Particle analyzing apparatus

A particle analyzing apparatus is provided with an irradiating optical system for applying a light beam to a particle to be examined flowing through a circulation portion in a flow cell, a photometering optical system for photometering the light from the particle to be examined irradiated by the irradiating optical system, and moving means for rendering a base bed on which the flow cell and the photometering optical system are placed movable relative to the irradiating optical system.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a particle analyzing apparatus in a flow 
sightmeter or the like in which simple adjustment of the positions of a 
photometering optical system and a flow cell relative to an irradiating 
optical system is possible. 
2. Related Background Art 
In a conventional particle analyzing apparatus used in a flow sightmeter or 
the like, as shown in FIG. 1 of the accompanying drawings, a parallel 
laser beam L from a laser light source, not shown, is applied to a 
particle S to be examined flowing at a high speed through the central 
circulation (or flowing) portion 2 of a flow cell 1 having a minute 
cross-section of 200 .mu.m.times.200 .mu.m, for instance, while being 
wrapped in sheath liquid, through the intermediary of a condensing lens 3, 
as shown in FIG. 2 of the accompanying drawings. Forward scattered light 
scattered by the particle S to be examined is condensed on a photoelectric 
detector 5 through an objective 4, whereby information chiefly about the 
size of the particle S to be examined is obtained. Also, the sidewise 
scattered light and fluorescence scattered light from the particle S to be 
examined are condensed on a photoelectric detector 7 through an objective 
6, whereby important information chiefly about the complexity of the 
interior of the particle S to be examined can be obtained. 
To accomplish accurate measurement in a flow sightmeter, the optical axis 
of the laser beam L must be coincident with the center of the flow cell 1 
and the scattered lights from the particle S to be examined must be 
accurately condensed by the photometering objectives 4 and 6. For this 
purpose, the axis of the flow of the particle S to be examined and the 
condensing lenses 4 and 6 must be accurately adjusted relative to the 
optical axis of the laser beam L, but in the conventional apparatus, the 
flow cell 1 and the photometering optical system are separate from each 
other and, when the axis of the flow of the particle S to be examined is 
adjusted relative to the optical axis of the laser beam L in a state in 
which the flow cell 1 has been finely moved, the position of the optical 
system for sidewise scattered light deviates and therefore, it is also 
necessary to adjust the optical system for sidewise scattered light and 
thus, operation becomes cumbersome and moreover, it is difficult to effect 
sufficiently accurate adjustment. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a particle analyzing 
apparatus in which a photometering optical system and a flow cell are 
fixed, whereby alignment can be easily carried out by mere adjustment of 
the axis of the flow of a particle to be examined relative to the optical 
axis of a laser beam and measurement of high accuracy is possible. 
It is a further object of the present invention to provide a particle 
analyzing apparatus in which a light beam transmitted through the edge 
portion of a flow cell can be detected to thereby automatically accomplish 
alignment of the flow cell and the irradiating optical system. 
It is still a further object of the present invention to provide a particle 
analyzing apparatus in which the positional relation between the reflected 
lights from the opposite wall surfaces of a flow cell can be detected to 
thereby automatically adjust or confirm the relative relation between the 
flow cell and the photometering optical system.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A first embodiment of the present invention will hereinafter be described 
with reference to FIGS. 3 to 7. 
