The microanalyzer

Technical Notes Journal of Nuclear Medicine and Biology, 1975,Vol. 2, pp. 67-72. PergamonPress. Printedin NorthernIreland

The Microanalyzer * (Received 14 March

1974;

in revisedform 21 June 1974)

A THREE-MICROSCOPE synchronous scanner makes possible a wide range of automatic, semi-automatic, or manual measurements and correlations of bone autoradiographs, and microradiographs. sections, Two densitometers with faster-than-millisecond response operate over a density range O-5 (a factor of light intensity of 105). A function generator corrects their outputs for film non-linearity, and automatic measurements of film background prevent zero-drift. Measurements of counts per unit area, integrals of density and density area, density, distributions are made by pulse circuitry and displayed on meters, dual-pen recorder, scalers, the oscilloscope of a multichannel analyzer and typewriter. Scanning speed can be varied continuously from 1 pm/set to 1 cm/set so that the operator can measure small features visually or scan a whole section automatically in minutes. The instrument is being used for the microscopic analysis of bone burdened with radium and other radioelements in order to determine microresorption, bone surface dose, damage, scopic hotspot/diffuse ratio, diffuse intensity, and the relation between endosteal or marrow dose and body burden. Its flexibility may make it useful in other fields of microscopic analysis. Introduction Microscopic measurements and correlations of microradiographs, and sections autoradiographs, of bone(l) have become so time-consuming that automation within close operator control is desirable. A few of such measurements are the rate of bone formation, the number of plugged canals, the ratio of hotspot to diffuse intensities, the distribution of diffuse activity, the activity in new and old bone, the dose rate distribution at bone surfaces, and resorption (the fractional area of bone which contains negligible radium long after intake). An increasing number of such quantitative microscopic analyses are required in the radium program of the Center for Human Radiobiologyt2) in order to relate endosteal and marrow dose to body burden. * U.S.

Work performed under the Atomic Energy Commission.

auspices

of

the

Such measurements require wide range densitometry on the autoradiographs because hotspots may have 100 times the intensity of the diffuse component. Thus, microscopes with collimators on both light source and photomultiplier tube are required rather than flying spot scanners in order to reduce light scattering. They also require correlation between two or three types of image of the same bone section: ultraviolet, X-ray, and autoradiograph. Such correlation is impractical with a single scanner because perhaps a million words of computer memory would be needed to store each picture, not to mention the subsequent problem of orientation and registration. The new instrument, the Microanalyzer, incorporates a number of novel solutions to these problems. Description

of Microanalyzer

A!S~croscopes Three Leitz Ortholux microscopes have been mounted together. Their stages have been removed, rotated through 180°, and mounted on a ballbearing scanning table so that three microscope slides can be scanned in synchrony. Trinocular heads allow each microscope to be viewed simultaneously by the operator (binocular) and by photomultiplier (monocular). Motor drives Metric screws monitored by Veeder Root and electronic counters drive the scanning table both in x and in y. Motion along the x-axis is controlled by a continuous motor with an extremely wide range of speeds so that one can scan from 1 ,om/sec to 1 cm/set in either direction. Motion along the y-axis is controlled by a stepping motor, which changesy in steps of 5 pm to 5 mm at each end of an x-axis scan. At slow speed the operator can measure areas or count small features such as osteocyte lacunae by pushing buttons. At high speed he can make hundreds of thousands of densitometer measurements covering the surface of a slide in a matter of minutes. Slide synchronization Synchronizing two or three slides is facilitated by an optical bridge which allows the operator to superimpose the images of two of the three microscopes. Each stage may be adjusted in x, y and z (focus) as well as in angle relative to the motordriven scanning table. When two slides are

