Flow device to measure γ radioactivity and size of microparticlesM. Annis, C. M. Cobb, P. J. Bjorkholm, E. E. Frederick, A. T. Ramsey, and D. H. Frisch Citation: Review of Scientific Instruments 47, 998 (1976); doi: 10.1063/1.1134811 View online: http://dx.doi.org/10.1063/1.1134811 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/47/9?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Size-sensitive sorting of microparticles through control of flow geometry Appl. Phys. Lett. 99, 034101 (2011); 10.1063/1.3610940 The Measurement of Size Distribution of Indoor Natural Radioactive Aerosols by Imaging Plate Technique AIP Conf. Proc. 1034, 137 (2008); 10.1063/1.2991191 Plasma dragged microparticles as a method to measure plasma flows Phys. Plasmas 13, 103501 (2006); 10.1063/1.2356316 Electrodynamic radioactivity detector for microparticles Rev. Sci. Instrum. 60, 414 (1989); 10.1063/1.1140393 Device for microparticle array preparation Rev. Sci. Instrum. 49, 1488 (1978); 10.1063/1.1135294

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Flow device to measure y radioactivity and size of microparticles

M. Annis, C. M. Cobb, P. J. Bjorkholm, E. E. Frederick, and A. T. Ramsey

American Science and Engineering. Incorporated. Cambridge. Massachusetts 02139

D. H. Frisch

Department of Physics. Massachusetts Institute of Technology. Cambridge. Massachusetts 02139

(Received 3 November 1975; in final form, 3 May 1976)

A description is given of the design and performance of an instrument which measures particle size and radioactivity of individual microparticles after uptake of a 'Y-emitting radioactive tracer. A flow system allows the activated particles to be measured quickly and individually, giving statistically significant frequency distributions in a short time. The purpose of the flow device is to measure some property of particles of a given type, provided this property is correlated with the uptake of the tracer. The instrument sizes particles in the 5-150-fLm-diam range, and detects 'Y rays of E> 30 keY.

INTRODUCTION

The instrument described in this paper measures (i) the amount of 'Y-ray radioactivity uptake in a micro­particle, and (ii) the size of the same particle. The purpose of measuring uptake is to evaluate some property of the particle which is a function of the af­finity of the particle for a particular ion if activated in a particular manner. This method is advantageous if direct measurements are more difficult or impossible, or data acquisition rate is important. The technique is intrinsically rapid; although the described instrument was used to measure an average of 20 particles/min, data acquisition rates 100 times faster are feasible.

The requirements for the flow device are (i) particles must be both isolable and suspendable in a fluid, and (ii) the particulate uptake of radioactivity must be measurable at several counts/sec or greater. Although radioactive R7Ga was used for our experiments, the instrument can be used with other 'Y-ray tracers (de­tails are given in Appendix A).

A practical display of the uptake data is by means of histograms in which the distribution of particles is plotted as a function of activity. Since the uptake for possibly all types of particles is also a function of particle size, this measurement is included. To incorpo­rate "particle size" within any analysis, a multiple set of histograms (or a three-dimensional histogram) is useful. In some situations, the uptake-correlated property would itself be the particulate size (propor­tional to surface area or volume).

Although this instrument was designed to measure the in vitro uptake of 67Ga in exfoliated epithelial cells from the human uterine cervix, it has other applications both in and remote from cytology. In the experiment referred to in the next section, it was found that nearly all materials become highly radioactive when submerged

998 Rev. Sci. Instrum., Vol. 47, No.9, September 1976

in concentrated o7GaCI:1 solution. This phenomenon was utilized to measure labelled foreign particles introduced by the various containers and filters which are used in handling biological cells.

DESIGN CRITERIA

A detailed description of the experiment for which the 67Ga uptake flow device was constructed is given hy Cobb et a/. I Only a summary of the experimental procedures is given here. This brief description serves to clarify the reasons for the design particulars.

The samples analyzed, taken from the human uterine cervi x, are scrapings which would normally be used for a Papanicolaou test. Each sample consists typically of 50 000 cells suspended in I ml of a fixing solution consist­ing of ~86% ethyl alcohol; the epithelial cells of interest generally range from 10 to 40 fLm diameter in the ethanol. The I ml sample is then added to a 1.5 ml solution of f17GaCI:l (assayed at ~ I mCi) to permit radioactive up­take by these cells. Equilibrium levels of cell radio­activity occur after ~20 min.

