Post on 30-Jan-2023
Evaluation of a Green Laser Pointer
for Flow Cytometry
Robert C. Habbersett, Mark A. Naivar, Travis A. Woods, Gregory R. Goddard,Steven W. Graves*
� AbstractFlow cytometers typically incorporate expensive lasers with high-quality (TEM00) out-put beam structure and very stable output power, significantly increasing system costand power requirements. Red diode lasers minimize power consumption and cost, butlimit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offerincreased wavelength selection but presumed emission instability has limited their use.A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristicsand then used as the excitation light source in a custom-built flow cytometer for theanalysis of fluorescent calibration and alignment microspheres. Eight of ten modulestested were very quiet (RMS noise � 0.6% between 0 and 5 MHz). With a quiet laserpointer module as the light source in a slow-flow system, fluorescence measurementsfrom alignment microspheres produced CVs of about 3.3%. Furthermore, the use ofextended transit times and �1 mWof laser power produced both baseline resolution ofall 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoery-thrin molecules per particle. Data collected with the transit time reduced to 25 ls (inthe same instrument but at 2.4 mW laser output) demonstrated a detection limit of�75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, andpower consumption of the tested laser pointer module suggests that it may be suitablefor use in conventional flow cytometry, particularly if it were coupled with cytometersthat support extended transit times. Published 2007 Wiley-Liss, Inc.�
� Key termsflow cytometry; DPSS laser; laser pointer; portable
FLOW cytometry is now an integral technology in many bio-medical disciplines
performing diverse biological assays in both clinical and research settings (1,2).
The ability of flow cytometry to assist in the diagnosis and assessment of specific in-
fectious diseases (especially HIV/AIDS) makes this technology very important in
global health care (3–6). Additionally, flow cytometry could become an important
analytical platform to perform biological point-detection, bio-surveillance, and
forensic analysis in support of homeland defense (7,8). However, the size and
expense of most flow cytometers currently restricts their use mostly to laboratory
environments.
The need to retain the essential capabilities of existing instruments—while redu-
cing their size, cost, and complexity—is driving the development of inexpensive flow
cytometers through reductions in the size, cost, use of consumables for the fluidics,
and complexity of data acquisition and optical systems. Fluidic reductions have con-
centrated on micro-fluidic flow cytometry adaptations (9–12), using unfocused flow
without sheath (13), or the use of alternative sample focusing forces such as acoustic
(14), dielectrophoretic (15), or aerodynamic sheath (16,17). Data systems have become
ever smaller and more capable as one more benefit of the general electronics and com-
puter revolution. Optical system reductions have used low-power light sources such as
LEDs (9,18), the incorporation of diode pumped solid state lasers (DPSS), and red
diode lasers (4,19,20). Although LEDs are available with impressive output intensity at
The National Flow Cytometry Resource,Bioscience Division, Los Alamos NationalLaboratory, Los Alamos, NM 87545
Received 15 November 2006; RevisionReceived 9 July 2007; Accepted 11 July2007
Grant sponsor: NIH; Grant number:RR020064; Grant sponsor: National FlowCytometry Resource, NIH; Grant number:RR001315.
*Correspondence to: Steven W. Graves,National Flow Cytometry Resource, MailStop M888, Los Alamos NationalLaboratory, Los Alamos, NM 87545.
Email: graves@lanl.gov
Published online 21 August 2007 inWiley InterScience (www.interscience.wiley.com)
DOI: 10.1002/cyto.a.20454
© Published 2007 Wiley-Liss, Inc.�This article is a US government workand, as such, is in the public domain inthe United States of America.
Original Article
Cytometry Part A � 71A: 809�817, 2007
very low cost, and have been demonstrated in flow cytometry
(18–20), the incoherent and highly divergent light emitted
makes them more difficult to incorporate into an instrument
and the low effective excitation power density limits their
applicability (1). Small efficient lasers are much simpler to
implement in a flow cytometer and the incorporation of diode
and DPSS lasers has led to a significant cost and size reductions
for many commercial cytometers such as the FACSAria and
Cyan, and, in particular, have already made numerous small
analyzers possible such as: Accuri C6, the Guava EasyCyte Mini,
Partec CyFlow, Luminex 100, the PointCare AuRICA and Opto-
Flow Microcyte (1,21,22). Of commonly used lasers, only the
red diode laser is inexpensive ($100–$500); but red light prohi-
bits the use of many common fluorophores. Conversely, DPSS
lasers can be obtained in a variety of interesting wavelengths
including 488 and 532 nm. These wavelengths have many opti-
mized fluorophores and assays, which makes these lasers among
the most useful in modern flow cytometers. However, for use in
conventional flow cytometry a variety of factors including: laser
lifetime, asymmetrical beam structure, and noise of inexpensive
DPSS modules (typically used in laser pointers), and stability,
have made the use of relatively expensive (typically >$2000 for
532 nM and >$5000 for 488 nM) single-longitudinal-mode
(SLM) stabilized DPSS lasers, or other approaches such as
VCSELs (Coherent Sapphire), preferable (1) to less expensive
possibilities.
