Optical arrangement for flow cytometer to facilitate large angle light-scattering measurement

An optical arrangement for a flow cytometer, wherein intense light is focused by a microscope objective having a numerical aperture Na.sub.i, onto the cells carried by a flow of water through the focal plane of the objective, with another microscope lens situated opposite the objective, with an optical axis and object plane coinciding with the objective and with a numerical aperture, NA.sub.O, which is significantly larger than that of the objective. The objective contains a circular field stop in, or close to, its secondary focal plane, with a diameter corresponding to a numerical aperture NA.sub.df, which is slightly larger than NA.sub.i, and much less than NA.sub.O. The fluorescence and scattered light from the stream of cells are separated by a dicroic mirror on basis of their different wavelength, so that they give rise to separate images in separate image planes of the objective. A telescope is situated behind the image plane and creates an image of the field stop in a plane with two concentric mirrors of different diameters, which separate light scattered from the cells according to the different scattering angles and direct them onto separate light detectors.

BACKGROUND OF THE INVENTION 
The present invention relates to an optical arrangement for flow 
cytometers. 
A flow cytometer is an instrument for measurement of the fluorescence and 
light scattering of individual biological cells and other types of 
microscopical particles. In the flow cytometer, the cells are carried by a 
laminar flow of water through the focus of a high intensity light source. 
The cells are typically stained with a fluorescent dye which binds 
specifically to one particular cell constituent. Each cell passing through 
the focus will thus emit a short pulse of fluorescence and scattered 
light. The intensity of the fluorescence will be proportional to the 
cellular content of fluorescent dye and thereby with the cellular content 
of the stained constituent. The intensity of the scattered light and its 
angular distribution is a complex function of the size, shape, structure 
and chemical composition of the cell. By measuring with separate detectors 
the light scattering at small and large scattering angles, respectively, 
it is thus possible to distinguish cells on the basis of size, shape, and 
structure. 
For some purposes, the cells may be stained by two or three different dyes 
which bind to different cellular constituents and fluoresce at different 
wavelengths. The corresponding spectral components of the fluorescence can 
be separated by dichroic mirrors and band filters and measured by separate 
detectors. Hence, each cell may generate several signals; typically two 
light scattering signals--low and large angle scattering--and two or three 
fluorescence signals. This technology is well known and has been published 
in many articles, e.g. in "Flow cytometry and sorting" (Melamed, M. R.; 
Lindmo, T; Mendelsohn, M. L., Eds.), Wiley-Liss, New York 1990. 
The cellular content of the constituent(s) to be measured may be quite 
small, that is down to about 1.multidot.10.sup.-18 g/cell. The demands on 
the sensitivity of the instrument are correspondingly high. In order to 
achieve such sensitivity the excitation light has to be concentrated into 
a very small and correspondingly intense focus. Furthermore, the optics 
which collects the fluorescence and scattered light must have the highest 
possible numerical aperture. It is essential also that any light from 
other sources than the cells, e.g. the background due to fluorescence and 
light scattering from optics and other components in the optical path, is 
as low as possible. 
There are two major types of flow cytometers: a) Instruments employing a 
laser as the source of excitation light, and b) instruments using a high 
pressure arc lamp with xenon or mercury. The laser-based instruments have 
the advantage that the excitation light can be focused into a very small 
and correspondingly intense focus. Furthermore, the beam of excitation 
light is near parallel, which simplifies the distinction of light 
scattered to different angles. Arc lamp-based instruments have the 
advantage that the spectrum of the light source contains all wavelengths 
from UV through the visible spectrum. Hence, by means of appropriate 
filters the proper wavelength for excitation of any fluorescent dye can be 
selected, thus making this type of instruments more versatile. 
All laser-based flow cytometers have essentially the same optical 
configuration, namely so that the vertical sample stream cuts through the 
focus of a horizontal laser beam and so that this focus is intersected at 
a 90 degree angle by the optical axis of the optics which collects the 
fluorescence and the light scattered to large angles, i.e. around 
90.degree.. Behind the light collecting optics fluorescence and light 
scattering are separated by a dichroic mirror and directed onto separate 
light detectors. The fluorescence may be further split into different 
spectral components by additional dichroic mirrors and measured by 
separate detectors. 
The light of the focused laser beam is near parallel, that is falling 
within a light cone of about 2.degree. or less. Hence, the light 
scattering at low scattering angles is measured through an other lens with 
its optical axis coincident with the laser beam. The laser beam is 
prevented from entering the lens by a field stop situated in front of the 
lens. 