Next the suspended cells are transferred from the radioactive liquid to pure ethyl alcohol by (i) filtering off the liquid through a 10 fLm polycarbonate filter and then replacing this waste solution with an equal volume of pure alcohol, and (ii) repeating this two­step process at least six times. The resulting sample of cells has a typical activity of ~ I nCi/celi suspended in alcohol whose activity is ~ 5 nCi/ml.

Now the sample is ready to be measured. The general scheme is to force the alcohol with the suspended cells through a flow tube which traverses the center of a scintillator crystal to detect radioactivity. The size of each particle is next measured within the flow tube by a particle detector. The cells must be sufficiently sepa­rated so that each radiation burst is correlated with a

Copyright © 1976 American Institute of PhYSics 998

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MOTOR

MINI­COMPUTER PLUS TAPE DRIVE

30 mL BEAKER WITH MAGNETIC STIRRING BAR

ORTEC 1 CHANNEL

PHA

NITROGEN GAS PRESSURE TANK

FIG. I. Flow device and data recording system. The stirred liquid containing the radioactive particles is forced by pressure from the sample beaker up the vertical flow tube, through the )I·ray de· tector and particle sizer. and into the constant rate withdrawing syringe.

particle size measurement. The accumulated data are then assembled into histograms which display the number of particles of a given size range as a function of the radioactive uptake per particle.

DESCRIPTION OF FLOW DEVICE

The complete data acquisition system consists of two subsystems: the flow device and some form of data re­corder and/or analyzer- Figure I shows the complete system with the flow device connected to a digital magnetic recording system. The flow device consists of

999 Rev. Sci. Instrum., Vol. 47, No.9, September 1976

four main sections: the pressure-injection system, radia­tion detector, particle sizer, and the flow system.

As shown in Fig. I, the pressure chamher is located beneath the lead shielding. Its inner dimensions are 6.4 cm high x 7.6 cm diam. The 30 ml sample heaker and stirring system are shown inside the pressure chamber. To minimize dust contamination, this chamber normally remains sealed and the beaker is filled and emptied via two I/O tubings. Tube A (tube B) consists of a 680 t-tm (300 t-tm) Teflon tubing outside of the pressure chamber, and a 760 t-tm stainless steel tubing inside the pressure chamber. The Teflon portion of tube A (tube B) is as long (short) as is convenient for loading; the stainless steel tubing terminates 0 mm (3 mm) from the bottom of the beaker.

To load the beaker with a sample, 30 ml of pure ethyl alcohol is first injected by a mechanically driven2 30 ml syringe through a 0.2 t-tm Teflon:; filter connected to tube A. While this ethanol is being stirred, the concentrated cellular sample is injected by a 5 ml syringe through tube B. This injection is followed by several milliliters of pure ethanol, also filtered by a 0.2 t-tm Teflon filter, to flush the remaining sample from tube B into the sample beaker- At the end of a run, the beaker is emptied and flush-cleaned utilizing tube A.

The cells are kept in suspension by constant stirring with a Teflon-coated magnetic stirring bar ( 1.9 cm long x 0.9 cm diam). The stirring bar is suspended by a stainless steel rod from the aluminum beaker cover­The bar and rod are supported by a sealed ball-bearing cage, and any particulate matter from this rotating connection is caught by a dirt trap at the top of the suspension rod. The stirring bar is driven by a coaxially rotating magnet placed below the pressure chamber- The spin axis is located D/3 from the beaker's edge, where D = 4 cm is the beaker's diameter; this place­ment reduces the formation of any pattern in the particle density. A suitable speed to maintain a uniform cellular distribution was found to be 300 rpm.

The pressure for the chamber is supplied by a tank of compressed nitrogen gas, regulated by a standard reduction valve. This gas is filtered by a 0.2 t-tm Teflon filter. A pressure gauge and release valve are connected to the output gas line. When gas pressure is ap­plied to the chamber, both alcohol and the suspended cells are forced up the vertical stainless steel flow tube. The lower end of the flow tube is located 1 mm from the bottom of the beaker- The tube is in one piece (to minimize turbulence) and extends from the pressure chamber, through the center of the scintillator, terminat­ing at the particle detector (see Fig. 1).