The purpose of this work is to evaluate the performance of
an inexpensive 532 nm DPSS laser pointer (LP) in conventional
flow cytometry. The perception has been that these lasers are
too noisy to be of use. Such DPSS 532 nm ‘‘laser pointers’’ have
been used in flow based detection systems, notably a viral parti-
cle counter (23), and a microfabricated flow sorter using optical
trapping (11). With regards to the viral counter, this system
used a 15 mW DPSS 532 nm LP without stabilization to inter-
rogate viral particles in a dual-channel fluorescence detection
system. However, insufficient quantitative data (precision or
sensitivity) was presented to evaluate the potential use of such
laser in conventional flow cytometry, and the limit of detection
was stated as �25,000 FITC molecules (a fluorophore not well
excited at 532 nm). In the case of the microfabricated flow sort-
ing application the LP was used simply to excite fluorescent
microspheres for imaging (11). In both of these publications no
effort was made to determine if the LP would be suitable for
use in conventional flow cytometry. The purpose of our work is
to determine if an inexpensive 532 nm DPSS LP is suitable for
use in flow cytometry. To perform this evaluation we adapted a
high sensitivity flow cytometer that has primarily been used in
the past for single molecule detection (24–26) to use the LP.
Here, we demonstrate, through the analysis of calibration
microsphere sets, that this LP is suitable for use in many flow
cytometry applications, and given proper system design (detec-
tors, transit times, etc. . .) can achieve sensitivity and resolution
comparable to that of flow cytometers using much more expen-
sive laser light sources. Also, we will briefly discuss the implica-
tions of this approach to low cost portable flow cytometers and
assays that could benefit from a reduction in laser power
requirements, cost, and size.
MATERIALS AND METHODS
Microspheres
We used the following microsphere products from Spher-
otech Inc. (Libertyville, IL): Rainbow Calibration Particles:
RCP-30-5A (8 peaks), RCP-20-5 (4 peaks), Nile Red Fluores-
cent Alignment Particles (FAP-3056-5), and 1.87 lm diameter
carboxylated polystyrene microspheres (CP-15-10) to provide
an unstained microsphere when used in conjunction with the
4 peak RCP-20-5 set. We also used 2 lm Nile Red alignment
microspheres (F-8825) from Molecular Probes (Eugene, OR).
Except for the F-8825 microspheres, the microspheres were
concentrated five to tenfold via centrifugation to compensate
for the low sample volumetric flow rate in this system (�0.3–
0.6 ll/min.) due to the narrow bore (40 lm ID) quartz capil-
lary tubing used to deliver the sample to the flow cell as
described in (24).
Light Source
The $159 OEM version of a commercially available LP
(532 nm, model GMP-532-5F3-CP) from LaserMate Group,
Inc. (Pomona, CA), shown in Figure 1 small inset photo, was
used in this study. We purchased 10 units of this LP module,
which has a rated optical output of 3–5 mW and operates on
2.1–3.0 VDC. Initially a Tektronix (Beaverton, OR) power
supply (model PS281) was used to provide the DC voltage
(typically 2.3 VDC @ 270 mA). Comparable system perform-
ance was obtained with an inexpensive dedicated power sup-
ply (SPC Technology power supply model 9698, 7.5 VDC out-
put @ 1 A with a simple three-terminal voltage regulator),
which was used for all experiments presented here. The LP
module itself is a very compact device with good advertised
specifications from the manufacturer (TEM00, <1.4 mrad
beam divergence, M2 < 2, and 5% output power stability).
Gaussian beam structure was verified via reflecting a magni-
fied image of the beam on a distant surface (data not shown).
Instrument Setup
The flow cell and sample delivery in this system is identi-
cal to that of the slow-flow system used for DNA fragment siz-
ing (24). For these experiments the sheath bottle was elevated
higher and pressurized to either �2 psi or �15 psi in order to
increase the flow velocity to reach a transit time of about 250
or 25 ls, respectively, as measured on an oscilloscope using
the listed microspheres, with the focused laser beam diameter
at �10 lm. For the 25 ls transit-time measurements the laser
power was set to 2.4 mW by rotating the half-wave plate with
respect to the polarizing beam splitter (Fig. 1). These devices,
in combination, limit the laser beam intensity that reaches the
flow cell by altering the amount of polarized light that can
pass through the beam splitter (27). For 250 ls transit time
experiments the laser power was nominally set to 1 mW in a
similar fashion. Thorlabs (Newton, NJ), 100 adaptor rings wereused to mount the miniature PMTs in 100 diameter lens tubes
supported by identical filter holder blocks on both sides of the
flow cell (Fig. 1). Side-scattered (orthogonal) light (SSC) was
collected through an Omega Optical (Brattelboro, VT)
ORIGINAL ARTICLE
810 Laser-Pointer Based Flow Cytometer
DF530/30 nm band pass filter and a ND 1.0 filter, and fluores-
cence was collected through a Chroma Technology Corp.