68

Technical notes

synchronized, the stage angle of the third microscope is set and one of the slides transferred. It is then easy to achieve x-y alignment of the third microscope using landmarks already known to match on the two slides. The operator can synchronize three slides of the average bone section to within a few The rigid scanning microns in about 20 min. table then maintains this synchronization throughout a run. Encode7 Motion of the scanning table in the x-axis is measured accurately by an encoder which is attached directly to the x-axis metric screw. This encoder gives one pulse for each micron of x-motion in either direction. These pulses can be routed to four different scalers according to operator or machine decisions in order to measure four different areas on a section. (In this mode the machine’ operates like the old area-integrating eyepiece that had micrometer screws to control its cross-hairs.) Each pulse is associated with an area of Ay ym2, where Ay is the number of microns between successive x-scans as set on the stepping motor control. Alternatively, the pulses from the encoder can be used to request automatic density measurements every 1, 10,20, 50 or 100 pm of x-axis travel. Densitometers Density measurements are made by either of two fast, wide-range densitometers. Each densitometer consists of a lP28 photomultiplier, high voltage supply, and a logamplifier.t3) Response time is less than 1 msec, so hotspots as small as 10 ,nm in diameter can be measured at a top scanning speed of 1 cm/set. Each microscope densitometer has apertures both above and below the slide being scanned so that the problem of light scattering around small hotspots is eliminated. Aperture diameter may be adjusted from 10 pm to 1 mm. A working density range of O-5 is obtained (a factor of lo5 in light intensity). Thus the densitometers can measure both hotspot and diffuse intensities at top scanning speed even when the hotspot/diffuse ratio is as large as 100. Correction of jilm non-linearity For densities greater than about 1.5, autoradiographic film becomes increasingly non-linear with radioactive exposure.(l) Correction for this nonlinearity is made by passing the densitometer output through a function generator which is calibrated for the particular film being used. Thus, the full density 5 range can be used in activity or dose rate analyses.

Light sources Light sources for the two densitometers utilize halogen filament bulbs which are more intense and more stable than conventional light sources. An ultraviolet light source is also available for observing tetracyline deposits in bone sections. Logic control The information derived from the encoder, the two densitometers, and manual buttons is correlated by pulse circuitry in any way the operator desires. A particular logic can be chosen in a matter of seconds by switches on the master control panel (Fig. 1). The machine can, therefore, analyze information almost instantaneously without recourse to magnetic or paper tape or to a computer. This direct approach to data acquisition, correlation, and display was chosen to give the average operator maximum control over his data without programming. It also makes it easy to substitute one component for another to check operation or enhance reliability. Density displays Densitometer

information

can

be

displayed

in

four different ways. (1) The logamplifier outputs can be read directly on density meters with ranges of either O-0.5 or o-5-0. (2) At low scanning speeds, the two densitometer outputs can be plotted by a dual pen recorder calibrated in distance by a third pen responding to pulses from the x-axis encoder. The recorder uses electrically sensitive paper to avoid the problems of ink recorders. (3) Whenever a density measurement request is made, a gate circuit at one of the densitometer outputs produces a single pulse whose height is proportional to density. Each density pulse is sent to a 512-channel pulse height analyzer. The oscilloscope face of the analyzer presents the distribution of density measurements from which it is easy to read diffuse or hotspot intensities or the areas of film associated with different ranges of film density. (4) For measurements of the total radioactivity associated with different regions of bone, one would like to total a series of density measurements made at a given Ax and Ay during a scan of a whole section or part of a section.(l) This total is essentially an area integral of density. It might be calculated by multiplying the number of counts in each channel of the analyzer by the corresponding density and summing. To do this automatically, the densitometer output voltages are sent to a voltage-controlled, variable frequency oscillator together with a millisecond timer and any of four scalers. When each

Technical notes

density measurement request is made, a train of pulses whose number is proportional to density is counted in the desired scaler. At the end of the scan, density integrals can be read directly from the scalers. Log log oscillosco@e disjlay For detailed analysis of hotspot and diffuse distributions in a single picture, a log log presentation of number versus density on the multichannel analyzer is useful. (Otherwise, the important diffuse intensities would be crowded into the first few channels.) Thii log log display is available directly on the oscilloscope face by switching the multichannel analyzer to log counts and by inserting a second logamplifier between the densitometer output and the gate circuit (logexpansion). Probably the best presentation, however, is linear counts against log density because this shows the diffuse distribution as a nearly symmetrical peak (see Fig. 3). Typewriter

An electric typewriter is provided to read out data from the multi-channel analyzer. Discriminators