The pressure of N2 gas required to force a laminar flow of liquid of viscosity TJ through a cylindrical tube at an average speed v is given approximately by the Hagen- Poiseuille law,

(I)

The dimensions of the flow tube are D = 250 t-tm and I = 27.5 cm long. The viscosity of the ethyl alcohol at

Radioactivity and size of particles 999

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20°C is 1.2 cP. An efficient average flow speed most often used in our experiments is v = 7.0 cm/sec. This would require a value of P = 0.14 kg/cm2 gauge. How­ever, for a constant p, v will change each time a cell (or any other particle) sticks to, or detaches from, the tubu­lar wall. To avoid this, we set P - 2 kg/cm2 gauge and then maintain f' = 7.0 un/sec by connecting the uptake syringe and its plunger to a synchronous-motor with­drawal pump2 shown at the top of Fig. I.

If regulation of l" by the uptake syringe were not needed, some back pressure would still be required to rcmove bubbling at the end of the flow path as the pres­sure on the liquid approached 0 gauge. For volatile liquids such as alcohol, this bubbling would occur as far as within the particle sensor cell (see Fig. I), causing the bubbles to be registered as particles.

Radiation from each particle in thc flow tube is de­tected by a Nal(TI) crystal 0.6 cm thick x 7.6 cm diam) attached to the face of an RCA 8054 photo­mUltiplier tube (PMT). An aluminum-lined holc (6.4 mm diam) and the inserted flow tube pass through thc center of the crystal. The anode signal is amplified and applied to an Ortec model 486 pulse-height analyzer (PHA). Radiation from H7Ga is listed in Appendix B. The PHA is tuned only for the 93 keY y rays: justification for not including the other y rays in the measurements is detailed in Appendix B. The logic pulses from the PHA are then applied to a data recorder and/or analyzer.

The end of the stainless steel flow tube connccts to the input of a particle sensor cell. The sensor cell (a modified 0-5-150), sensor preamp, and five-channel PHA are manufactured by HIAC." Inside the sensor cell. a light beam traverses perpendicularly across a 150 x 500 p,m rectangular fluid passage and through a 150 x 150 p,m window to the detector. The width of the fluid passage matches the window width so that all particles are fully measured: the depth of the fluid passage is chosen so that its cross-sectional area approximately matchcs that of the stainless steel flow tube. A smooth transition is made from the circular to rectangular cross section.

PARTICLE SIZING

Each particle is sized according to its measured cross­sectional area CIf and recorded as a diameter of an equivalent disk. The value of C\I is based on the reduction of the light beam intensity as the particle passes in front of the 150 x 150 p,m window. The light source is a tungsten lamp and the detector is a photo­diode (HP5082-4207): the peak spectral response of the optical system is .\.) = 0.9 p,m. The refractive index of alcohol or water is 11., (0.9 p,m) = 4/3. so that within these fluids, the peak spectral response is at Ai = .\./n, = 2/3 p,m. The incident light is collimated to within ~ 1° (half-intensity), and it is estimated that the equiv­alent half-angle aperture size for a light source of plane waves is (} = 4°.

For the measurement ofa sphere in a given apparatus,

C\I = e(d,/ll,8)Q(d,m)(71"d 2/4),

1000 Rev. Sci. Instrum., Vol. 47, No.9, September 1976

where d is the diameter. In is the refractive index of the sphere relativc to the fluid, Q is an efficiency factor for the light extinction (= absorption + scattcring). and E is that fraction of the extinguished light which does not enter the light detector. To a high dcgrcc of accuracy. Q = 2 for a nontransparent sphere: half of Q is due to absorption and reflection. and the other half is due to dif­fraction. Likewise. for partially or fully transparcnt spheres, Q = 2 if the diametric phase shift p ~ (271"/'\) X (d)lm - II satisfies the condition p ~ I. At low values. more noticeably at say p < 30. interference bctwccn the diffracted and rcfracted light. callcd "'anomalous diffrac­tion," will occur.;' For a 2: I range in wavelengths. Q 2: 2 for 6 < P < 30. For p < 4. Q rapidly vanishes a\ p --> O.

The dependence of C ll upon thc optical system is due to E( d,I11,(J). Hodkinson and Greenleavcs fi show that for visible light (2: I range in A). half of the diffractcd light is scattered beyond 4° of the forward direction for d = 5 p,m spheres and decreases to- 2<Y/r for £I < I () p,m. Thus for a blackbody spherc, E( d,111 ,40)Q( £1.111)

= 1.5 for 5 p,m sphercs and 1.2 for £I -> 10 p,m. Since nearly no light is reflected less than 4° for a mctal sphere. these values for EQ are true for any nontransparcnt sphere.