(Rockingham, VT) HQ585/40 nm band bass filter. For experi-
ments using 4 peak or 8 peak microspheres, a Raman edge fil-
ter (538ALP–Omega Optical) was added to the fluorescence
filter holder to ensure that no scattered laser light reached the
detector.
Laser Noise Measurements
For noise measurements, mirror (M) in Figure 1 was
removed and the LP output directed onto a high-speed photo-
diode (Thor Labs PDA-36A-EC variable gain photodiode ampli-
fier module) set to 0 dB gain and, therefore, operating with a
bandwidth of 17 MHz. The output of the PDA module was con-
nected to one input channel of a Tektronix 300MHz TDS 3034
Digital Oscilloscope equipped with a FFT math module. The
FFTof the PDA output signal was performed by the oscilloscope,
and the data were saved in comma separated value (CSV) for-
mat, and imported into Excel. Initial measurements on all ten
laser modules were made by providing 2.3 V from a laboratory
power supply (PS-281 Tektronix,) to the LP modules using the
manufacturer’s voltage regulation circuitry. After the initial
measurements the voltage regulator circuitry of one LP module
was removed and the module was directly driven using the labo-
ratory power supply. Additionally, identical comparison noise
measurements were made by replacing the LP in the system with
a high quality ‘‘microGreen’’ 20 mW DPSS 532 nm single-longi-
tudinal-mode (SLM) laser (JDS Uniphase, Milpitas, CA).
Detectors and Detection Electronics
PMT preamps were designed and built for the miniature
Hamamatsu (Bridgewater, NJ) 6780 PMTs, with the PMT type
used in each experiment indicated in the figure legends. These
multi-alkali metal-package detectors typically have a gain of
about 106 and cathode radiant sensitivity of 60–80 mA/W in
the 400–700 nm range, depending on the specific model used.
Hamamatsu’s design for the PMT control circuit was followed
explicitly. The preamp circuit, shown in Figure 2, was designed
to provide high input impedance and low-pass filtering, with
a cutoff at approximately 15 kHz for the 250 ls transit time
measurements. For the 25 ls transit time measurements the
pre-amplifier was modified by changing the PMT load resistor
to 200 kOhm and reducing the feedback resistor on the gain
stage to 10 kOhm giving a cutoff frequency of approximately
50 kHz (Fig. 2). A power supply was assembled to provide 65
VDC (200 mA) for the pre-amps, and 115 VDC (200 mA) to
power and control the PMT high-voltage. The PMT control
voltage was typically set to approximately 0.3 VDC for SSC
and 0.4–0.5 VDC for fluorescence. For the higher sensitivity
measurements using the RCP-20-5 microspheres (with added
nonfluorescent bead) the control voltage on the fluorescence
PMT was raised to 0.53 VDC, still well below the rated maxi-
mum of 1.0 VDC.
Figure 1. The slow-flow cytometer optics breadboard, flow cell, and sample holder. H, half wave plate; P, polarizer; S, shutter; M, mirror;
FL, focusing lens; CL, collection lens; F1, filter 1; F2, filter 2; PMT, photomultiplier tube. Inset 1. A close up of the laser pointer OEM module
with a metric scale ruler. Inset 2. A simplified schematic of the system.
Figure 2. This pre-amplifier circuit presents a 1M Ohm load to the
PMT. The values shown without an asterisk represent the pre-
amplifier used for extended transit times. The first op-amp is a high
input impedance voltage follower, which passes the signal to an
inverting op-amp which has a gain of 3.3 and a low-pass cutoff fre-
quency of 15 kHz. The pre-amplifer was modified by replacing com-
ponents with those shown marked with an asterisk. This version of
the pre-amplifer has a gain of 1.0 and a cutoff frequency of 50 kHz.
ORIGINAL ARTICLE
Cytometry Part A � 71A: 809�817, 2007 811
Data Acquisition
A prototype version of a new data acquisition system was
used to collect conventional flow cytometric data files in which the
event-based parameters of height, width, and area were collected.
Full details of this system are given in a separate publication (27),
but one aspect of this system, essential to the work presented here,
is that it accepts event durations ranging from 1 ms to >10 ms.
Custom hardware boards convert the analog pre-amp output (2 V
peak-to-peak) into a 14-bit digital data stream using a free running
14-bit, 40 MS/s, ADC (Analog Devices (Norwood, MA)—
ADS5421Y). A field programmable gate array on each custom
board captures the correlated digitized waveforms and sends them
to an OrSys (Markdorf, Germany) digital signal processor board
(Micro-line C6211CPU). The digital signal processor (Texas
Instruments (Dallas, TX) – TMS320C6211) extracts the pulse
height, area, and width parameters, sending the list-mode results
to the host computer over FireWire (IEEE1394). The pulse param-
eters are recorded in FCS 3.0 data files with 24 bits for area, 16 bits
for peak, 12 bits for width, and 28 bits for time (1 ls resolution).
