A sensitive frontal approach fluorometer which is suitable for measuring the fluorescence of samples in open top microtest wells and which has an optical system for (a) directing an exciting light downwardly into the well's open top to fluorescently excite the sample and (b) detecting the sample's emitted light which passes upwardly through the well's open top.

FIELD OF INVENTION 
This invention relates to fluorometers and is particularly concerned with a 
novel optical system for a fluorometer of the frontal approach type. The 
fluorometer of this invention is particularly suitable for measuring the 
fluorescence of substances in microtest wells (or microtiter wells, as 
they are also called) and other similar vessels. 
For purposes of this specification, a fluorometer of the frontal approach 
type is one in which the exciting light or radiation enters through the 
open top of a sample-holding vessel and in which the detected emitted 
light (resulting from fluorescent excitation of the material) exits 
through the vessel's open top. 
The term "light" as used in this specification refers to both non-visible 
light (e.g., ultraviolet light) and visible light, that is, light visible 
to the naked eye. 
BACKGROUND 
Prior to this invention various fluorometers have been used in a wide 
variety of applications for measuring the fluorescence of fluorescently 
excitable materials. For example, fluorometers are used in junction with 
fluorescent assays to detect and measure the quantities of immunological 
and non-immunological substances. 
In carrying out fluorescent assays, microtest plates (or microtitration 
plates, as they are also called) and strips of microtest wells are often 
used. Microtest plates are formed with a multiplicity of wells which are 
joined together in a molded one-piece structure for containing microliter 
quantities of fluid samples in liquid or solid form. Examples of a 
microtest plate and microtest wells in strip form are described in U.S. 
Pat. No. 4,154,795 which issued to A. C. Thorne on May 15, 1979. 
The use of microtest plates and microtest strips of wells in fluorescent 
and other types of assays offers several important advantages. First, they 
permit the mass preparation of a large number of test sample solutions at 
the same time. Second, they are more convenient to handle as compared with 
individual test tubes. Third, they can easily and inexpensively be washed. 
Fourth, they are inexpensive and disposable. Fifth, they are customarily 
formed from plastic materials which are not fragile like glass. Sixth, 
they can be made from a material having an ability to attract certain 
molecules such as protein molecules so that they can serve as a solid 
phase in an immunoassay. 
Some fluorometers are not sufficiently sensitive to measure the 
fluorescence of the small, microliter quantities of the relatively low 
fluorescent samples which are prepared with the microtest plate and strip 
equipment described above. Other fluorometers, while having sufficient 
sensititvity, are usually unsuitable for measuring the fluorescence of 
substances in microtest wells because they are designed to direct the 
exciting light and/or the sample's emitted light through a wall of the 
sample-holding vessel. As a result, the microtest plates and strips, which 
are customarily molded from plastic materials having a substantial level 
of native fluorescence, are excessively excited to produce spurious light 
emissions which interfere with and impair accurate measurements of the 
intensity of the light emitted by the fluorescently excited test sample 
itself. 
SUMMARY AND OBJECTS OF INVENTION 
With the foregoing in mind, the general aim and purpose of this invention 
is to provide a novel fluorometer which meets both of the foregoing 
requirements, namely high sensitivity and suitability for measuring the 
fluorescence of microliter quantities of test samples in microtest plate 
wells and similar vessels. 
A more specific object of this invention is to provide a frontal approach 
fluorometer with a novel optical system which utilizes a special focusing 
technique to provide for a highly efficient transfer of radiation energy 
from an excitation light source to the sample and also to provide for a 
highly efficient transfer of emitted light from the fluorescently excited 
sample to a detector. 
In accordance with this invention, the optical system comprises a pair of 
bi-convex lenses or double-convex lenses, as they are also called, one for 
transmitting the exciting light to the test sample, the other for 
transmitting the light emitted by the fluorescently excited material to a 
photodetector. 