Thc above values for EQ are also valid for partially or fully transparent sphercs if 111 :> 1.3. However. for lower values of 111, a measurable amount of rc­fracted light will fall within 4° of the forward direction. rcaching 2if/r for 111 = I. I: thus for no absorption and d> 10 p,m. Cll = mP/4. For 1111 - I I < - O. I. most of both the diffracted and refractcd photons are scattercd within 8 = 4°, causing a significant rcduction in E.'

Since the boundary values of any PHA can be varied with regard to a C lI voltage responsc, if the HIAC is calibrated with monosized spheresH of II > 1.3 11/. thc calibration will be valid for all particles with 111 ~ 1.3. For 1.1 < 111 < 1.3. all diameter measurcmcnts arc ac­curatc if recalibrated for thesc valucs or Ill.

For 1111 - I I < O. I. small values of ell can often be avoided by substituting a fluid with a sufficicntly dif­fcrcnt 11/. Howevcr. values of I < III < 1.1 are often un­avoidable when analyzing biological cells. sincc they ab­sorb some of the surrounding fluid. Mie calculations for various simple cellular models. complcmented in many cases by experimental data, are given in the litcra­ture!l-l:; for differential light scattering: such calcula­tions show that 0.1 < E < 0.2 (8 = 4°) for many cells of. say, 8- 40 p,m. with some influence by the nucleus. We have observed that the measured diameters in ethanol of exfoliated cells from the human uterine cervi x register approximately one-half to thrce-fourths of the true equivalent diameters if the HIAC is calibrated with standardizcd polystyrene spheres (Ill = 1.18 in ethanol).

DATA RECORDING

The simplest method of recording is by chart re­corder. Recordings on an ultraviolet light-sensitive

Radioactivity and size of particles 1000

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FIG. 2. Oscillograph recording of data from the flow device. Traces were recorded left to right with increasing time. The bottom trace displays standardized Nal pulses from individual 93 keY photons. These pulses are applied to a typical count-rate circuit (0.06 sec time constant) which generates the trace displayed im­mediately above. The output of the HIAC sensor preamp is shown in the very top trace; the ampli­tudes (neglecting undershoot) should be proportional to the aspect cross section of the par­ticles. Ct. These pulses are ap­plied to a five-channel PHA; each channel response is represented by a unique pulse height in the trace immediately below. The channel boundaries are at 5. 7. 10. 15.25. and 150 j..tm diameter.

paper by an oscil\ograph14 (±5% flat frequency galva­nometer response, 0-2.2 kHz) are shown in Fig. 2. The lower two traces represent the response from the logic output of the scintillator's PHA. The width of each isolated, strong burst of radiation approximately repre­sents the transit time (tNal ~ 0.6 sec) of a radioactive particle through the scintillator. The upper two traces represent particle sizes. The various dimensions are such that each particle should be detected by the HIAC particle sizer approximately 2tNa i ~ 1.2 sec after it passed the center of the scintillator.

The first (leftmost) radioactive burst (double-peaked) becomes saturated on the second peak; saturation of the galvanometer driver occurs for a periodic (e.g., oscil­lator output) count rate> 100 counts/sec. This double­peaked burst indicates that the first two particles are too close to each other to be analyzed. Radiation from the third particle is barely separable; the fourth particle is clearly isolated. The next four particles are each re­corded in all but the largest HIAC PHA size channel. The last (rightmost) small burst of radiation is un­doubtedly due at least in part to the two (nearly un­resolvable) <5 /-Lm particles seen without any HIAC PHA responses.

A versatile method of data analysis can be realized if the recording system shown in Fig. 1 is used. A Data General'5 Nova minicomputer is simply used to sort the digital data for recording onto a digital nine-track magnetic tape. The six outputs of digital PHA data are recorded as 16 bits every 10 msec. With this scheme, raw data are preserved, and the analysis can be per­formed many times; each run can vary in the method of analysis and the effects resulting from a change in any analytical parameter can be tested; these parameters will be discussed after the following section.

HYDRODYNAMICS

The traces in Fig. 2 were obtained during a typical run: cells containing 67Ga and suspended in alcohol were recorded at Valcohol = 7.0 cm/sec. The flow be-

1001 Rev. Sci. Instrum., Vol. 47, No.9, September 1976

comes laminar at a distance of I ~ R W4 from the entrance of the flow tube, where R is the tube radius and :Yl is the Reynolds number (here, :Yl = 12). rhus laminar flow oc­curs after a distance of only I ~ 375 /-Lm, which is well before entering the scintillator.