Data Analysis and Visualization
FCS data files were displayed and analyzed using FCS
Express v.3.0 (De Novo Software, Thornhill, Ontario). Graphics
were converted for publication in Corel (Ottawa, Ontario)
PHOTO-PAINT Version 11. Data were fit using SigmaPlot Ver-
sion 10 (Systat Software, Inc., Richmond CA).
RESULTS AND DISCUSSION
LP Noise
The noise content of ten LP modules was evaluated by
clamping each module on the system breadboard and directing
the laser beam into a high bandwidth photodiode module. The
DC output of the photodiode was displayed by the TDS 3034
oscilloscope, which was used to perform the Fast Fourier Trans-
form (FFT) of the output of each LP (the 0 dB reference point
for the FFT corresponds to 0.5 V). The FFT is an efficient means
of transforming the time-varying amplitude of an analog signal
into a frequency vs. amplitude graph, revealing the magnitude
of every frequency component of the original analog signal.
Eight of the 10 LPs (1–5 and 7–9) were remarkably quiet at all
frequencies tested, with a measured RMS noise of 0.6% or less
for all eight, and a median RMS noise of 0.07% (Table 1). A
graph of the FFT for LP no. 3 is shown in Figure 3A, and is rep-
Table 1. Laser pointer noise measurement values
LASER NUMBER
AC RMS
(mV)
RMS DC
LEVEL (mV)
%RMS
NOISE
1 0.67 1,010 0.07
2 0.52 1,010 0.05
3 0.50 880 0.06
4 0.66 931 0.07
5 0.53 725 0.07
6 183 610 30
7 2.50 632 0.4
8 0.48 1,060 0.05
9 3.90 696 0.6
10 432 1,000 43
10 DD 1.85 1,610 0.1
Uniphase 1.25 1,320 0.09
Figure 3. This figure is the result of connecting the Thor Labs high-speed photodiode module output to one input channel of the Tektronix
3034 oscilloscope to calculate the FFT of the noise in the laser output. Panel A is the FFT of LP no. 3 which is representative of the 8 quiet
LPs. Panel B is the FFT of LP no. 10 which is representative of the two noisy LPs. Panel C is the FFT of LP no. 10 after removing the manufac-
turer’s voltage regulation circuitry and driving the laser-diode directly with a lab power supply. Panel D is the FFT of a 20 mW SLM micro-
Green laser from JDS Uniphase.
ORIGINAL ARTICLE
812 Laser-Pointer Based Flow Cytometer
resentative of the 8 quiet LPs. It is clear that at frequencies
between 0 and 5 MHz all noise is at least 280 dB, which was
found to be true for the seven other quiet LPs (data not shown).
This performance was judged likely to be suitable for flow
cytometry.
However, two of the LP modules (no. 6 and no. 10) were
clearly very noisy, with measured RMS noise values of 30%
and 43%, respectively (Table 1). A graph of the FFT for LP
no.10 is shown in Figure 3B and is similar to the FFTobtained
for LP no. 6 (data not shown). This graph contains a high
magnitude noise component only 215 dB down within the
bandpass of the pre-amp. This performance was not accepta-
ble for flow cytometry (data not shown).
To understand the source of the noise, we removed the
manufacturer’s voltage regulation circuitry (Fig. 1, inset) from
LP module no. 10 and directly powered the infra-red laser-
diode itself (at 1.8 VDC, 311 mA) using a laboratory power
supply. Operated in this manner, LP no. 10 was much quieter,
with a RMS noise value of 0.1% (Table 1—laser number 10
DD). When the FFT of the output was plotted it was seen that
the noise was once again at about 80 dB (Fig. 3C), sufficiently
quiet for flow cytometry. Therefore, it seems clear that the pri-
mary source of noise in these low-cost DPSS lasers is due to
the voltage regulator circuit, which is designed to limit the LP
output power to �5 mW (allowing these modules to be sold
as eye-safe) and to compensate for voltage drop as the output
of the battery (the intended power source) drops.
To compare the performance of the LP modules with a
high-quality laser (�$7k), we inserted a 20 mW SLM micro-
Green laser from JDS Uniphase and performed identical noise
measurements. This laser has been used extensively for single-
molecule and DNA fragment sizing measurements by flow
cytometry (24–26). This laser produced RMS noise measure-
ments (0.09%) well within the specifications (0.5%) of the
manufacturer (Table 1). The graph of the FFT obtained with
the JDS Uniphase laser is shown in Figure 3D, with similar
noise characteristics to that of the quiet LPs (Figure 3A),
which is supported by the RMS noise measurements (Table 1).
These levels of noise were judged to be sufficiently low to use
this type of LP modules in a flow cytometer, as demonstrated
below.
