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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