Some laser-based flow cytometers employ two lasers emitting at different 
wavelengths and focused to separate foci, so that the cells are excited 
sequentially with two different wavelengths. Thus, it becomes possible to 
measure two different dyes which cannot be excited by the same wavelength 
or which interfere in ways which are not compatible with the measurement. 
Such "two focus excitation" has many interesting biological applications. 
All arc lamp-based flow cytometers employ epi-illumination, which is to say 
that the optics which concentrate the light in the excitation focus, also 
collects the fluorescence. In order to achieve the highest possible 
excitation intensity as well as optimal fluorescence collection 
efficiency, this optics should have the highest possible numerical 
aperture (NA). Hence, an oil immersion microscope lens, having 
NA.apprxeq.1,3, is used for this purpose. 
The large field angle of the illumination field of such a lens makes it 
impossible to distinguish the light scattering at small and large 
scattering angles by the same type of optical configuration as that usede 
in the laser-based instruments. The Norwegian patents Nos. 145.176 and 
156.917, as well as U.S. Pat. Nos. 4,408,877 and 4,915,501 disclose how 
light scattering can be measured in the epi-illumination type of optical 
configuration used in arc lamp-based instruments. By means of a central 
field stop close to the back focal plane of this lens, a dark field is 
created which allows measurement of light scattering at both small and 
large scattering angles through a second microscope lens situated opposite 
to the first and with its aperture within the dark field produced by the 
field stop in the first lens. 
However, this configuration has certain shortcomings. Thus, it allows the 
light scattering at large angles to be measured only within a very small 
aperture, i.e. NA.gtoreq.0,04. This small aperture limits the sensitivity 
of the measurement of this parameter. Furthermore, with this configuration 
the "large angle" range has a lower limit which does not exceed about 
20.degree.. 
Another disadvantage of the epi-illumination configuration of current arc 
lamp-based flow cytometers is that it does not allow excitation in two 
separate loci of different wavelength, thus, limiting to some extent the 
range of applications of such instruments. The epi-illumination also 
implies that the optics which collects the fluorescence, i.e. the 
microscope objective, is exposed to very high intensities of excitation 
light. Even with microscope objectives of the very highest quality this is 
causing some fluorescence from the elements of the objective which adds to 
the background on which the cell fluorescence is detected, and thereby to 
a reduction of the signal to noise ratio, which is equivalent to a 
reduction of the sensitivity. 
SUMMARY OF THE INVENTION 
The present invention is a novel optical configuration which eliminates 
some of the above mentioned limitations of current designs of are 
lamp-based flow cytometers. Thus, the present invention facilitates large 
angle light scattering measurement at considerably higher scattering 
angles and with a much higher numerical aperture than was feasible with 
the previous configuration. Hence, the light scattering sensitivity is 
considerably increased relative to current designs. It also produces less 
background light in the fluorescence light path, and allows "two focus 
excitation". 
More specifically, the present invention provides an optical arrangement 
for flow cytometer, wherein intense light is focused by a microscope 
objective or similar lens having a numerical aperture NA.sub.1 onto a 
stream of cells carried by a laminar flow of water through the focal plane 
of said objective; and wherein another microscope lens is situated 
opposite to the objective, and with its optical axis and object plane 
coinciding with those of said objective, and with a numerical aperture, 
NA.sub.O, which is significantly larger than that of said objective; 
wherein said objective contains a circular central field stop in, or close 
to, its secondary focal plane, said field stop having a diameter 
corresponding to a numerical aperture, NA.sub.df, which is slightly larger 
than NA.sub.i, while it is much less than NA.sub.O, so that the 
illumination field of the objective falls entirely within said field stop, 
and hence so that the image of the stream of cells created by the 
objective contains only fluorescence and scattered light from the stream 
of cells; wherein the fluorescence and scattered light from the stream of 
cells are separated by a dichroic mirror on basis of their different 
wavelength, so that the fluorescence and scattered light give rise to 
separate images of the stream of cells in separate image planes of the 
objective; and wherein a telescope, situated immediately behind the image 
plane creates an image of said field stop in a plane, where is situated 
two concentric mirrors, of different diameter, which separate light 
scattered from the stream of cells to different scattering angles and 
direct the scattered light of different scattering angles onto separate 
light detectors. 
According to a further feature of the invention, the stream of cells 
coincides with the object plane of the objectives, and the stream of cells 
is illuminated through the objective in one or two adjacent foci of 
different wavelength emitted by two separate light sources. 
