscopy or time-resolved fluorescence in flow cytometry devices.

Literature Cited

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

Additional Supporting Information may (MIFlowCyt item location) be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22065_sm_SuppInfo.doc47KSupporting Information: MIFlowCyt

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