Abstract:
A laser beam is periodically deflected before being directed into a sample volume. The beam is deflected at a frequency such that the beam makes one or more passes through the sample volume while data are collected from the sample volume. The amplitude of motion of the beam, the dwell time of the beam at any given point, and the Gaussian intensity profile of the beam cooperate to produce an effective flat topped illumination profile for the light that is incident on specimens in the sample volume. The total photon exposure at any given point in the sample volume is a function of both the beam intensity and the dwell time at that location. Therefore, a longer dwell time and lower intensity at the edge of the profile are in balance with a shorter dwell time and higher intensity at the center of the profile.

Description:
RELATED APPLICATIONS 
     This application is based on a prior copending provisional application, Ser. No. 61/246,919, filed on Sep. 29, 2009, the benefit of the filing date of which is hereby claimed under 35 U.S.C. §119(e). 
    
    
     BACKGROUND 
     Lasers are an ideal source to illuminate cellular samples for analysis due to their high brightness, monochromaticity and coherence. Although there are different spatial modes for laser light, the most common is the TEM00 mode in which laser light is generated with a Gaussian intensity profile. The Gaussian intensity profile is governed by the equation I=I o *e −2 (r/w) 2 , where “I o ” is the peak intensity point, “w” is the waist radius (the point where the intensity is 13.5% of the peak intensity) and “r” is distance from the peak. Per the equation, the intensity of the beam begins to fall off immediately on either side of the peak. Assuming a beam waist 85 microns in diameter, the intensity drops to 97% of the peak value at a point just 5.0 microns from the center of the beam. At a point 10 microns from center, the intensity drops to 89% of the peak value. Therefore, when illuminating a core stream containing cells in motion, as is done in flow cytometry, a cell disposed just 5 to 10 microns from the center of the core stream will see a significantly lower level of irradiation and therefore, generate less fluorescence. Because the location of cells in the core stream can vary by these amounts in normal operation of a typical flow cytometer, the laser intensity profile across the beam is a source of measurement variation in these instruments. 
     In flow cytometry, the laser beam diameter is maintained large relative to the cell and core size, in order to minimize cell-to-cell fluorescence intensity measurement variation. The typical beam size is about 85 microns in diameter for a theoretical core stream size of 10 microns. If the core stream is made larger, then the beam must also be made larger to maintain illumination uniformity. It should be noted that making the core larger may lead to a much higher amount of coincident events in a standard flow cytometer. Although coincidence is not an issue in an imaging flow cytometer, an increase in core size may lead to defocus, which may degrade the imagery. Although making the beam approximately ten times larger than the cell decreases measurement variation, it also wastes a considerable amount of light, thereby decreasing the sensitivity of the instrument, unless this decrease is offset with a higher power and concomitantly higher cost laser. It is therefore desirable to use a higher proportion of the laser light to illuminate a core stream in a flow cytometer without incurring an increase in measurement variation, which should decrease instrument cost by enabling the use of a lower-power laser, or alternatively, increase sensitivity if the same power laser is used. It would also be desirable to actively enable tailoring of the beam profile to further increase sensitivity, where higher amounts of variation are acceptable. 
     There have been many attempts over the past two decades to generate what are commonly called “flat top” laser profiles to reduce intensity variation near the center of the laser beam. One elegant approach uses diffractive optics, which can be designed to generate a wide array of profiles, including a flat top. Although this approach is effective at generating a flat top, it suffers from the drawback of light loss, reducing overall intensity, and therefore, negating much of the benefit relative to simply increasing beam size. 