System Precision
With Nile Red alignment microspheres (FAP-3056-5,
Spherotech) passing through the flow cell, the sheath flow rate
was adjusted to produce Gaussian pulses (fluorescence PMT
output displayed on the oscilloscope) with an average dura-
tion of �250 ls (FWHM – data not shown) and the laser
power (measured after the beam splitter) set to � 1 mW out-
put. The pulse data for these microspheres were collected
using our custom data acquisition system (27), which
recorded height, width, and area measurements for both the
SSC and fluorescence detectors (Fig. 4). When gated only on
the SSC pulse height and area parameters, to eliminate doub-
lets and other noise (Fig. 4A), the fluorescence CV was 3.3%
for peak and 3.4% for area (Figs. 4B and 4C), which compares
relatively well with the 2.16% fluorescence that Spherotech
lists as the intrinsic fluorescence CV for this set of micro-
spheres. Nile Red alignment microspheres (F-8825, Molecular
Probes), recorded at 25 ls transit time (FWHM), laser power
� 2.4 mW, and gated on SSC peak vs. area (Fig. 4D) produced
CV measurements of 2.7% for peak and 2.6% for area (Figs.
4E and 4F), which compares well with the 2.2% CVs for these
microspheres measured using the PE channel and 488 nm ex-
citation on a Becton Dickinson FacsCalibur (data not shown).
The increased CVs we observed (compared to that of the com-
mercial flow cytometer or manufacturer’s specifications) could
be due to noise within the laser (though that seems unlikely
given the low noise observed in Fig. 3), but is probably due to
Figure 4. Nile Red Alignment microspheres. The PMTs used in
this experiment were the 6780-00 for SSC (control voltage of
0.273 VDC A–C or 0.388 VDC D–F) and the 6780-02 for fluorescence(control voltage of 0.509 VDC A-C or 0.394 VDC D-F). Transit times
were 250 ls (A–C) or 25 ls (D–F) A: A bivariate contour plot of SSCpeak vs. SSC area. The contours were drawn at 1, 5, 10, 30, 50, 70,
and 90% of event levels. The rectangular gate shown was used to
gate fluorescence values. B: A histogram of gated fluorescence
peak values. The CV of all events in this histogram is 3.3%. C: A
histogram of gated fluorescence area values. The CV of all events
in this histogram is 3.4%. D: A bivariate contour plot of SSC peak
vs. SSC area. The contours were drawn at 1, 5, 10, 30, 50, 70, and
90% of event levels. The rectangular gate shown was used to gate
fluorescence values. E: A histogram of gated fluorescence peak
values. The CV of all events in this histogram is 2.6%. F: A histo-
gram of gated fluorescence area values. The CV of all events in
this histogram is 2.7%.
ORIGINAL ARTICLE
Cytometry Part A � 71A: 809�817, 2007 813
fluidic instabilities in our slow-flow system or electronic noise
within our prototype data acquisition system. Clearly, for the 25
ls measurements we are achieving better precision, which is
probably attributable to the increased flow rates through the
flow cell and the resulting smaller sample stream diameter, but
could also be related to the intrinsic variations between the two
sets of microspheres used. Regardless, the observed precision is
likely to be suitable for most flow cytometry applications.
System Linearity, Sensitivity, and Resolution
The 8-peak fluorescent Rainbow microspheres (RCP-30-
5A) were used to demonstrate system linearity, sensitivity, and
resolution. Initial measurements were made at 250 ls transittime and LP output power set to �1mW. System settings were
as described in the methods section and fluorescence data
were gated on SSC peak and area to provide doublet discrimi-
nation (Fig. 5A). The fluorescence peak and area histograms
(Figs. 5B and 5C) demonstrate excellent peak-to-peak resolu-
tion. System linearity for fluorescence quantification can be
estimated by plotting the mean area values as a function of
known microsphere fluorescence values. To provide MESF
value estimates, we cross calibrated the 8 peak Rainbow
microspheres against BD Quantibrite PE microspheres by run-
ning them on the instrument at the exact same settings, as
described in Chase and Hoffman (28). This resulted in the
estimated MESF-PE values shown in Table 2. When analyzing
the RCP-30-5A bead set, the system displayed linearity over
four decades (Fig. 5D). While this aspect of system perform-
ance is primarily due to the PMT linearity, this data clearly
demonstrates that the low-cost laser used here produced linear
measurements comparable to any commercial flow cytometer
(over four decades of linearity).
To measure the fluorescence sensitivity of this instrument
(250 ls transit time), the detection efficiency (Q) and back-
ground (B) were calculated using the rapid method and iden-
tical minimal bead set, described by Chase and Hoffman (28).
Two modifications were made to their method. (1) MESF-PE
values were used. (2) The standard deviation of the least fluo-
rescent microsphere, which was well resolved from back-
ground and represented a normal population (Figs. 5B and
5C), was calculated straight forwardly. Chase and Hoffman
used a standard deviation calculation approach intended for
non-normal distributed populations because their least-fluo-
rescent microsphere was not well resolved above background.