Two discriminators are provided, one integral and one differential. They can be switched to the output of either of the densitometers and used for counting features of interest. Alternatively, the discriminator outputs can be used to route information to different scalers or to different quadrants of the multichannel analyzer’s memory. The integral discriminator can also be used to request density measurements. An audible tone is available to inform the operator of discriminator state while he adjusts or checks the discriminator level through one of the microscopes. Requests for den.@

measurements

Requests for density measurements can be made in three ways: (1) The operator may simply press a button on the manual control box. (2) Density measurements may be made at definite distances along the x-axis scans as determined by pulses from the encoder. (3) Density measurements may be made whenever the integral discriminator changes state. This integral discriminator may be switched to the other densitometer so as to scan a microThe autoradiograph’s densitometer radiograph. will then make a measurement whenever the microradiograph’s densitometer indicates that a bone surface is being crossed. One may thus obtain the dose distribution at bone surfaces (on the oscillo-

69

scope) and the average dose to bone surfaces on the scalers (one scaler totalling surface density measurements and another counting bone surface crossings). If one desires a measurement of the total *“Ca or =Ra activity at bone surfaces, one may total density measurements from the autoradiograph made at regular intervals within about 100 pm of each bone surface. This can be done by requesting autoradiographic density measurements every micron or 10 pm of x-axis travel and routing these measurements to a particular scaler and quadrant of the pulse height analyzer only when the output of the microradiograph’s densitometer is within the window of the differential discriminator. With a 200~pm aperture on the microradiograph’s densitometer, this window signal will be given whenever the scan is within 100 ,um of a bone surface. Routing

In many microscopic measurements one would like to separate area or density information into categories such as bone surface, bone volume, new bone, old bone, etc. Accordingly, four scalers are provided into which one can rout encoder pulses (for area), variable frequency oscillator pulses (for density integrals), or simply operatorproduced pulses (for counting features of interest). The routing decisions can be made directly by the operator using the four buttons on the manual control box, or they can be made by the machine using one or both discriminators. The same routing signals can be used to direct density pulses from the gate to any one of four quadrants of the multichannel analyzer. In this mode of operation, 128 channels of memory are available for each of four categories of density pulses. (It was decided to limit the total number of channels in order to reduce the time necessary for typewriter readout. Accuracy of both diffuse and hotspot analysis can be achieved by recording log density versus number using logexpansion.) Feedback

Finally, in order to stabilize density measurements against changes in line voltage, light intensity, or gain in a photomultiplier or logamplifier, an automatic background-measuring circuit is provided. The operator chooses a region of the autoradiograph which will be crossed at the end of each x-axis scan and which represents a uniform film background. The border of this region is set on the middle thumb wheel of the electronic x-axis position counter (Fig. 1). Then whenever the x-axis scan reaches the indicated setting, the machine stops taking data and the densitometer reading is averaged with a time constant chosen by the operator. When

Technical

70

the scan again leaves the chosen background region, the average light intensity of the background is fed back to the densitometer as the reference value I0 Density is defined as log IO/1 for the next scan. where 1, is the reference light intensity and I is the measured intensity. Thus the autoradiographic densitometer can measure small densities relative to film background, being well stabilized against zero drift. There is also a button to call for the background averaging process (“density zero”) if the slide is imperfect and greater operator control is desired. Finally, direct control over densitometer zero is available by potentiometer. Calibration Density calibration in terms of activity per gram of bone is achieved through the use of Plaster of Paris radiators whose film exposures are scanned It is in the same way as autoradiographs. convenient to reserve one quadrant of the multianalyzer for calibration densities. A block diagram of the rnicroanalyzer is shown in Fig. 2.

Some Applications Density distributions Initial experience been most gratifying

with and

the microanalyzer has indicates that measure-

FIG. 2. Block diagram

note5

ments which used to take days or weeks can now be made in minutes or hours. A linear count, log density plot of the diffuse distribution of a dog injected with radium is shown in the oscilloscope photograph in Fig. 3. There were no hotspots within the area scanned. The ordinate shows the number of locations on the autoradiographic film The abscissa shows the log with each density. of the density (the output of the photomultiplier viewing the autoradiograph after it has been passed through two log amplifiers, the first to convert light intensity to density, the second to convert density to log density). On the abscissa, each 12 channels (points) corresponds to a factor of 2 in density. The peak corresponds to a density 0.4. A similar plot of an autoradiograph from a human radium case shows both diffuse component The diffuse peak lies at and hotspots (Fig. 4). density 0.2 and the maximum hotspot at density 4.2. When the hotspot density is corrected for nonlinearity of the film, density 4.2 is increased to density 6.5, so the hotspot/diffuse ratio is 33 (as seen on Type A autoradiographic film). The autoradiographic scan was made wGth a 25-pm aperture and the densitometer measurements were made with both delta x and delta y equal to 20 pm. A synchronized microradiograph of the same bone section was viewed by the second densitometer