     Another approach disclosed in U.S. Pat. No. 4,826,299 uses a double wedge-shaped optic that is disposed in the laser beam path. The laser beam is then significantly expanded before being imaged into the flow cell. The net effect is the generation of a nearly flat top intensity profile. This method does not suffer the intensity loss associated with diffractive optics and therefore generates a higher intensity than the Gaussian intensity profile with a flat top over the region of interest. When properly designed, this technique can generate a flat top which is 1.5 to 2 times more intense than the standard Gaussian intensity profile, over a 10 micron region around the center of the beam. However, this technique also has several drawbacks. First, the method uses highly specialized optics and requires a substantial expansion of the beam prior to imaging into the flow cell, which adds cost and poses additional constraints on the optical and mechanical design of the instrument. While these issues may theoretically be overcome at a lower cost than using a higher powered laser, this technique relies on superimposing one part of the beam with another in order to smooth out the profile. Since laser light has a high degree of coherence, in practice, the overlapping beams can constructively and/or destructively interfere with each other, generating significant perturbations in the profile. Once the beam is aligned, and a uniform profile is established, very small changes in the position of the beam on the final imaging lens may lead to significant perturbations in the intensity profile. Therefore, very small thermal changes or pointing errors can lead to a loss of uniformity and negate the benefits of the method. Accordingly, an alternative approach is needed to produce a laser beam having a flat top Gaussian beam intensity profile that avoids these problems, and thus, to achieve the benefits of using such a laser beam, as noted above. 
     SUMMARY 
     This application specifically incorporates herein by reference the disclosures and drawings of the patent application identified above as a related application. 
     An exemplary approach has been developed that overcomes the issues associated with the prior art in order to generate a stable, uniform profile at very low cost. The profile optimizes the use of the laser&#39;s energy to increase the photon dose experienced by objects flowing through a flow cytometry instrument, thereby increasing the sensitivity of the instrument. Further, the apparatus to implement this approach is easy to manufacture and can be simultaneously applied to multiple lasers within a given system. Further still, the present novel approach provides the ability to actively tailor the beam profile to match various applications after it has been installed in the instrument. The beam may also be tailored to provide for a larger flat top region, at the expense of intensity, to reduce instrument variation when compared to a standard Gaussian intensity profile. The present novel approach can be applied to illuminate cells that are either stationary or in motion, whether in flow or on substrates, but is described below primarily in the context of cells in a flow moving along a one dimensional path. 
     The present exemplary approach employs a laser beam that is reflected from a mirror, which is in turn, supported on a structure enabling precise rotation of the mirror. The structure rotates about an axis substantially parallel to the axis of cell flow, resulting in a beam scanning motion in a plane substantially perpendicular to the axis of cell flow. It should be understood that other alternative mechanisms can be employed to deflect the laser beam so that it scans in the desired manner. In one exemplary embodiment, the mirror is forced to rotate with a sinusoidal velocity profile at a frequency such that the reflected beam makes one or more passes through the cell core stream during the time in which light from a cell is integrated by the instrument. The mid point of traversal of the laser beam is aligned coincident with the center of the core stream. It is important to note that an amplitude of the scan for the laser beam at the point where the laser beam is incident on a particle is less than the diameter (or less than the largest other cross-sectional dimension) of the laser beam. For example, if the beam waist or cross-sectional dimension of the beam is 25 microns at the point where the laser beam is incident on a particle in the flow stream of the particle analyzer, the beam might scan or deflect across the flow stream by only 9 microns at that point. 
     The amplitude of the scanning motion, the dwell time of the beam at any given point, and the Gaussian intensity profile of the laser beam all are selected to cooperate in forming an effective flat top illumination profile over the period of integration. The total photon exposure of the cell at any given point in the profile is a function of both the beam intensity and the dwell time at that location. At the edges of the motion, the photon dose is increased, because the beam moves more slowly, offsetting the lower photon dose due the Gaussian intensity profile. Therefore, the longer dwell time and lower intensity at the edge of the profile are in balance with the shorter dwell time and higher intensity at the center of the profile. If the moving beam had a uniform intensity profile instead of a Gaussian intensity profile, the effective profile would provide a higher photon dose at the edges where the beam moves slowly. 