FIG. 3 is a plan view of an optical system and an alignment apparatus. A 
circulation portion 2 for passing sample liquid therethrough in a vertical 
direction perpendicular to the plane of the drawing sheet is provided at 
the center of a flow cell 1, a laser light source 10 is disposed in a 
direction orthogonal to the flow of the sample liquid, and an imaging lens 
11 for adjusting the imaged shape of a laser beam L is disposed on an 
optical axis 01 to direct the light emitted from the laser light source 10 
to the circulation portion 2. Also, on the forward scattered light side 
from a particle S to be examined by the laser beam L, a beam splitter 12, 
an objective 13 and a photoelectric detector 14 are disposed in succession 
from the flow cell 1 side. An objective 15 and an array-like photoelectric 
detector 16 are disposed on an optical axis opposite to the beam splitter 
12 to detect the distributed state of the light beam divided by the beam 
splitter 12. The output of the photoelectric detector 16 is connected to a 
monitor 17 for observing the distribution of intensity of light. On an 
optical axis 03 orthogonal to the axis of flow of the particle S to be 
examined and to the optical axis 01, a photometering objective 18, a 
half-mirror 19, a condensing lens 20, a stop 21, a condensing lens 22, 
dichroic mirrors 23, 24 and a mirror 25 are disposed in succession from 
the flow cell 1 side. A barrier filter 26 and a photoelectric detector 27 
are disposed in the direction of reflection of the dichroic mirror 23, a 
barrier filter 28 and a photoelectric detector 29 are disposed in the 
direction of reflection of the dichroic mirror 24, and a barrier filter 30 
and a photoelectric detector 31 are disposed in the direction of 
reflection of the mirror 25. Photomultipliers for increasing a weak light 
and making it detectable are used in these photoelectric detectors 27, 29 
and 31. On the reflection side of the half-mirror 19, there is provided an 
auto-focus unit 32 which is used to adjust or confirm the focuses of the 
flow cell 1 and an optical system for photometering the sidewise scattered 
light and fluorescence. 
The laser light source 10 to the photoelectric detector 14 on the optical 
axis 01 and the objective 15 and photoelectric detector 16 on the optical 
axis 02 are fixed on a base plate 40 after adjustment of their axes. Also, 
the flow cell 1 and the objective 18 to the mirror 25 on the optical axis 
03, the barrier filters 26, 28, 30, the photoelectric detectors 27, 29, 31 
and the auto-focus unit 32 are disposed on a stage 41 movable in Y 
direction parallel to the optical axis 03 after adjustment of the focus, 
and a stage 42 movable in X direction parallel to the optical axis 01 is 
interposed between the base plate 40 and the stage 41. The amount of 
movement of the stage 41 in Y direction may be measured by a dial gauge 
43, and the amount of movement of the stage 42 in X direction may be 
measured by a dial gauge 44. 
Referring to FIG. 4 which is a cross-sectional view taken along line IV--IV 
of FIG. 3, two rails 50 are laid on the upper surface of the stage 42 in Y 
direction, and by the rails 50 being fitted to rails 51 on the lower 
surface of the stage 41, the stage 41 is parallel-movable in Y direction 
relative to the stage 42. A bearing 53 is provided at the right end of the 
stage 42, and a cam shaft 54 is rotatably supported on the bearing 53. 
Further, a cam shaft 55 is fitted to the outer side of the cam shaft 54 
and is rotatable relative to the cam shaft 54. Eccentric cams 54a and 55a 
are provided around the cam shafts 54 and 55, respectively, and the stages 
41 and 42 are biased by springs, not shown, so that these cams 54a and 55a 
are normally in contact with a guide 56 fixed to the base plate 40 and a 
guide 57 fixed to the stage 41 as shown in FIG. 5, respectively. Further, 
an anti-slippage member 58 and a rotatable knob 59 for rotating the cam 
shaft 54 are mounted on the upper portion of the cam shaft 54, and a 
rotatable knob 60 is mounted on the upper portion of the cam shaft 55. 
Referring now to FIG. 5 which is a side view taken in the direction of 
arrow V of FIG. 3, two rails 61 are laid on the base plate 40 in X 
direction and are fitted to two rails 62 on the lower surface of the stage 
42 so that the stage 42 is parallel-movable in X direction relative to the 
base plate 40. 
The laser beam L emitted from the laser light source 10 enters the 
circulation portion 2 of the flow cell 1 through the imaging lens 11, and 
part of the forward scattered light by the particle S to be examined 
travels rectilinearly through the beam splitter 12 and is condensed on the 
photoelectric detector 14 through the objective 13, whereby the intensity 
of light thereof is metered. The remainder of said forward scattered light 
is reflected by the beam splitter 12 and condensed on the array-like 
photoelectric detector 16 through the objective 15, whereby the positional 
relation of the particle S to be examined to the optical axis 01 is 
detected. 