Because of laminar flow, the velocity profile of the alcohol within the scintillator is parabolic, with

(3)

where r is the radial distance from the cylindrical axis. The maximum fluid velocity Vmax = v(O) is twice its aver­age velocity V.

The 93 keY l' rays from 67Ga can be detected by the scintillator only when the carrier cell is located between the lead sleeves,16 a separation of INa! = 6.4 cm (see Fig. 1). Thus the minimum scintillator transit time is INaJ/2v = 0.45 ~ec. The widths of all radiation bursts in Fig. 2 satisfy the relationship INal < 0.65 sec; this is ob­served for ~95% of all transits at v = 7.0 cm/sec. This tendency for restricted transit times implies [from Eq. (3)] that the cells normally travel within the central quarter area of the flow tube through the scintillator.

This tendency for cells to concentrate within the central region of the flow tube is due to a radially inward cellular migration. For neutrally buoyant, easily deformable particles in Poiseuille flow, as given in Eq. (3), the steady state radial position is on axis. The amplitude of the radial restoring force depends in a complicated way upon many factors such as radial distance, particle and tube size, central velocity and viscocity of the fluid, buoyancy, deformability. etcY If neutrally buoyant rigid particles are used in the flow device, the radial restoring force is much smaller, with equilibrium not at r = 0, but at r = R/2.18

To correlate the amount of radioactivity associated with each particle with the size of the same particle, the transit time between the center of the scintillator and the HIAC sensor (tNal-HIAd must be known. In addition, to maximize the efficiency of the instrument, we must minimize the fluctuations in INa! and in INal-HIAC' To quantitatively understand the various ef-

Radioactivity and size of particles 1001

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fects on these transit times, the following analysis is made.

Since the particle velocity is a function of its radial position, the transit time for a given axial distance is also a function of the radial position of the particle. Therefore the transit time distribution for a set of particles reflects the radial distribution of the particles, assuming no radial motion. The time to travel an axial distance 1 is [from Eq. (3)]

(4)

The number of particles with a transit time between t

and t + dt is simply the number of particles between the appropriate I' and I' + dr. Denoting this number by dN, and assuming that there are No particles homogeneously distributed across the tube, we have

dN(r) = (No! A)dA(r) = (No! A)21Tr dr. (5)

Substituting into Eq. (5) the expression for I' dr as ob­tained from Eq. (4), we have

flJl' t 2: tmin = II Vma '.

Figure 3(a) represents dN Idt for the case where £1MI') in Eq. (5) is restricted to I' 5 R12. Figures 3(b) and 3(c) are histograms representing values of t;-i,I1

and tNal-HL\(', respectively, for 20 prominent events (>200 counts/sec recorded at v = 7.0 cm/sec. as meas­ured from high-speed (2.54 cm/sec), time-marked oscil­lograph recordings. Since of the 40 transit times meas­ured, only one exceeds (4/3)tmin, there is a strong indication that the particles generally flow at r 5 RI2 through the scintillator and between the scintillator and HIAC sensor.

Two points should be noted here: (i) The oscillograph recordings reveal no correlation between the magnitudes of tNal and hal-HL\(' for each particle. This implies some radial fluctuation in r 5 R12. (ii) The final 230 of the flow path, I:-.Ial-IIL\(· = 12.5 cm, is not cylindrical: this is the circular-to-rectangular transition from the junction at the top of the flow tube to the light beam in the HIAC cell. However, the similarity of Fig. 3(c) to 3(a) indicates that a good transition was made.

DATA ANALYSIS

On-line analysis can be performed without an inter­mediate recording of data. One method extensively used was to analyze the digital data by a Nova minicomputer. Data for each particle in a sample were analyzed and assembled into a histogram: evolution of the histogram in real time was displayed on a CRT. A second method briefly used was to analyze data with a multichannel analyzer: histogram data for all five particle diameter ranges can be displayed simultaneously by some ana­lyzers as an isometric three-dimensional plot on a CRT.

Most of the data analysis for studying the cellular uptake of fl7Ga was done off-line by an IBM System/370 computer. Data were prerecorded on digital tape by the method previously described. The primary program

1002 Rev. Sci. Instrum., Vol. 47, No.9, September 1976

(a)

1.0

0.8

~ 1=0 0.6

'~I-: 0.4 - 0.2

~ 4/3 t min

No t min

---0.0 '7 )'-----1'-----+-----,---,.---,.----r--

2 t min TIME

10

8 en t min UJ .....I 6

~ u i=

4 c:: < a..