     It will be readily apparent to those skilled in the art that the means for deflecting the beam may include devices other than a mirror, such as other types of reflective devices, an acousto-optical modulator (AOM), an acousto-optical deflector (AOD), diffractive devices, and other devices that can be controlled to deflect light through a very small angle at a frequency of a few thousand Hertz. Likewise, different variants of this novel approach may employ different beam sizes, motion amplitudes at the point where the beam is incident on particles, motion profiles and the like, without departing from the scope and spirit of the invention. In the context of cells in flow, the photon dose normalization is required in only one axis orthogonal to flow because the motion of the cell in the axis of flow results in a uniform integration of illumination across the cell in the axis of flow. However, those skilled in the art will recognize that stationary cells or other objects can be illuminated uniformly in both axes by implementing the present invention in two axes, either with a second rotating mirror system that acts in series with the first, or by scanning a single mirror (or other appropriate deflecting device) relative to two orthogonal axes. 
     This Summary has been provided to introduce a few concepts in a simplified form that are further described in detail below in the Description. However, this Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. 
    
    
     
       DRAWINGS 
       Various aspects and attendant advantages of one or more exemplary embodiments and modifications thereto will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: 
         FIG. 1  schematically illustrates a Gaussian laser beam being reflected from a movable reflective surface, where the motion of the surface sweeps the beam transversely through the sample volume during data collection, effectively producing a flat top illumination profile during the sampling period; 
         FIG. 2A  graphically illustrates a conventional Gaussian intensity profile characteristic of an unmodified laser beam, and a flat top Gaussian intensity profile obtained using the technique illustrated in  FIG. 1 , where the reflective surface vibrates through a small angle so as to deflect the laser beam repetitively with an amplitude of about 9.6 microns (at the point where the light beam is incident on a particle); 
         FIG. 2B  graphically illustrates an intensity profile as seen by a cell when the cell is illuminated using the technique of  FIG. 1 , and data are integrated over a period of time; 
         FIG. 3  is a table indicating factors involved in the empirical model used to generate the graphs of  FIGS. 2 ,  4 ,  5 , and  6 ; 
         FIG. 4  is similar to  FIG. 2 , but graphically illustrates the changes when a laser beam with a 30 micron waist is scanned at an amplitude of 12 microns; 
         FIG. 5  is similar to  FIG. 2 , but graphically illustrates the changes when a laser beam with a 15 micron waist is scanned at an amplitude of 6 microns; 
         FIG. 6  is similar to  FIG. 2 , but graphically illustrates the changes when a laser beam with a 25 micron waist is scanned at an amplitude of 4.5 microns; 
         FIG. 7  graphically illustrates a conventional Gaussian intensity profile characteristic of an unmodified laser beam, and a flat top Gaussian intensity profile obtained using the technique illustrated in  FIG. 1 , where the reflective surface is moved linearly, rather than in a sinusoidal fashion; 
         FIGS. 8A-8D  schematically illustrate different views of an exemplary system including two piezoelectric crystals for moving the reflective surface of  FIG. 1 , to produce a flat top illumination profile during the sample acquisition, wherein  FIG. 8A  is an isometric top-side view of the frame, reflective surface and flexure stage,  FIG. 8B  is an isometric bottom-side view of the same,  FIG. 8C  is an isometric bottom-side view of the flexure stage and a transducer, and  FIG. 8D  is an isometric top-side view of a first and second transducer, the reflective surface and the flexure stage; and 
         FIG. 9  is a schematic illustration showing the focused and scanned laser beam incident on a particle in an interrogation region of a particle analyzer. 
     
    
    
     DESCRIPTION 
     Figures and Disclosed Embodiments are not Limiting 
     Exemplary embodiments are illustrated in referenced Figures of the drawings. It is intended that the embodiments and Figures disclosed herein are to be considered illustrative rather than restrictive. No limitation on the scope of the technology and of the claims that follow is to be imputed based on the examples shown in the drawings and discussed herein. Further, it should be understood that any feature of one embodiment disclosed herein can be combined with one or more features of any other exemplary embodiment that is disclosed, unless otherwise indicated. 