In the system described here, the 1920 MESF-PE bead had a
standard deviation more than three times the standard devia-
tion of the dimmest bead (the unstained microsphere in Chase
and Hoffman’s work), and a CV more than three times greater
than the CV of the brightest bead. (The 790 MESF bead also
meets these criteria, but not as well.) Therefore, the variations
in the fluorescence measurement of the 1920 MESF-PE bead is
presumably dominated by photon statistics, and Q and B were
calculated from those data. Using the data for the 8 peak
microsphere set, shown in Table 2, Q 5 0.04, 0.07 (peak and
area, respectively), and B 5 68, 34 (peak and area, respec-
tively) MESF-PE. The area value for B indicates that at the
Table 2. Fluorescence peak and area measurements for the 8 peak rcp-30-5a microspheres analyzed with 250 ls transit time
BEAD MESF-PE PEAK MEAN PEAKCV(%) PEAK SD AREA MEAN AREA CV(%) AREA SD
1 60 5.91 47.89 2.83 346.21 25 86.55
2 790 53.39 18.03 9.63 2,155.56 18.08 389.64
3 1,920 148.78 8.88 13.22 7,469.32 8.72 651.15
4 6,340 526.2 4.24 22.29 33,408.9 3.37 1,127
5 19,830 1,684.75 2.28 38.4 119,620.44 2.03 2,426.5
6 50,326 4,397.3 1.89 83.01 325,596.1 2.03 6,597.3
7 208,058 17,878.33 1.62 289.41 1,357,471.63 1.71 23,167
8 535,963 45,845.64 1.49 680.83 3,508,256.78 1.62 56,826
Figure 5. 8 peak RCP-30-5A microspheres run at 250 ls transittimes. The PMTs used in this experiment were the 6780-00 for
SSC (0.258 VDC control voltage) and the 6780-02 for fluorescence
(0.396 VDC control voltage). A: A bivariate contour plot of SSC
peak vs. SSC area. The contours were drawn at 1, 5, 10, 30, 50, 70,
and 90% of event levels. The gate shown was used to gate fluores-
cence values. B: A histogram of gated fluorescence peak values.
C: A histogram of gated fluorescence area values. D: Fluorescence
area values vs. MESF-PE values. The error bars for each value
represent two standard deviations from the mean. The data were
fit to a linear function (log(y) 5 m * log(x) 1 b). The R2 for this fitwas 0.992.
ORIGINAL ARTICLE
814 Laser-Pointer Based Flow Cytometer
extended transit time the LP produces a detection limit well
below 50 molecules of PE per particle. The values for Q pre-
dict that the LP supports excellent resolution of dimly fluores-
cent particles on the system, which is also visually confirmed
in the histograms (Fig. 5).
For an additional evaluation of the system sensitivity at
250 ls transit time, the dimly fluorescent 4 peak RCP-20-5
microspheres were spiked with an unstained 1.87 lm micro-
sphere (CP-15-10), and analyzed with the fluorescence PMT
control voltage raised to 0.533 VDC. The RCP-20-5 MESF-PE
values were estimated to be 36, 644, 19,100, and 67,000 by
cross calibration of the 4 peak microspheres against BD Quan-
tibrite PE microspheres (28). When these microspheres were
analyzed on the slow-flow cytometer (using the LP), with the
fluorescence values gated on SSC peak and area to exclude
doublets (Fig. 6A), it was apparent that the 4 peak Rainbow
microsphere set was not monotonic in SSC. The brightest
bead exhibited slightly higher SSC area (Fig. 6A), which was
confirmed by the manufacturer (Brian Shah, Spherotech Inc.,
personal communication). The additional unstained micro-
sphere had a very similar SSC profile to the dimmest three
microspheres of the rainbow set and was not resolved by scat-
ter (data not shown). The lowest stained microsphere (36
MESF-PE) was well resolved from the unstained microsphere
in both the fluorescence peak and area histograms (Figs. 6B
and 6C) further supporting the Q value presented above.
When the fluorescence area histogram is plotted and the mean
Figure 6. 4 peak RCP-20-5 microspheres with an additional blank
polystyrene microsphere run at 250 ls transit times. The PMTsused in this experiment were the 6780-00 for SSC (0.283 VDC con-
trol voltage) and the 6780-02 for fluorescence (0.533 VDC control
voltage). A: A bivariate contour plot of SSC peak vs. SSC area.
The contours were drawn at 1, 5, 10, 30, 50, 70, and 90% of event
levels. The gate shown was used to gate fluorescence values.
Notably this gate had to be enlarged to account for the two differ-
ent sizes of microspheres within the 4 peak Rainbow set (see
text). B: A histogram of gated fluorescence peak values. C: A his-
togram of gated fluorescence area values. D: Fluorescence area
values vs. MESF-PE values. The error bars for each value repre-
sent two standard deviations from the mean. The data were fit to
a linear function (log(y) 5 m * log(x) 1 b). The R2 for this fit was0.984. A dotted line was drawn from the mean value plus two
standard deviations of the blank (0 MESF-PE) microsphere (run in
the same sample as the 4 peak RCP-20-5 microspheres) to the lin-
ear function and down to the MESF-PE axis to provide an estimate
of minimal detection capability, which was 18 MESF-PE.