According to another feature of the invention first and second slits may 
cover the image of each of the adjacent foci from said light sources in 
the object plane, so that fluorescence measured behind the first slit 
originates from only one of said foci whereas fluorescence measured behind 
the second slit originates only from the other of the foci. 
According to yet another feature of the invention, the mirrors in the image 
plane of the telescope are flat, polished end planes that are cut at an 
angle of 45.degree. of two concentric tubes having their common axis 
coinsiding with the optical axis of the telescope.

DETAILED DESCRIPTION 
The invention, shown schematically in FIGS. 1, 2 and 3, is a device which 
contains a light source 1 which, through a lens 2, illuminates an 
excitation slit 3, which is situated in the image plane 4 of a microscope 
objective or similar lens 5 which concentrates the excitation light from 
the light source 1 in an excitation focus 6 in the object plane 7 of the 
objective 5. An interference band filter 8 is situated in the light path 
behind the objective 5 in order to isolate the appropriate wavelength of 
excitation. 
The device can also include a secondary light source 9 which, through a 
lens 10, illuminates an excitation slit 11. An image of this slit 11 is 
formed by the lens 5 in the image plane 7 via a dichroic mirror 12. An 
interference filter 13 isolates a band of excitation wavelength, 
preferably not overlapping that of the band filter 8. The excitation slits 
3 and 11 are situated so that their images in the object plane 7 do not 
overlap, but are closely adjacent on each side of the optical axis 14 of 
the lens 5. 
The sample stream, containing cells or other microscopical particles to be 
measured, is conducted by the measuring chamber 15 in the object plane 7 
through the optical axis 14 of the lens 5. 
Another microscope objective 16, preferably of the oil immersion type with 
a numerical aperture of approximately NA=1,3, is situated opposite the 
lens 5 so that the two objectives 5 and 16 have their respective optical 
axis 14 and respective object plane 7 coinciding. 
Inside said objective 16 is a central, circular field stop 17, with its 
center in the optical axis 14 and in a plane which is close to the back 
focal plane of the objective 16. The field stop 17 covers the central part 
of the aperture of the objective 16, thus stopping light falling within a 
solid angle corresponding to a numerical aperture, NA.sub.df, which is 
just slightly larger than the numerical aperture, NA.sub.i, of the lens 5. 
Hence, excitation light focused onto the object plane 7 by the lens 5 is 
not transmitted by the objective 16. Consequently, the light collected by 
the objective 16 will contain only fluorescence and scattered light from 
the sample stream through the measuring chamber 15. Behind the objective 
16 is situated a dichroic mirror 18 with a characteristic wavelength so 
that said scattered light is reflected to form an image of said sample 
stream in an image plane 19 of the objective 16, whereas the fluorescence 
is transmitted to form a corresponding image in the image plane 20 of the 
objective 16. 
Behind the image plane 19 is a telescope 21 which forms an image, as shown 
in FIG. 2, of the plane containing the field stop 17 in a plane 23. 
Outside the dark field 22, which is the image of the field stop 17, is 
light scattered from cells in the sample stream. It will be understood 
that light falling at a given distance, r, from the center of the image in 
the plane 23 is emitted with scattering angles exceeding a certain limit, 
.alpha..sub.1 (Eq. 1) and below an upper limit, .alpha..sub.2 (Eq. 2). 
EQU .alpha..sub.1 .apprxeq.arcsin (r/r.sub.O)(NA.sub.df /n)!-arcsin(NA.sub.i 
/n) Eq.(1) 
EQU .alpha..sub.2 .apprxeq.arcsin (NA.sub.O +NA.sub.i)/n! Eq.(2) 
where n is the refractive index of the sample stream, usually water, and 
r.sub.O the radius of the image 22 of said field stop 17, as determined by 
the magnification of the telescope 21. 
It can be seen that the lowest scattering angle which can be detected in 
the image plane 23, that is, at the periphery of the image 22 of the field 
stop 17 where r=r.sub.O, is given by: 
EQU .alpha..sub.1 (min).apprxeq.arcsin(NA.sub.df /n-arcsin(NA.sub.i /n) Eq.(3) 
The largest scattering angle that can be detected, i.e. at the outer 
periphery of the image (FIG. 2) in the image plane 23, where: 
EQU r=r(max)=r.sub.O (NA.sub.O /NA.sub.df) Eq.(4) 
is given by: 
EQU .alpha..sub.1 (max)=arcsin(NA.sub.O /n)-arcsin(NA.sub.i /n) Eq.(5) 
The theory of light scattering from microscopical particles as well as 
experimental data on this phenomenon shows that the intensity of the 
scattered light falls off very rapidly with increasing scattering angle 
over the entire range from 0 to about 60.degree.. Hence, a light 
scattering signal collected over a certain range of scattering angles will 
be strongly dominated by scattering from angles close to the lower limit 
of this range. Thus, a light scattering signal collected just outside the 
periphery of the image 22 of the field stop 17, to a good approximation 
will represent low scattering angles, that is angles just above 
.alpha..sub.1 (min); whereas light collected close to the outer periphery 
contains only light from large scattering angles, that is, upwards from 
about .alpha..sub.1 (max). 