     In  FIG. 1 , an exemplary embodiment  10  of the present novel approach employs a laser beam  12  with a Gaussian intensity profile, and therefore, no specialized optics are required. In this exemplary embodiment, Gaussian profile beam  12  originating from a laser source  11  has a 1/e 2  diameter of 700 microns. The laser beam strikes and is reflected from a mirror  14  that is supported on a frame  18  by a flexure stage  16 . The reflected laser light is then imaged through at least one optical component  22  (comprising one or more lenses) having a focal length of about 40 mm. The resulting reflected and focused laser beam has a waist of about 25 microns at a point  26  where it is coincident on a core stream  24  passing through a flow cytometer, at a position approximately 54 mm from the lens. 
     In this exemplary embodiment, mirror  14  is supported on a mount comprising frame  18  with flexure stage  16  allowing a small rotation of the mirror in an axis that is perpendicular to the core stream. In a conventional illumination system for a flow cytometer, the mirror is fixed after alignment and remains motionless during sample acquisition. However, in the present novel approach, flexure stage  16  is driven with a piezoelectric crystal transducer (not shown in this Figure) in order to effect movement of the mirror. Mirror  14  moves so that the reflected laser beam traverses an arc transversely through the flow cell, i.e., in an arc that is at a right angle relative to the direction of travel of a cell or other object carried with flow through the flow cytometer. In one exemplary embodiment, the varying mirror angular position in the optical train and the amount of deflection of the mirror and thus, of the reflected laser beam effected by the piezoelectric crystal transducer cause reflected laser beam  20  to traverse the flow cell in a sinusoidal pattern with about a 9.6 micron amplitude and at a rate that completes at least one cycle during the time the cell traverses the interrogation region of the instrument, where the cell is illuminated by this reflected scanned laser beam. In this and in each of the other exemplary embodiments discussed herein, it must be emphasized that the waist (or diameter, or the largest cross-sectional dimension) of the laser beam at the point where the laser beam traverses the flow cell and may be incident on a particle, is always substantially greater than the extent of scan deflection of the laser beam at that point (e.g., a laser beam with a waist of about 25 microns is scanned with an amplitude at the flow cell of about 9.6 microns in the above example). 
     The combined effect of the Gaussian beam profile and the sinusoidal motion profile of the reflected laser beam as it traverses the flow cell operate in concert to equalize the photon dose experienced by any object such as a cell at point  28 , where the cell is disposed while flowing through an interrogation region. This procedure effectively generates a flat top profile for the laser beam intensity, around the center of core stream  24  in the flow cytometer, as shown for a scanned laser beam solid line curve  32  in a graph  30  of  FIG. 2A . Because of the reduced beam diameter caused by one or more optical components  22  focusing the reflected laser beam, the flat top profile has twice the intensity of a conventional 85 micron diameter Gaussian laser beam, as indicated by a dash line curve for the conventional (i.e., non-scanned) laser beam, while maintaining an intensity uniformity equivalent to or better than the 85 micron Gaussian beam within +/−5 μm of the peak intensity. The focusing of the beam thus produces a laser beam with a smaller waist and higher intensity, but the scanning of the laser beam by the mirror effectively flattens the intensity profile of the beam as the reflected and focused laser light fully illuminates a cell or other particle in the interrogation region of the flow cytometer with the scanned light. 
     The intensity profiles of both the scanned flat top (solid line  32 ) and conventional Gaussian (dash line  34 ) intensity profile laser beams are shown in  FIG. 2A . Data (i.e., light rays) are collected from the sample volume using, for example, a time delay integration detector, so that the data are integrated over the sample time. Since data are integrated during the entire traversal of the sample passing through the interrogation region before being read off the time delay integration detector, it appears to the cell as if a single non-scanned beam with a flat topped intensity profile (as shown by flat peak  38  in graph  36  of  FIG. 2B ) were being imaged into the sample volume of the flow cytometer. 
     Note that in the example of  FIG. 2A , the laser beam reflected from the moving reflective surface of mirror  14  and focused by one or more optical components  22  has a beam waist of only about 25 microns, yet provides the same range and 195% of the intensity obtained using a conventional laser beam having a larger 85 micron beam waist. Because the diameter of the laser beam waist is related to power, the smaller 25 micron scanned laser beam is a more efficient illumination source than the 85 micron conventional laser beam. 