Figure 7. 8 peak RCP-30-5A microspheres run at 25 ls transittimes. The PMTs used in this experiment were the 6780-00 for
SSC (control voltage of 0.349 vdc) and the 6780-02 for fluores-
cence (control voltage of 0.460 vdc). A: A bivariate contour plot of
SSC peak vs. SSC area. The contours were drawn at 1, 5, 10, 30,
50, 70, and 90% of event levels. The rectangular gates shown
were used to gate fluorescence values as described in the text. B:
A histogram of gated fluorescence peak values from the gate with
the higher SSC peak values. C: A histogram of gated fluorescence
peak values from the gate with the lower SSC peak values. This
histogram compared with (B) clearly shows that the unstained
microspheres have reduced fluorescence compared to the first
population of the eight peak Rainbow microspheres. D: A histo-
gram of gated fluorescence area values from the gate with the
higher SSC peak values. E: Fluorescence peak values vs. MESF-PE
values. The error bars for each value represent two standard
deviations from the mean. The data were fit to a linear function
(log(y) 5 m * log(x) 1 b). The R2 for this fit was 0.992. A dottedline was drawn from the mean value plus two standard deviations
of the blank (0 MESF-PE) microsphere (run in the same sample as
the 8 peak RCP-30-5A microspheres) to the linear function and
down to the MESF-PE axis to provide an estimate of minimal
detection capability, which was 52 MESF-PE. F: Fluorescence area
values vs. MESF-PE values fit in identical fashion as E. The R2 for
this fit was 0.997. The estimate of minimal detection capability
was 64 MESF-PE.
ORIGINAL ARTICLE
Cytometry Part A � 71A: 809�817, 2007 815
values (of the five populations) fit to a linear function, the
lower estimate of detection can be evaluated by extrapolating
the 95% confidence interval (derived from the mean and two
times the standard deviation) of the unstained microsphere
back to the linear function and interpolating the number of
MESF-PE that this represents (Fig. 6D). This approach is anal-
ogous to that used to establish the detection limit in early mo-
lecular flow cytometers (29,30). In our case, the extrapolated
detection limit is approximately 18 MESF-PE for area mea-
surements (Fig. 6D). The increased sensitivity of the instru-
ment for this experiment is predicted as the PMT voltage has
been raised to increase its gain and is in good agreement with
the values determined via the Q and B method (above). Using
the fluorescence peak the extrapolated detection limit is �14
MESF-PE (data not shown). These measurements indicate
that at these PMT voltages the LP can support detection limits
of �20 MESF-PE. In addition to the demonstration of sensi-
tivity this data also demonstrates that the system was linear
over four decades at these settings.
As the extended transit time of the slow-flow system was
expected to be the major factor responsible for the high-sensi-
tivity of this system, we also evaluated the system sensitivity at
25 ls transit time (FWHM) and laser power set at 2.4 mW.
For this measurement we again used the 8-peak fluorescent
Rainbow microspheres (RCP-30-5A) that are 3 lm in diame-
ter, but we also added an unlabelled 2 lm microsphere (CP-
15-10). A bivariate contour plot of SSC pulse height vs. SSC
area demonstrates that two populations are easily discrimi-
nated (Fig. 7A). When the population with the higher SSC
amplitude is gated on it can be seen that we achieve resolution
of all eight peaks of the Rainbow microsphere set in the fluo-
rescence peak measurement (Fig. 7B). When the population
with the lower SSC amplitude is gated on we are able to sepa-
rate out the fluorescence values for the unstained microsphere
(Fig. 7C). Although the unstained microsphere is visually
shifted when compared to the dimmest stained microsphere,
the two beads are not well resolved when they are separated
using SSC. This is expected because the predicted detection
limit is approximately the same as the fluorescence level of the
dimmest stained bead. We also achieve resolution for all eight
peaks using fluorescence area measurements derived from gat-
ing on the higher amplitude SSC populations (Fig. 7D). The
fluorescence CVs resulting from fluorescence peak and area
measurements are shown in Table 3. For the peak values, the
Q and B can again be calculated as described above (the area
values do not have a microsphere that meets the requirements
described above). Using the fluorescence peak data for the 8
peak microsphere set, shown in Table 3, Q 5 0.05 and B 5 73
MESF-PE. Despite the much shorter transit time these num-
bers indicate a similar resolution and sensitivity as those
found at the 250 ls transit time. This excellent sensitivity is
also supported by plotting the fluorescence means of either
fluorescence peak (Fig. 7E) or area (Fig. 7F) and by extrapolat-
ing the 95% confidence interval (derived from the mean and
two times the standard deviation) of the unstained micro-
sphere back to the linear function and interpolating the num-
ber of MESF-PE equivalents that this represents (Figs. 7E
and 7F). This is analogous to the approach taken above and
results in a detection limit estimate of either 52 (peak) or 64
(area) MESF-PE. The sensitivity and resolution data demon-
strate that this LP (at 2.4 mW and using transit time as short
as 25 ls) can support detection limits and resolutions compa-
rable to commercial cytometers.