On the exciting light side of the optical system, the distance of the light 
path followed by the exciting light between the exciting light source and 
the sample in the microtest well or other sample-holding vessel is set to 
equal a multitude (e.g., four times) the focal length of the lens used for 
transmitting the exciting light, and the lens for the exciting light is 
located at the midpoint of the exciting light's path. The light path 
distance between the exiting light source and the lens for the exciting 
light is therefore equal to twice the lens' focal length. Likewise, the 
light path distance between the exciting light's lens and the sample in 
the well is also equal to twice the lens' focal length. The image of the 
light source will therefore be focused sharply on the sample in the well 
to maximize the fluorescent excitation of the sample for a given intensity 
of the exciting light. A corresponding focusing technique is applied to 
the lens for the sample's emitted light. 
Thus, on the emitted light side of the optical system, the total length of 
the light path between the sample in the well and the fluorometer's 
photodetector is set to equal a multiple (e.g., four times) the focal 
length of the lens used for transmitting the sample's emitted light, and 
the lens for the emitted light is located at the midpoint of the light 
path followed by the emitted light. The light path distance between the 
lens for the emitted light and the sample in the well is therefore equal 
to twice the lens' focal length, while the light path distance between the 
lens for emitted light and the photodetector is also equal to twice the 
lens' focal length so that the full image of the sample is sharply focused 
on the photodetector to maximize the intensity of the light detected by 
the photo- detector. 
Because of the foregoing focusing conditions, the sensitivity of the 
optical system is significantly enhanced as compared with systems using 
unfocused light. 
According to another feature of this invention the total length of the 
light path for the exciting light and the total length of the light path 
for the emitted light are selectively and independently adjustable to 
compensate for imperfections in the lenses which cause the lenses' focal 
lengths to deviate from a design or ideal value. The length of the light 
path for the exciting light may be adjusted by adjusting the position of 
the object, namely the light source or an optical stop lying in front of 
the light source. On the emitted light side of the optical system, the 
light path for the emitted light is adjusted by adjusting the position of 
the photodetector. 
In addition to the foregoing, the optical system of this invention includes 
a novel mirror and mask arrangement which lies between the two lenses. 
This mirror and mask arrangement performs a number of important functions. 
First, it downwardly reflects the exciting light to cause it to enter the 
sample-holding well through the open top thereof. Second, it works in 
conjunction with the lens for the exciting light to direct the exciting 
light beam through the well's open top without striking the well's side 
wall or the surface regions around the open top of the well. Third, it 
reflects the sample's emitted light, which passes upwardly through the 
well's open top, to cause it to pass through the lens for the emitted 
light. Fourth, it keeps the sample's emitted light from entering the 
exciting light channel lying between the mirror arrangement and the 
exciting light source, and it also keeps the exciting light from entering 
the emitted light channel lying between the mirror arrangement and the 
photodetector. Finally, it reduces the amount of scattered radiation in 
the emitted light channel to reduce the noise signal level in the 
fluorometer's photodetector. 
Further objects of this invention will appear as the description proceeds 
in connection with the below-described drawings and the appended claims.

DETAILED DESCRIPTION 
Referring to the drawings and particularly to FIGS. 1 and 2, the 
fluorometer incorporating the principles of this invention comprises an 
outer cabinet 10, a support base 12, an optical system 14, and a movable 
carriage 16 for supporting a microtest plate 18. Cabinet 10 is preferably 
light tight. Base 12 supports carriage 16. 
Microtest plate 18 contains a multiplicity of open form. Plate 20 may be of 
the type shown in the previously top wells 20 for receiving and holding 
test samples in liquid mentioned U.S. Pat. No. 4,154,795 or it may be of 
the type shown in U.S. Pat. No. 3,356,462 which issued to N. M. Cooke et 
al on Dec. 5, 1967. The disclosures of these patents are incorporated into 
this specification by reference. 