Also, the sidewise scattered light by the particle S to be examined enters 
the dichroic mirrors 23, 24 and the mirror 25 through the photometering 
objective 18, the half-mirror 19, the condensing lens 20, the stop 21 and 
the condensing lens 22, and the reflected lights of respective wavelength 
ranges by these mirrors 23, 24 and 25 are condensed on the photoelectric 
detectors 27, 29 and 31, respectively, through the barrier filters 26, 28 
and 30, whereby the intensity of light is metered. 
To adjust the flow cell 1 and the photometering optical system with respect 
to X direction and Y direction, the rotatable knob 59 of the cam shaft 54 
may first be rotated, whereupon the cam 54a may rotate and slide on the 
guide 56 and therefore, the stage 42 can parallel-move in X direction 
relative to the base plate 40 by an amount corresponding to the amount of 
lift of the cam 54a. Likewise, the rotatable knob 60 fixed to the cam 
shaft 55 may be rotated, whereupon the cam 55a may rotate and slide on the 
guide 57 and therefore, the stage 41 can parallel-move in Y direction 
relative to the stage 42 by an amount corresponding to the amount of lift 
of the cam 55a. 
The amounts of parallel movement in X and Y directions can be in a 
considerably slight range because the amount of lift relative to the angle 
of rotation can be set arbitrarily by the cams 54a and 55a, and these 
amounts of movement can be read by the dial gauges 43 and 44 fixed on the 
base plate 40. 
The procedure of adjusting the present particle analyzing apparatus will 
now be described. The sidewise photometering optical system is 
axis-adjusted relative to the optical axis 03, whereafter it is fixed to 
the stage 41 and alignment of the flow cell 1 relative to the 
photometering optical system is effected. For this purpose, by the use of 
the auto-focus unit 32, the flow cell 1 is parallel-moved independently in 
X and Y directions and, in a position wherein the focus is adjusted to the 
center of the flow cell 1, the flow cell 1 is fixed to the stage 41. 
Accordingly, the flow cell 1 and the sidewise photometering optical system 
can be parallel-moved together with each other on the stage 41 in X and Y 
directions relative to the base plate 40. 
To effect alignment of the flow of the particle S to be examined with the 
optical axis 01 while moving the adjusted flow cell 1 on the optical axis 
01 by moving the stages 41 and 42, use is made, for example, of 
quasi-sample liquid absorbing a light of the wavelength range of the laser 
beam L, instead of the particle S to be examined. Part of the laser beam L 
emitted from the laser light source 10 is absorbed by the quasi-sample 
liquid, and the distribution of intensity of light during the absorption 
is measured by the array-like photoelectric detector 16, the output signal 
of which is observed by the monitor 17. When the optical axis 01 and the 
center of the flow of the quasi-sample liquid are coincident with each 
other, the distribution of intensity of light observed is a bilaterally 
symmetrical wave form in which the central portion of a Gaussian 
distributed wave form is concave as shown in FIG. 6. However, when the 
optical axis 01 and the center of the flow of the quasi-sample liquid are 
not coincident with each other, the central concave portion will deviate 
to the left or the right and the distribution will not exhibit a 
symmetrical wave form. In such case, adjustment may be effected by turning 
the rotatable knob 60 to parallel-move the flow cell 1 in Y direction 
until the wave form on the monitor exhibits bilateral symmetry. 
Also, to confirm the coincidence between the focus position of the laser 
beam L from the laser light source 10 and the center of the flow of the 
quasi-sample liquid, the rotatable knob 59 may be turned to parallel-move 
the flow cell 1 in X direction, thereby effecting adjustment so that the 
concave valley portion of the Gaussian distribution on the monitor becomes 
lowest in level and narrowest in width. By the use of such an adjusting 
method, alignment of the flow of the quasi-sample liquid with the optical 
axis 01 can be accomplished. 
In the present embodiment, the optical system for photometering the 
sidewise scattered light of the applied light on the optical axis 03 by 
the particle S to be examined and the fluorescence is placed on the stages 
41 and 42 to which the flow cell 1 is fixed, and these stages 41 and 42 
are parallel-moved in Y direction or X direction to thereby accomplish the 
alignment of the laser beam L with the optical axis 01, but a similar 
effect may be obtained by moving the base plate 40 on which the laser 
light source 10 and the optical system for photometering the forward 
scattered light are placed relative to the optical system for 
photometering the sidewise scattered light and the fluorescence to thereby 
effect alignment. In the present embodiment, a method using the cam shafts 
54 and 55 is used for fine adjustment of the stages 41 and 42, but of 
course, other driving mechanisms may also be used to effect adjustment of 
the optical axis. 