       FIG. 2A  thus compares the Gaussian beam intensity profiles of a standard laser beam having an 85 micron beam waist with that of a smaller 25 micron beam waist laser scanned rapidly with mirror in the horizontal axis to achieve ˜10 scan cycles while data are being collected (i.e., where the sample volume is a flow cell and the sample is a biological cell, data are collected during the time required for the biological cell to pass through a field of view in the sample volume (i.e., a flow cell/sample cuvette)). The beam waist as the term is used herein refers to the cross-sectional dimension of the beam at the point where the beam is incident on a sample, e.g., the biological cell, in a particle analyzer. Scanning the laser beam with the mirror effectively generates a broader, flat topped Gaussian beam intensity profile. Since the laser beam used in the scan has a smaller waist, its peak intensity is higher than the Gaussian peak of the standard (non-scanned) laser beam and therefore, the scanned laser beam generates more fluorescence output from the cell or other target specimen on which it is incident. Computational analysis can be used to compute the effective profile for various waist sizes, different amounts of scan amplitude, and different scan profiles (sinusoidal, linear, binary, etc.) to determine how the range and intensity of the scanned beam compares to a fixed beam of a given size (i.e., a given beam waist). Thus, the 85 micron conventional beam and the 25 micron scanned beam discussed above are simply exemplary and are not in any way intended to be limiting on the present novel approach. 
     Because the relationships between beam waist, scanning parameters and intensity are predictable, computational analysis enables the effect of changing variables such as beam waist and scanning parameters to be determined using a computer model. Such an empirical computer model was developed using a conventional spreadsheet application. The empirical model was developed using the following concepts. With a fixed beam, the photon dose at a given point in the flow cell is strictly a function of the beam&#39;s intensity profile. When the beam is scanned through the sample volume, the photon dose at any location in the flow cell is the product of the intensity of the beam at that location (which changes as it scans), multiplied by the dwell time of the beam at that location. If the beam has a Gaussian intensity profile and the scan function is sinusoidal, the effective beam intensity profile is the product of those two functions, with the specific shape of the curve being dependent on the width of the Gaussian profile and the amplitude of the sinusoid.  FIG. 3  is a table indicating factors involved in the empirical model used to generate graph  30  in  FIG. 2A , a graph  36  in  FIG. 2B , graphs  40  and  46  in  FIG. 4 , graphs  50  and  56  in  FIG. 5 , and graphs  60  and  66  in  FIG. 6 . 
       FIG. 4  is similar to  FIGS. 2A and 2B , respectively, but graph  40  illustrates the changes when a laser beam with a 30 micron waist at a point where the laser beam may be incident on a particle, is scanned at a scan amplitude of 12 microns at that point. The effective flat topped peak laser beam that is achieved is 140% wider (a solid line  42 ) than a non-scanned laser beam having the 85 micron waist (a dash line  44 ), while the intensity of the scanned beam is 157% greater than that obtained using a non-scanned laser beam having the 85 micron waist. Graph  46  illustrates a flat topped peak  48  in  FIG. 4 . Such a modification would be useful if a wider beam is desired to reduce cell-to-cell exposure variation for critical measurements, and the trade off of a more modest increase in the intensity can be accepted. 
     Graphs  50  and  56  in  FIG. 5  are similar to  FIGS. 2A and 2B , respectively, but graph  50  illustrates the changes when a laser beam with a 15 micron waist is scanned at an amplitude of 6 microns. (Again, the waist of the laser beam and the scan amplitude are measured at the point where the laser beam might be incident on a particle in the flow stream or other designated region of a particle analyzer.) The effective beam achieved is 30% narrower (as indicated by a solid line  52 ) than a non-scanned laser beam having the 85 micron waist (as indicated by a dash line  54 ), while the intensity of the scanned beam is 314% greater than that obtained using the non-scanned laser beam having the 85 micron waist. Graph  56  indicates a flat topped peak  58  for the scanned laser beam. Such a modification would be useful if a larger cell-to-cell exposure variation is desired, in return for a relatively large increase in the intensity. 