2

0 0.5 0.6 0.7 0.8

t Nal [~ ]

10 ~ t min en 8 UJ .....I u 6

?,~moo~,o, i= c:: 4/3 t min

4 < a.. (c)

2

0 1.2

t Nal . HIAC

I iii

1.3 1.4

[.4 ]

i I 1.5

+

FI(;. 3. (a) Expected transit time distrihution for laminar flow of nonmigrating particles uniformly spread within the central quarter area of the flow tuhe. The transit time (413)1",,,, corresponds to a trajectory at r = R12. (h) Ohserved distrihution of 1,,,, for 20 particles. (c) Distrihution of 1,,,,-IlIA" for the same 20 events. The diameters of the 20 particles analyzed range from 40 to 60 /Lm.

used would first scan the tape buffer for a HIAC particle response. When found, a further scan for a second particle would be made within a tape buffer length equal to t,IPad (dead time) of data. If not unique, tdpad of data beyond the second particle is skipped and the search recommences for an isolated particle. When found, i.e., when no other particle exists within ±trlea,h all of the radiation counts are tallied over the time segment

tlll .\(' - t:-;,I1-IIL\(' - tWind'", 12 to

tlll.\(' - t"al-III.\(' + t\\'indo\\'/2,

where tHL\C is the HIAC responding time and tWindow is the temporal window width preset for tallying radiation counts. The chosen value of twindow should be large enough to include most values of t'lal, plus half of most of the peak-to-peak jitter in tNal-IIIA(' (see Fig. 3). The dead time should be set to tdead 2: twindow; a large in­equality allows minimal cross talk but lowered economy. For a fixed value of tdead , the optimum density of flow particles for a maximum number analyzed in a given time period is when c- I = 0.37 of the particles are accepted. 1~

Radioactivity and size of particles 1002

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THE FCllC"I'G HISTCGRA~ REPRESENTS T~E RACIATIC' CISTRleUTION CORRESPONDING TO PARTICLES RETWEEN ~ AND 150 U~ CIA"ETER.

T~ERE "ERE lOll, PARTICLES ANALYZED BELOW. THIS ECUALS 100 t OF TOTAL PARTICLES ANalYZED BETWEEN 5 AND I~O U" (TOTal' 10341; ALSO FOUALS 67 t OF TOTAL PARTIClFS DffECTED ~fTwHN ~ ANC 150 U~ !TOTAL' 15411.

TOTAL SCI~TIllATlnN COUNTS BEla" • 14142 • 100 • OF TrTAl FCR All PARTICLES ANALY1EO (TOTAL' 141421.

EVE,TS I C~AN'EL RAO I A TlON COUNT5IC~

10 IS 20 25 30 35 40 4~ 50 t4 100 160 250 400 640 1000 1600 2~00 4000 6400 10000 I I I I I I I I I I I I I I I I I I I I I I I

o I 1 l 4

5 & 7

q

10 II 12 Il 14 IS 16 17 18 Iq 20 21 22 2~

24 25 26 11 28 2q ~o

l I 32 33 34 35 H 37 ]e 3q

40 41 42 43 44 45 4t 41 4~

40 <0 <;1

l2 100001l000010000100C,01000010000100 lq 100ccerCCOICCCClcorCICCCC'CCCCCCOCC~OOOC 37 ICOOO~OoOOloOOO'Oo~o.oOOOIOOOOlooor.co 21 100COICCOOlrCCOlrccccc 77 100001100001100001000010000100 30 10CCCICCCCCCCCClccrcccccccccccc 32 1000011000010000100001000010000100 41 IOoCCICCCOICCCCICoCCICccccccccccccc.coooln 44 100001l00001000010000t0000100001oocr.CCOCIOCCC 55 100COIOCCOIOCCCIcnccccorcccrCOCCOGCICoOr,loC(,OI00001l0n 4& InnnOllrnnOloooooooroeOOOOlonOOIOOOClcoccccroccc &G ICOGOICccocccrrlcocccrocCCCCCCICCCCICOCCCOOOOIOOOOlonoODO 57 10000moooomoooomocOOOOOOOIOOOOIOOOOIOOOOIOOOOICOOOIOC 4e loonOOOOOOIOOOOlonOOIOCOOCCCCCCCCCCCCCCCICCCCICCC 44 IrccrICCCCICOCOllc~noooooo.onoOlooonoooooIOOOO 56 IOOOOIOOOOIOOOOlonrolnnOOICCCCCCCCCCCCCClccrCICCCClrc 40 IcccrCCCCOlrornarorraoonOllocooaOOOODOOOOD 2R loonn~OOOOloooo~rUCOIIOOOOIIOOO