of microanalyzer.

Technical notes and was used to insure that the autoradiographic measurements were made only over bone (microradiograph white). The peak in each frequency distribution provides a clearcut and accurate measurement of the most frequent density of the diffuse component over the bone section. Figure 5 gives a detailed view of the upper end of the hotspot distribution in Fig. 4 and suggests that a linear intercept of the distribution with the x-axis provides an accurate way of specifying maximum hotspot intensity. Surface~volume rataos

71

ment of white areas on the autoradiograph would include not only areas of bone remodelling but also cavities which contained no bone. So a synchronized microradiograph is scanned by the second densitometer to signal the presence of bone. Then, one scaler counts encoder pulses when the microradiograph is white to give total bone area. A second scaler counts encoder pulses only when both autoradiograph and microradiograph are white; this yields the desired area of resorption. Activity analyses

Classification of the radioactivity of a bone Microradiographs (x-rays) of bone have been section according to mechanism uses the full capability analyzed to determine the ratio of bone surface of the analyzer. A bone section, labelled with to bone volume. This ratio enters calculations of tetracycline to show areas of bone formation, is the total exchangeable calcium of bone and calcula- viewed by the operator in ultraviolet light under one tions of the relative biological effectiveness for microscope. The corresponding autoradiograph tumor induction by bone surface seekers like plu- is placed under a second microscope and viewed tonium as compared to that by bone volume seekers by a densitometer. The operator presses button 1 The integral discriminator (D) is whenever he wishes to rout autoradiographic like radium. set to change state whenever a bone surface is density information to channel 1 of the multichannel crossed. The number of crossings is counted in analyzer (MCA) and scaler 1. The MCA receives one scaler and used to calculate the bone surface. such density measurements for, say, every 10 pm Micron pulses from the encoder are counted in of stage travel. The density information for scaler another scaler whenever the same discriminator 1 is converted to pulse trains by the voltage-tois in the low density state (DL), indicating that the frequency-converter (VFC), so the sum of all microradiograph is white and the scan is over bone; density measurements made while the operator this count gives the bone area. presses button 1 is recorded in scaler 1; this count gives the total activity associated with new bone Measurements of area by operator and MCA channel 1 gives its density distribution. Some measurements, such as the rate of bone The corresponding microradiograph is viewed by the formation using tetracycline markers, require judg- second densitometer on the third microscope; it indicates when bone surfaces are being crossed ment on the part of the operator. Accordingly, a bone section, whose areas of new bone growth and automatically routs the autoradiographic inhave been marked by injected tetracycline, is formation to, say, MCA channel 2 and scaler 2; observed under ultraviolet light during the scan. if the operator is not pressing button 1 when a bone When the cross hain of the microscope are moving surface is crossed, then that autoradiographic across new bone, as outlined by tetracycline, the information will correspond to non-growing bone operator presses a button on the manual control surface and will be recorded in MCA channel 2 box and routs to one of the scalers the micron and scaler 2. A discriminator on the autoradiopulses from the encoder; this scaler therefore records graph’s densitometer may be set at, say, five times the area of new bone. The total area of bone in the diffuse intensity and used to rout the VFC pulses for hotspots to scaler 3; total activity in the section may be recorded by a second scaler. hotspots. Total diffuse activity can similarly be Measurements of resorption recorded in scaler 4; activity of old bone. The When an autoradiograph of a bone section from areas of bone associated with each of these components a radium case is made several decades after radium can be obtained by counting all the density pulses intake, there will often be regions of bone which routed to each of the MCA channels. (The “data/ have been resorbed and replaced by new bone reduce/integrate” module on the Nuclear Data containing almost no radium. These “white spots” analyzer conveniently sums the number of pulses in any group of channels and types this area inforin the otherwise gray regions of the autoradiographSo in one scan, one the diffuse component-indicate the cumulative mation on the typewriter.) amount of bone that has been resorbed and replaced can record the total radioactivity, the area, and the during the interval between radium intake and dose distribution associated with new bone, old bone autoradiographic observation. However, measure- and resting bone surface.