Detailed description will now be made of an embodiment in which a light 
beam transmitted through the edge portion of the flow cell is detected, 
whereby alignment of the flow cell with the irradiating optical system is 
accomplished. 
FIG. 7 shows the construction of the optical system and the signal 
processing system. A circulation portion 102 through which sample liquid 
pass in a direction perpendicular to the plane of the drawing sheet is 
provided at the center of a flow cell 101. A laser light source 103 is 
disposed sidewise of the flow cell 101, and an imaging lens 104 is 
interposed between the flow cell 101 and the laser light source 103, and 
on the side of the extension of the optical axis thereof which is opposite 
to the flow cell 101, there are disposed in succession a beam splitter 
105, a convex lens 106 and a photoelectric detector 107. A convex lens 108 
and a one-dimensional light array sensor 109 comprising, for example, CCD 
(charge coupled device) are provided on the reflection side of the beam 
splitter 105. Further, a sidewise photometering system 110 is disposed 
sidewise of the flow cell 101 orthogonal to the laser beam L and the 
direction of circulation of sample liquid. A connecting part 111 having a 
gear is attached to the flow cell 101 so that the flow cell 101 may be 
driven in a direction orthogonal to the irradiation optic axis by a motor 
112 through the connecting part 111. Revolution of the motor 112 is 
detected by an encoder 113, the output pulse of which is put out to a 
counting part 114. The output of the light array sensor 109 is connected 
to an edge detecting part 115 and a monitor 116 and the output of the edge 
detecting part 115 is connected to a control part 117, the output of which 
may revolve the motor 112 through a driving circuit 118. Transmission and 
reception of a signal may be effected between the control part 117 and the 
counting part 114. 
The forward scattered light from the particle to be examined by the laser 
beam L is condensed on the photoelectric detector 107 through the beam 
splitter 105 and the convex lens 106. 
The cross-sectional distribution of intensity of the laser beam L from the 
laser light source 103 presents a Gaussian distribution, and the laser 
beam L is imaged on the circulation portion 102 of the flow cell 101 
through the imaging lens 104. The distribution of intensity of the laser 
beam L at this imaging position is likewise a Gaussian distribution. When 
the sample liquid is flowing through the circulation portion 102 in the 
flow cell 1 for the purpose of measurement while being wrapped in sheath 
liquid, it is necessary that the center of the Gaussian distribution of 
intensity by the laser beam L be aligned with the center of the 
circulation portion 102. That is, to maintain good measurement accuracy, 
it is required for the center of the flow of the sample liquid to be 
coincident with the peak position of the imaged beam. 
Part of the forward scattered light from the particle to be examined passed 
through the beam splitter 105 and the convex lens 108 is imaged on the 
one-dimensional light array sensor 109. If the center of the flow cell 101 
is coincident with the optical axis in a plane perpendicular to the 
irradiation optical axis, the output wave form of the light array sensor 
109 exhibits a perfect Gaussian distribution as shown at 109a, and if it 
is enlarged, it will be such as shown in FIG. 8. 
However, when there occurs a deviation in the plane perpendicular to the 
irradiation optical axis between the optical axis and the center of the 
flow cell 101 and as shown, for example, in FIG. 9, the laser beam L 
enters the edge portion 102a which is the inner wall of the circulation 
portion 102, the output wave form of the light array sensor 109 assumes a 
disturbed Gassian distribution including a saw-tooth-like wave as shown at 
109b. In the present embodiment, the disturbance of such Gaussian 
distribution is detected, whereby alignment of the center of the flow cell 
101 with the irradiation optical axis is accomplished. 