~o IOCOCICCCCCCcccccoccccccccccccc 27 100001000010000lnOOOl00001l00 26 IrC(CICCCOCCCCCCCCCCCCCCCCC 34 10000100001000010nOOl00001000010000 LG IflCCCICCCOCCCCCCCCCC IR I ['OOG~oorOI(JoC'nlonr 18 IrOCCICC(CCCC(CCC([ Il 10000100001000 16 IOCCOICCCCCCCCCCC 10 Iroce~oooo.

16 IGCCCC((CCCCCCCC[ <; 100001 6 IC[CCIO

IrCCOle I[Ccrcccc Iroo

4 I [eee ICCCC Ire ICC Icr ICC ICC loee I ( I Ie Ie

2 ICC a I C I o I I Ir' a I

c:? I rn <;; Ie 54 I S' I r:t 1 I ~ 51 I 58 I SO 0 I 60 G I &1 0 I 62 I 10 &1 0 I t4 C I 65 a I 66 0 I 67 a I t8 C I ,q 0 70 0 7L a 72 C 13 0 74 C l' a 16 C 77 0 78 0 7q a 80 I 0

FIG. 4. Typical histogram plotted by the line printer. The distribution represents the number of isolated (tdead = 0.8 sec) particles (range 5-\50 /lom diam) as a function of radiation recorded per window width (wIndow = 0.8 sec (tNal - 0.6 sec). where the window center was set at (Nal-HIAC = 1.25 sec prior to the HIAC response.

When all of the data recorded on tape for a given run were analyzed. histograms for the various particle size ranges would automatically be plotted by the line printer. An example of such a histogram is shown in Fig. 4.

1003 Rev. Sci. Instrum., Vol. 47, No.9, September 1976

NOISE BACKGROUND

The average background radiation in Fig. 4 is only 0.4 count/twindow = 0.5 count/sec; this determination is made from counts remote from the detected particles

Radioactivity and size of particles 1003

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as recorded on an oscillograph. However. the radiation recorded by a scaler before and after this particular run was also 0.5 count/sec. This is due to 0.3 count/sec of PMT dark current and 0.2 count/sec of 67Ga contamina­tion on the wall of the flow tube deposited from the numerous prior runs. The 67Ga in the suspension alcohol contributes only 0.03 count/sec; the liquid volume within the scintillator is 3.1 ILl. The 67Ga uptake by the wall of the flow tube within the scintillator was typically 0.1 count/sec per 10 h of continuous running.

After each run, the flow device is cleaned with 120 ml of 0.2 fLm filtered alcohol. Since the sample beaker con­tains most of the contaminates (particle noise), cleaning is facilitated by twice injecting, then ejecting, 40 ml of alcohol through tube A (see Fig. I). After such cleaning, only abollt I particle/ml is detected for 0.2 ILm filtered alcohol (stirring on). Ifit were not for the injection tubes, the sample beaker would have to he filled outside the pressure chamber; at best >3 particles/ml would he detected for o.~ fLm filtered alcohol (stirring on).

APPENDIX A: TRACER SELECTION

The lise of6'Ga radioactivity as a tracer is not unique. Any 'Y emitter can he utilized whose emission energy is > 30 ke Y . provided of course that the carrier element or molecule possesses such characteristics (usually chemi­cal) that its uptake is a function of the microparticle property to be studied.

The value of 30 keY is dictated hy the absorption of 'Y rays in the thinnest stainless steel wall tubing com­mercially available. 20 Lower energies could be used with lower 7. material, hut a lower limit of 20 keY is set by the scintillator's 0.5-mm-thick aluminum hole liner. There i" no upper limit, although the absorption ef­ficiency of a Nal(Tl) scintillator drops to a minimum at 5 MeY. In addition, the shielding ability of lead is a minimum at 3.2 MeY; the effect on the measured scintillator transit times would be such that (assuming the lead sleeves in Fig. I removed)

t~al(3.2 MeY) = 3t:-la,(93 keY).