     Graphs  60  and  66  in  FIG. 6  are similar to graphs  30  and  36 , respectively, in  FIG. 2A  and  FIG. 2B , but graph  60  illustrates the changes when a laser beam with the same 25 micron waist is scanned at an amplitude of 4.5 microns (a solid line  62 ). In this case, the intensity is three times that of the conventional 85 micron Gaussian beam (a dash line  64 ), but the uniform region has also decreased by about a factor of three. Changing the scan amplitude is very easy to do in situ to tailor the laser beam profile for a specific application. In this case, the user is willing to sacrifice measurement consistency in order to increase sensitivity. Graph  66  in  FIG. 6  illustrates a flat topped profile peak  68  for the scanned laser beam. 
     A graph  70  in  FIG. 7  shows the results for an alternative embodiment of the invention, where the laser beam has a Gaussian profile with an 8 micron waist in the scan axis. The motion profile in this case has been changed to linear with an 8 micron amplitude. In this exemplary embodiment, the intensity (a solid line  72 ) is more than three fold that of the standard 85 micron Gaussian shown as a dashed line  74 , while maintaining the same range of uniformity. It will be understood by those skilled in the art that generation of a something close to a linear profile may be accomplished with a piezoelectric transducer (PZT) and a mirror. However, generation of such a profile takes a significant amount of energy to reverse motion at the ends of the scan. Those skilled in the art will also appreciate that a linear profile may also be generated with an acousto-optic modulator (AOM) or other deflecting devices. However, use of such a device is costly and may lead to energy loss due to loss of the various diffracted orders. Nonetheless, tailoring of the beam size and motion profile may lead to intensity profiles that are advantageous for some applications. 
     Beam tailoring can be accomplished by changing the amplitude of the beam scanning motion, as well as the specific waveform with which the beam traverses the interrogation region of the instrument. Likewise, the profile may also be affected by the waveform or dimension of the non-scanned beam. In general, the smaller the non-scanned beam diameter before the beam is reflected to scan it, the greater will be the gain in intensity over the conventional Gaussian intensity profile. These Figures demonstrate both sinusoidal beam motion profiles that may be applicable to a resonant scanner, as well as linear profiles suitable for non-resonant scanners. The profile may alternatively also be a ramp, a saw tooth, or a modified sine wave. The frequency of traversal may also be changed, but it is desirable to maintain a sufficiently high scan frequency such that at least one pass is made over the flow cell or region where the beam may be incident on a particle during the time in which a particle traverses the cross section of the beam in the direction orthogonal to the scanning movement of the laser beam. In general, a higher number of passes of the laser beam over the particle is desirable to maintain a more consistent photon dose per particle, as individual particles traverse the illuminated region of the particle analyzer. 
     As noted above, there are a number of different methods to induce a scanning motion in the beam, to deflect the laser beam over a relatively small amplitude (i.e., an amplitude less than the size of the largest cross-sectional dimension of the laser beam—at the point where the beam might be incident on a particle). One low cost approach is to employ a PZT in conjunction with a mirror on a flexure stage. The piezoelectric crystal of the PZT changes its length in a linear fashion as a function of the voltage applied across the crystal. When affixed to a flexure stage as shown in  FIGS. 8A-8D , a change in the length of the PZT causes a shift in the angular attitude of the mirror, thereby deflecting the laser beam through a corresponding arc. If the voltage applied to the crystal changes in a sinusoidal fashion with time, the corresponding angular change in the mirror position will vary accordingly and produce a sinusoidal sweep of the laser beam. Likewise, if the change in the voltage is linear with time, the change in angular attitude will be linear, causing a linear time variant sweep of the beam. In all cases, for the purposes of this novel approach, the amplitude of the sweep or scan of the laser beam is less than the diameter or cross-sectional dimension of the laser beam, where both the size of the laser beam and the amplitude are measured at the point where the laser beam might be incident on a particle. 