Assuming that the laser beam L enters the edge portion 102a of the 
circulation portion 102 as shown in FIG. 9, the output signal from the 
light array sensor 109 exhibits a disturbed Gaussian distribution as shown 
at 109b in FIG. 9, and this wave form is input to the edge detecting part 
115. The edge detecting part 115 is a signal processing circuit comprised 
of a band-pass filter or the like, and it takes out only a saw-tooth-like 
wave as shown in FIG. 10A, generates a pulse as shown in FIG. 10B 
correspondingly to this wave form, and puts out binary signals "1" and "0" 
to the control part 117 when this pulse is generated. That is, when the 
laser beam L enters the edge portion 102a of the circulation portion 102, 
the signal "1" is put out from the edge detecting part 115 to the control 
part 117 having a circuit comprised of a microcomputer or the like, and 
when the laser beam L does not enter the edge portion 102a, the signal "0" 
is put out from the edge detecting part 115 to the control part 117. 
When during adjustment, the flow cell 101 is moved in the direction of 
arrow A by the control signal from the control part 117 to the driving 
circuit 118, the laser beam L impinges on the edge portion 102a and the 
output wave form of the light array sensor 109 becomes such as indicated 
by a'. The control part 117 recognizes the signal "1" from the edge 
detecting part 115, stops the movement of the flow cell 1 through the 
driving circuit 118 and resets the content of the counting part 114 to 0. 
When the flow cell 101 is now moved in the direction of arrow B, the 
output pulses from the encoder 113 are input to the counting part 114 and 
each one pulse is added. When the flow cell 1 is thus moved in the 
direction of arrow B, the output of the edge detecting part 115 assumes 
the output condition of the normal Gaussian distribution shown at c' in 
FIG. 11 and the laser beam L now enters the edge portion 102b, and the 
edge detecting part 115 again puts out the signal "1", and the control 
part 117 recognizes this signal "1" and puts out a stop signal to the 
driving circuit 118. 
In this case, the pulse number N obtained in the counting part 114 
corresponds to the amount of movement of the flow cell 101 in FIG. 11. 
Here, the counting part 114 is again reset, and the control part 117 puts 
out to the driving circuit 118 an instruction for moving the flow cell 101 
in the direction of arrow A, and at a point of time whereat the pulse 
number counted by the counting part 114 has become N/2, the control part 
puts out a stop instruction to the driving circuit 118. The then position 
of the flow cell 101 is the medium distance of the initial amount of 
movement, i.e., the position in which the irradiation optical axis and the 
center of the flow cell 101 are coincident with each other. In this case, 
the output wave form obtained by the light array sensor 109 exhibits a 
normal Gaussian distribution free of disturbance as shown at c' in FIG. 
11. 
In the present embodiment, the range of movement of the flow cell 101 is 
mechanically limited and, if switches S1 and S2 are mounted at the 
opposite ends of the range of movement as shown in FIG. 7 so that the 
arrival of the flow cell 101 can be detected, the flow cell 101 will not 
deviate entirely from the laser beam L and excess movement of the flow 
cell 101 can be easily prevented. Also, by the output wave form of the 
light array sensor 109 being displayed on the monitor 116, the alignment 
condition can be observed. Further, in the present embodiment, the flow 
cell 101 and the sidewise photometering system 110 are made integral with 
each other so that the sidewise photometering system 110 is moved with the 
flow cell. 
Also, as shown in FIG. 12, the encoder 113 of FIG. 7 may be replaced by a 
potentiometer 120 and the counting part 114 may be replaced by an A/D 
converter 121 so that the output of the potentiometer 120 may be read, 
whereby the position of the flow cell 101 can be recognized. Again in this 
case, as in the illustrated embodiment, the center of the flow cell 101 
can be positioned at the midpoint and automatic alignment is possible. 
Reference numeral 122 designates an amplifier for amplifying the output of 
the potentiometer 121 and inputting it to the A/D converter 121. 
FIGS. 13 to 16 show a embodiment in which the relative relation between the 
flow cell and the photometering optical system can be adjusted or 
confirmed by detecting the positional relation between the reflected 
lights from the opposite wall surfaces of the flow cell. 