Implications for Low Cost Portable Instruments
Although the data presented here is compelling, further
evaluation will be required to fully characterize the noise,
long-term performance, pointing stability, and lifetime of the
low cost LP modules. All of the ‘‘quiet’’ modules performed
very well, and 1 LP module was used in daily operation for
more than a year, which suggests that these LP modules may
have appropriate lifetimes for use in low-cost flow cytometers.
We have demonstrated performance sufficient for many appli-
cations including operation in a system with the transit time
as short as 25 ls. While the full sensitivity and resolution of
any system is a complex combination of fluidics, optics, detec-
tors and light sources, the data presented here suggest that this
LP may be a suitable light source for instruments in which the
transit time is �25 ls. Furthermore, given improved collection
optics and fluidics more optimally designed for short transit
times (the flow cell used here has an obstructed flow cell – the
sample delivery capillary is inserted into the flow channel –
that may produce sample stream instabilities at higher flow
rates), we expect that such a LP based system would have
Table 3. Fluorescence peak and area measurements for the 8 peak rcp-30-5a microspheres analyzed with 25 ls transit time
BEAD MESF-PE PEAK MEAN PEAKCV(%) PEAK SD AREA MEAN AREA CV(%) AREA SD
1 60 6.15 60.84 3.74 70.17 70.69 49.6
2 790 106.34 10.82 11.51 787.31 13.5 106.25
3 1,920 235.23 6.51 15.31 1,745.82 6.99 121.96
4 6,340 711.46 3.87 27.52 5,324.5 3.58 190.41
5 19,830 2,157.02 3.27 70.51 16,178.37 2.67 431.79
6 50,326 5,472.52 3.12 170.47 40,976.2 2.65 1,087.3
7 208,058 22,118.29 2.94 649.23 16,6924.94 2.46 4,101.1
8 535,963 55,799.95 2.28 1273 431,511.53 2.26 9,753.9
ORIGINAL ARTICLE
816 Laser-Pointer Based Flow Cytometer
improved resolution and sensitivity over what was shown
here.
The sensitivity and resolution of this LP driven flow cy-
tometer (particularly with the transit time at 250 ls) using a
low cost DPSS 532 nm LP module is demonstrably at least as
good as any current commercial instrument. Having demon-
strated nearly equivalent performance in precision, sensitivity,
and resolution at 25 ls transit time makes this approach more
viable. The very high sensitivity seen in �slow-flow� conditionsis assumed to be primarily due to the extended transit time,
which has been very significant with regard to the fluorescence
detection of single molecules (24,25). Here, it also facilitates
the use of a low cost DPSS laser that costs at least an order of
magnitude less than the highly stabilized DPSS lasers com-
monly used to obtain excellent flow cytometry data. The excel-
lent performance of this laser module with relatively inexpen-
sive and small detector components (this test system used
miniature PMTs costing between $400 and $900) raises the
possibility of the use of similar LP modules being implemen-
ted in a low-cost portable instrument. One aspect that is criti-
cal if this system were to be used in a portable instrument is
the effect of varying environmental conditions on the per-
formance of the LP and detectors (most notably the tempera-
ture). No attempt was made to characterize this system under
such conditions; however the specifications for the laser
pointer indicate an optimal operating temperature range of 22
to 28�C.To obtain suitably high particle analysis rates for the
hydrodynamically focused system used here, the microspheres
were usually concentrated via centrifugation just prior to analy-
sis to compensate for the inherently low volumetric flow rate,
which would have resulted in untenably long analysis runs
using conventional particle concentrations. Considering the
performance and cost benefits of an instrument configured
with ‘‘slow-flow’’ hydrodynamic particle focusing, this
approach is relatively compelling for research use even with an
additional centrifugation step and provides a pathway for
researchers to cheaply and quickly assemble a high-sensitivity
instrument. However, other methods of particle focusing
including: acoustic focusing for flow cytometry, which con-
centrates particles as it focuses them and allows long transit
times (0.1–1 ms) as no acceleration is involved (14), or DEP
focusing approaches having similar properties (15), are likely
to provide a more effective path to a truly low cost portable
flow cytometer. Such instruments could have great impact in
many areas of biomedical research and diagnostics, but most
importantly it could be a part of a solution to bring the most
accepted mode of CD41 and CD81 counting for AIDS pro-
gression assessment to resource poor areas of the world, which
is a highly sought after goal (3,4,6,31).
ACKNOWLEDGMENTS
The authors thank Jim Parson for excellent technical
assistance. We also thank John Martin and Mark Wilder for
their assistance and comments. We also thank Dr. James Jett
and Dr. James Freyer for numerous helpful suggestions and
comments.
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Cytometry Part A � 71A: 809�817, 2007 817