Consequently, the preset dead time toearl would have to be tripled to avoid including counts from an adjacent particle.

Another consideration is the value of the decay constant T of the radioactive tracer. If the measured activity is sufficient for any value of T, then the ideal choice is the largest value of T possible. Otherwise, if only an average uptake of M = (N* + N)/n molecules by each microparticle can occur, where N* and N are the number of tracer and nontracer nuclei, respectively, and 11 is the number of atoms per molecule, then the optimum value of T will depend upon T, the elapsed time between particle inoculation and the subsequent measurement in the flow device. The measured activity is

1004 Rev. SCi. Instrum., Vol. 47, No.9, September 1976

Maximizing /(T) with respect to T, we find that T = T: of course, T should be kept to a practical minimum.

APPENDIX B: R7Ga DATA

The decay of 67Ga is by electron capture, producing principal 'Y rays at 93 keY (38%), 185 keY (24%). 300 keY (16.1%). and 394 keY (4.37r). The half-life of this decay is 77.9 h = 3.25 d, which is "hout a minimum value for routine utilization on a weekly h""is. For detection. a single-channel PHA is u'>ed whose window is narrowly set for the 93 keY photon". Tn include the 185 keY photons, the optimum "ign;d-lo­noise ratio would be increased to

5 5 + (24/38)5 ---4-------,~--

N V2N S

1.15 - . N

However, this would require a second narrow window PHA with the two PHA outputs logically oRed. Adding a third 'Y-ray line would decrease the S/ N.

ACKNOWLEDGMENTS

The authors wish to thank Dr. R. Gajewski for his many valuable suggestions and critical support of thi" project. W. Fondation contributed technical assi-;t,tnce.

, C. M. Cohh. M. Annis. E. E. Frederick. R. Gaiew'iki. J. Zuckerman. and R. Richart (unpuhlished). .

2 Harvard Apparatus Co., Inc .. Millis. MA 02054. ;] All filter holders and Teflon filters used in conjunction with

this instrument are manufactured hy Millipore Corr .. Redford. MA 01730.

4 High Accuracy Products Corp .. Montclair. CA 917h3. , H. C. van de Hulst, Light Scattering 17.1' .'111/,,/1 P"rtid",

(Wiley, New York. 1957). "J. R. Hodkinson and I. Greenleaves. J. Opl. Soc. Am.

53, 577 (1963). 7 The value of EO (for 111 --> I) cannot he properly determined hy

the analysis of diffracted and refracted light as separate entities hecause of their small relative phase shift: here Mie calculations are required. A general discussion of light scattering hy small particles is given in two text hooks: Ref. 5. and M. Kerker. The Scattering (d Light {[lid Other Electromagnetic Rcu/iaTion (Academic. New York, 1969).

x Spheres grouped according to size and material are availahle from various sources. For example. monosized spheres of polystyrene. metals. pollen. and spores are availahle from Duke Standards Co .. P"lo Alto, CA 94306.

"P. F. Mullaney and P. N. Dean. Biophys. J. 10,764 (1970). II) A. Brunsting and P. F. Mullaney. App!. Opt. ll, 675 (1972). II A. Brunsting and P. F. Mullaney. Biophys. J. 14,439 (1974). '2 A. F. Fercher, Optlk 43, 129 (1975). ]:, P. F. Mullaney and R. J. Fiel, App!. Opt. IS, 310 (1976) 14 Consolidated Electrodynamics Corp., subsidiary of Bell and

Howell, Pasaden". CA 91 109. " Data General Corp .. Southboro, MA 01772. 16 Since a space must exist between the lead shielding and the

scintillator crystal. lead sleeves of 2.3 mm thickness around the flow tube are interposed at each end of the crystal to sharpen the radiation transition region. For 93 keY photons. the sleeve transmission is lO-x•

17 P. R. Wohl and S I. Rubinow. J. Fluid Mech. 62,185 ( 1974).

1H A. Karnis. H. L. Goldsmith. and S. G. Mason. Nature 200, 159 (1963).

If' For any paralyzable counter. the maximum count rate occurs when lie of the true count rate is measured. See. e.g., R. D. Evans. The Atomic Nllc/ells (McGraw-Hill. New York. 1955). p.786.

211 Both stainless steel and Teflon small i.d. tubing are availahle from Hamilton Co .. Reno. NY 89502.

Radioactivity and size of particles 1004

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