FIGS. 13 and 14 show the construction of a photometering optical system and 
an illuminating system. FIG. 13 is a view as seen from a direction 
parallel to the flow of sample liquid flowing through the flow cell 201, 
and FIG. 14 is a view as seen from a cross-sectional direction of the flow 
of sample liquid. A sample to be examined is poured into the flow cell 201 
from the direction of arrow A and at the same time, sheath liquid is flows 
into the flow cell 201 from the direction of arrow B. In the circulation 
portion 201a of the flow cell 201, the sheath liquid is flowing in the 
state of a layer flow while wrapping the flow of sample liquid therein, 
and hydrodynamic focusing is effected, and the sheath liquid is discharged 
as a drain in the direction of arrow C. A laser beam emitted from an 
Ar.sup.+ laser light source 202 is imaged near the circulation portion 
201a of the flow cell 201 through an imaging lens system 203. An objective 
204 for condensing the forward scattered light by the particle to be 
examined and a photodetector 205 are disposed in the direction of 
rectilinear travel of the laser beam. On an optical axis perpendicular to 
the direction of incidence of the laser beam, there are disposed in 
succession a convex lens 206, a concave lens 207, an infrared light 
reflecting and visible light transmitting dichroic mirror 208, an imaging 
lens 209, a stop 210, a convex lens 211, dichroic mirrors 212, 213 and a 
reflecting mirror 214. 
The dichroic mirror 212 has a characteristic of transmitting therethrough a 
light of longer wavelength than the wavelength 488 nm of the Ar.sup.+ 
laser light, and an imaging lens 215, a barrier filter 216 transmitting 
the wavelength of 488 nm therethrough and an optical guide 217 are 
disposed on the reflection side of the dichroic mirror 212. The dichroic 
mirror 213 has a characteristic of reflecting green light and transmitting 
red light therethrough, and an imaging lens 218, a band-pass filter 219 
and an optical guide 220 are provided on the reflection side of the 
dichroic mirror 213. An imaging lens 221, a barrier filter 222 passing red 
light therethrough and an optical guide 223 are disposed on the reflection 
side of the reflecting mirror 214. 
A focus detecting part comprising a projecting optical system and a 
measuring optical system is provided on the reflection side of the 
dichroic mirror 208, and on the optical axis, there are disposed, as the 
projecting optical system, a convex lens 224, a half-mirror 225, aperture 
masks 226, 227 and an infrared light source 228. Further, on the 
reflection side of the half-mirror 225, there are provided, as the 
photometering optical system, a barrier filter 229 and a light array 
sensor 230. 
The forward scattered light of the laser beam by the particle to be 
examined is detected by the objective 204 and the photodetector 205. The 
reflected light from the particle to be examined which is dyed so as to 
emit fluorescence usually presents green or red fluorescence by the 
Ar.sup.+ laser light of wavelength 488 nm being applied thereto. It is 
known that PI used to detect the amount of DNA (deoxyribonucleic acid) 
emits red fluorescence and FITC used for the detection of cellular film 
surface antigen emits green fluorescence. Besides, the complicated 
structure of the interior of the particle to be examined is reflected in 
the sidewise scattered light by the application of the Ar.sup.+ laser 
light. 
In order to obtain the information from such sidewise scattered light, the 
sidewise scattered light from the flow cell 201 passes through the 
objective system comprising the convex lens 206 and the concave lens 207 
and through the imaging lens 209 and is once imaged at the focus position 
of the stop 210. The sidewise scattered light further passes through the 
convex lens 211 to the dichroic mirror 212, while light of shorter 
wavelength than the wavelength 488 nm of the Ar.sup.+ laser light is 
reflected by this dichroic mirror 212 and only the light of wavelength 488 
nm passes through the imaging lens 215 and is imaged on the end surface of 
the optical guide 217 by the barrier filter 216, and is transmitted to and 
measured by a photodetector, not shown. 
Of the light beam passed through the dichroic mirror 212, green light is 
reflected by the dichroic mirror 213 and passes through the imaging lens 
218 and the barrier filter 219 passing only green light therethrough and 
enters the optical guide 220. Further, the light beam passed through the 
dichroic mirror 213 is reflected by the reflecting mirror 214 and passes 
through the imaging lens 221 and the barrier filter 222 passing only red 
light therethrough and enters the optical guide 223. 