     Those skilled in the art will appreciate that a PZT can be used in resonant as well as non-resonant scanners. If the sweep frequency of the PZT is several times higher than the resonance frequency of the structure, the mirror may move with diminished amplitude and there may be a phase lag in the movement with respect to the voltage profile applied to the PZT. At frequencies well below the resonant frequency, the movement of the mirror should follow from the motion of the PZT. As the scanned frequency approaches the resonant frequency of the scanning structure, there will be an amplification in the displacement of the mirror when compared to the same amount of energy applied to the structure at a frequencies well above or below the resonance frequency, which illustrates the major benefit of the resonant scanning structure, i.e., very little energy is required to drive the mirror to effect the desired beam deflection if the structure is driven at its resonant frequency. In an exemplary resonant scanning embodiment of the present novel approach, cost is further reduced due to the simplification of the drive circuitry by eliminating the high currents and current reversals required to drive the structure. 
       FIGS. 8A-8D  thus illustrate different views of an exemplary embodiment of a system in accord with the present novel approach, where mirror  14  is driven by a first PZT (PZT 1 )  90 , with a second PZT (PZT 2 )  92  used as a means to preload the first PZT, and also as a means to monitor the amplitude of movement of the first PZT. Flexure stages  16  supporting the mirror are approximately 4 mm by 4 mm in cross section and 3 mm long. They support glass mirror  14 , which is sized to be about 25 mm wide by 15 mm tall by 3 mm thick. PZT 1  is used to drive the structure, while PZT 2  is used as a preload and monitoring device and is adjustable with a preload screw  94 . Considering the mass and stiffness of all elements in the structure, the resonance frequency was computed to be approximately 20 KHz. Empirical measurement of the resonant frequency confirmed the computed value. 
     In resonant structures, changes in temperature may induce changes in the modulus of elasticity of the materials used in the structure. Likewise changes in temperature may also induce changes in the size of the structure. Any changes in the modulus or size can affect preloading and ultimately the stiffness of the structure which in turn can change the resonance frequency of the structure. Very small shifts in the resonance frequency of the structure may have a large impact on the motion of the structure for a given amount of energy input into the structure. This in turn may substantially reduce the amplitude of the scanned beam. Therefore, it may be advantageous to monitor the amplitude of motion of the driving member of the structure to ensure it is moving the prescribed amount. In the exemplary embodiment shown in  FIGS. 8A-8D , the signal from PZT 2  is fed into a proportional-integral-derivative (PID) controller. The PID controller monitors any difference between a desired scan amplitude and the actual scan amplitude as measured by PZT 2 . The PID controller then actively changes the amplitude of the voltage supplied to PZT 1  in order to minimize any difference between the desired and actual amplitude of the scan motion. Those skilled in the art will appreciate there are many other options for deflecting the beam and controlling the amplitude of the deflection, which when employed, will not depart from the spirit and intent of the present novel approach. 
       FIG. 9  is a schematic illustration  100  of a scanned and focused laser beam  102  incident on a particle  104  in a core stream  24  of a particle analyzer (not fully shown). This Figure illustrates how the exemplary scanned and focus laser beam having a waist of about 25 microns is scanned with an amplitude of about 9.6 microns, which is substantially less than the waist or diameter of the laser beam where it is incident on particle  104 . Scanned and focused laser beam  102  completes at least one scan cycle as the particle is illuminated by the laser beam, and subsequent particles passing through the interrogation region of core stream  24  are then similarly scanned. The illumination of the particles by the laser beam can be used for analyzing the particles, e.g., based on a fluorescence emitted from the particles in response to their illumination by the laser light, but it will be understood that many other forms of analysis and imaging of the particles can be implemented in such a particle analyzer. 
     Although the concepts disclosed herein have been described in connection with the preferred form of practicing them and modifications thereto, those of ordinary skill in the art will understand that many other modifications can be made thereto within the scope of the claims that follow. Accordingly, it is not intended that the scope of these concepts in any way be limited by the above description, but instead be determined entirely by reference to the claims that follow.