A suitable value for NA.sub.i is 0,60, whereas NA.sub.df =0,62 and NA.sub.O 
=1,3. According to Eqs. 3 and 5, these values give: .alpha..sub.1 
(min)=0,97.degree. and .alpha..sub.1 (max)=51.degree.. 
The two light scattering components representing low and large scattering 
angles, respectively, are directed onto separate light detectors by means 
of two concentric mirrors 24 and 25 (FIG. 3) formed by the plane, polished 
front surfaces of two cylindrical tubes which are cut at 45.degree. to 
their axis and which are coaxial with the optical axis of the telescope 
21. Said mirrors 24 and 25 face in opposite directions, as shown in FIG. 
3. The inner tube 26 has an inner diameter equal to r.sub.O, while the 
inner diameter of the outer tube 27 is a little less than r.sub.max. Said 
mirrors 24 and 25 both have their center in the image plane 23 The outer 
tube 27 has an opening 28 in that side which is facing the mirror 24, so 
that the light reflected by the mirror 24 can pass through the opening 28 
and through a lens 29 to reach the detector 30. The light reflected from 
the mirror 25 is directed through a lens 31 onto a detector 32. 
Between the dichroic mirror 18 and the image plane 20 is another dichroic 
mirror 33 which directs certain wavelengths of fluorescence, usually 
shorter wavelengths, to form an image from the objective 16 in the plane 
34, whereas fluorescence of other wavelengths, usually longer, is 
transmitted to form an image in the plane 20. Thus, the device exhibits 
three separate image planes 19, 20 and 24 for the objective 16, wherein 
the same image is formed in three different regions of wavelength. In each 
of the image planes 19, 20, and 24 is situated a rectangular slit, the 
size of which can be varied so as to match the size of the image of the 
illuminated part of the stream of cells in the flow chamber 15 in order to 
eliminate light from other parts of the object plane 7 and thereby 
suppress background light which otherwise reduces the signal to noise 
ratio of the light detection and thereby the sensitivity. 
Dichroic mirrors 38 and 39 and optical hand filters 40, 41, 42 and 43 are 
situated behind the slits 36 and 37 in order to separate different 
spectral components of the fluorescence and direct these spectral 
components onto separate detectors 44, 45, 46 and 47. 
The dichroic mirror 18 is chosen so as to separate the scattered light, 
which is reflected, from the fluorescence which is transmitted because of 
its longer wavelength. The dichroic mirror 33 separates the fluorescence 
into two different spectral components, each of which is further separated 
by the dichroic mirrors 38 and 39. Thus, the present device can measure 
four different fluorescence components. This method of separating 
different spectral components of fluorescence is well known from the 
literature, e.g. "Flow cytometry and sorting", Melamed et al, Wiley-Liss, 
New York 1990. It is trivial to increase the number of fluorescence 
spectral components further by the addition of more dichroic mirrors and 
band filters. 
An important feature of the invention is that it facilitates so-called "two 
focus excitation". Light from two separate light sources 1 and 9 is passed 
through different band pass filters 8 and 13 which transmit two different 
spectral bands of excitation light. The optical axis of these two spectral 
bands are somewhat shifted relative to each other so that the objective 5 
forms two adjacent excitation foci in the object plane 7. Hence, the cells 
will pass sequentially through the two excitation foci. The slits 36 and 
37 are situated so that they cover the image of each of the two excitation 
foci. Hence, the fluorescence emitted from each of said excitation foci is 
separated from each other and can thus be measured by separate detectors. 
In the case that such "two focus excitation" is employed, one of the 
fluorescence detectors, for example 44 or 47 may be used to measure the 
scattered light from cells excited in that of the excitation loci which 
has the largest excitation wavelength. The invention thus facilitates 
measurement of scattered light at two different wavelengths and may 
thereby provide further information about the cells that are being 
measured.