In the focus detecting part, the infrared light source 228 is emitting a 
light of the infrared range, and the irradiating light transmitted through 
the aperture mask 227 shown in FIG. 13(a) arrives at the dichroic mirror 
208 via the aperture mask 226 having the pattern shown in FIG. 13(b), the 
half-mirror 225 and the convex lens 224, and is reflected to the left by 
the dichroic mirror 208 and is projected onto the flow cell 201 through 
the concave lens 207 and the convex lens 206. The width of the pattern of 
the mask 227 is more or less smaller than the width of the circulation 
portion 201a of the flow cell 201, and the projected light is reflected by 
the front and rear surfaces of the flow cell 201. However, if the 
reflection efficiency is neglected, the width of the pattern may be 
greater than the width of the circulation portion 201a. 
The pattern of the aperture mask 226 is such that the slit portion is 
eccentric from the optic axis, and this pattern is for causing the index 
mark by the mask 227 to enter the flow cell 201 from an oblique direction, 
but the projected light beam passes through this aperture mask 226 and 
therefore, if the flow cell 201 is in its normal position, i.e., a state 
in which the central portion thereof is optically conjugate with the 
photometering optical system, the state as shown at d in FIG. 15 will be 
obtained on the light array sensor 230 which is in a position optically 
equivalent to the convex lens 224. FIG. 15A shows the relation of the 
optically conjugate position of the entire flow cell 201, FIG. 15B shows 
the positions of the reflected lights from the front and rear surfaces of 
the flow cell 201, and FIG. 15C shows the photoelectrically converted 
output wave forms of the reflected lights from the front and rear surfaces 
of the flow cell 201. When the position of the focus thus deviates 
forwardly or rearwardly as indicated at a - c and e - g in FIG. 15A, the 
light beam is imaged at the positions shown in FIG. 15B and the signal 
wave forms shown in FIG. 15C are put out from the light array sensor 230. 
The signal wave forms thus obtained in the focus detecting part are 
analyzed by a signal processing part and, when the focus position 
deviates, focus adjustment is effected automatically. The focus adjustment 
thus effected is adjusting the deviation of the focus relative to the 
direction of emergence of the sidewise scattered light in FIGS. 13 and 14 
and moving the focus to the focus position of the optical system, but of 
course, a similar focus detecting part may also be disposed for the 
forward scattered light. 
FIG. 16 is a block diagram of a circuit for automatic focus adjustment. The 
output signal from a light array sensor 230 is analyzed by a peak position 
detecting part 231. In this peak position detecting part 231, there are 
obtained a peak position P1 of the signal corresponding to the reflected 
light from the front surface of the flow cell 201 and a peak position P2 
corresponding to the reflected light from the rear surface of the flow 
cell 201, and P1+P2 is calculated in an adder 232 and further, P=(P1+P2)/2 
is found by a divider 233. This P is indicative of the current in-focus 
state of the projecting optical system, that is, the focus position of the 
objective system comprising the convex lens 206 and the concave lens 207. 
Accordingly, the output S of the optical focus position data preset in a 
reference position setting part 234 is compared with the output value P 
from the divider 233 by a comparator 235, and the output is sent to a cell 
driving part 236 so that P=S, whereby the position of the flow cell 201 is 
controlled. When P&gt;S or P&lt;S in the comparator 235, the driving direction 
for the cell driving part 236 is changed and the center of the flow cell 
201 is controlled to the optical focus position and thus, automatic 
focusing is accomplished. 
In the focus detecting part of the present embodiment, CCD is used as the 
light array sensor 230 and a signal wave form is extracted by causing the 
reflected light from the flow cell 201 by the projected light to scan 
electrically, but alternatively, a similar signal wave form may be 
obtained by attaching a mechanical driving mechanism to the photoelectric 
element and effecting the scanning. Also, if a microprocessor is 
introduced into the signal processing part so that each operation is 
carried out by a software, the circuit construction will become simple and 
a similar performance can be obtained. Of course, focus adjustment may 
also be accomplished by adjusting the objective system instead of moving 
the flow cell 201. 
In the present embodiment, after the adjustment of the photometering 
optical system in the direction of the optic axis with respect to the flow 
cell or after the confirmation thereof, the flow cell and the 
photometering optical system are made integral and movable relative to the 
irradiating optical system as already described in connection with the 
previous embodiment.