72

Technical notes

S-rY The Microanalyzer is designed with maximum flexibility to assist the operator in any microscopic measurement. Any machine decision can be directly monitored by the operator looking through one of the microscopes at low scanning speed, and any machine function can be assumed by the operator or overridden to the extent necessary for a given measurement. The resulting instrument, we believe, has achieved a better combination of speed, wide-range densitometry, stability, flexibility, correlative capability and operator control for the purpose of microscopic analyses of bone than previous instruments. Its flexibility may make it useful in other fields of microscopic analysis. J. H.

MARSHALL

P. G. GROER R. F. SELMAN

Radiologicaland Environmental Research Division Argonne National LaboratoryArgonne, Illinois 60439, U.S.A.

BBmTC-MAA from @BmTC--albumin, generally result in products containing both relatively high concentrations of particles and a relatively wide range of particle sizes. With such products, it is not unusual for patients to receive numbers of particles well in excess of lOs/dose. Described below is a method for preparing MAA by which virtually all particles produced fall within 110-15 pm of the average size. From stock MAA suspensions, individual kit vials can be prepared, each containing specified numbers of particles. The MAA production method is sufficiently flexible to allow preparation of either large or small stock suspensions. Kit vials can be prepared to contain different (but known) numbers of particles which more closely match the expected daily clinical needs. For kit vials stored at low temperatures, high tagging yields have been obtained after eight months storage. Materials

D. J.

KEEPE

J. M.

PAUL

ElectronicsDivision Argonne National Laboratory Argonne, Illinois 60439, U.S.A.

Referencea MARSHALL J. H. Mineral Metabolism, Vol. 3, Calcium Physiology, pp. 1-122. Academic Press, New York (1969). Center for Human Radiobiology, Radiological and Environmental Research Division, Argonne National Laboratory, Argonne, Illinois. MCDOWELL W. P., PAUL J. M. and BOBIS J. P. Rev. Sci. Instrum. 39, 1068 (1968).

and Methods

Materials : N,-purged normal saline Human serum albumin, 25% (HSA) 0.2% Stannous chloride solution, freshly prepared (SnCl,.ZH,O in either 0.1 or 1 N HCl) 2 N HCl and 1 N NaOH pH Meter Combination heater/magnetic stirrer. Pre-formed MAA(basic

procedure

To a beaker containing Ns-purged saline (Nssaline), is added, with stirring, sufficient HSA to give a 0.5% albumin concentration. Stannous chloride is added to give a concentration of O-02%, after which the resulting solution is adjusted, if necessary, to pH 2.0 ( f0.2) using 2 N HCl. After the solution has stood 15 min at room temperature, the pH is brought to 5.5 using 1 N NaOH. InternationalJournalof Nuclear Medicineand Biology 1975.Vol. 2, Sterilization of the HSA(Sn) solution is accomplished pp. 72-74. PergamonPm. Printedin NorthernIreland by Millipore filtration, with the sterilized solution being added to a capped vessel containing a magnetic stirring bar. If desired, at this point, a portion A Method for Prepahg MAA Particles of of the HSA(Sn) solution may be set aside for subsequent pyrogen testing. Narrow Size D&xi-: Preparation A water bath is brought to 45-50°C using a of Kit vials chldhg Fixed combination heater/magnetic stirrer. The vial Numbers of Partides containing the HSA(Sn) solution (plus stirring bar) is placed in the bath, and with stirring of the (Received 10 April 1974) albumin solution, the bath temperature is increased htl-OdUCtiOlb at a rate of l”C/min. Unless a sophisticated hating HEAT coagulation methods currently used for unit is used, the bath temperature can be held preparing pre-formed MAA, or for preparing adequately to the specified heating rate by periodic