Wells 20 are uniformly spaced apart in twelve parallel spaced apart rows of 
wells with eight wells in each row to provide the standard total of 96 
wells. Each of the wells 20 is formed with a cylindrical side wall and a 
suitable bottom wall. Wells 20 depend from a top wall 22 of the plate. 
The carriage 16 together with plate 18 and optical system 14 are all 
mounted in cabinet 10. Carriage 16 lies below the optical system 14 as 
shown. 
Referring now to FIGS. 3 and 4, optical system 14 comprises an elongated 
housing 24 of rectangular cross-section, a suitable source of exciting 
light or radiation such as an ultraviolet lamp 26, a suitable 
photodetector such as a photomultiplier 28, a pair of bi-convex lenses 30 
and 32, a first pair of filters 34 and 36, a second pair of filters 38 and 
40, and a mirror and optical mask assembly 42. 
As shown in FIG. 3, lens 30 is mounted in a suitable holder 44. Filters 34 
and 36 are mounted side by side in another suitable holder 46 and are 
releasably retained in place by a leaf spring 48. 
Lens 32 is also mounted in a suitable holder 50. Filters 38 and 40 are 
mounted side by side in another suitable holder 52 and are retained in 
place by a leaf spring 54. 
Holders 44, 46, 50 and 52 are all mounted in housing 24 as shown. Holder 46 
and filters 34 and 36 are removable as a unit through an opening in the 
top of housing 24. Similarly, holder 52 and filters 38 and 40 are also 
removable as a unit through another opening in the top wall of housing 24. 
Still referring to FIG. 3, lamp 26 is mounted exteriorly of housing 24 at 
one end thereof, and photomultiplier 28 is mounted in a holder 56 at the 
opposite end of housing 24. The mirror and mask assembly 42 is mounted in 
housing 24 centrally between the housing's opposite ends. As shown, mirror 
and mask assembly 42 comprises a pair of light-reflecting mirrors 60 and 
62 and a pair of optical masks 64 and 66. Masks 64 and 66 are rigidly 
mounted on a support frame 68 which in turn is mounted in housing 24. 
Mirrors 60 and 62 are mounted on a support member 70 which in connection 
72, which permits selective vertical adjustment of the turn is mounted on 
frame 68 by means of a screw and slot assembly of mirrors 60 and 62 and 
support member 70. 
The assembly of mirrors 60 and 62 defines a V-shaped configuration in which 
one of the mirrors forms one leg of the V-shaped configuration and the 
other mirror forms the other leg of the V-shaped configuration. Mirrors 60 
and 62 abut against each other at the apex of the V-shaped configuration. 
The angle included between mirrors 60 and 62 is preferably 90.degree.. 
Mirrors 60 and 62 are symmetrical about a vertical plane passing through 
the interface between the apex-defining, abutting edges of the mirrors. 
The apex defined by the abutting ends of the mirrors 60 and 62 is 
indicated at 110 and lies vertically above an aperture 76 which is formed 
through the bottom wall of housing 24 above plate 18 in carriage 16. The 
wall region defining aperture 76 constitutes an optical stop. 
Still referring to FIG. 3, a further optical stop 80 is mounted on the 
outer end of a support block 82 which is slidably received in the end of 
housing 24 adjacent to lamp 26. The optical stop 80 is formed with a 
central opening 84 lying along an axis which normally intersects the 
longitudinal axis of lamp 26. Aperture 84 is axially aligned with and 
opens into an enlarged aperture 88 which is formed through support block 
82. Optical stop 80 lies on the outer side of housing 24 between lamp 26 
and support block 82. Aperture 84 lies closely adjacent to lamp 26 as 
shown. The diameter of the support block's aperture 88 is substantially 
larger than that of aperture 82 to allow the rays of the exciting light 
passing through aperture 84 from lamp 26 to diverge in the manner shown in 
FIG. 4. 
Still referring to FIG. 3, the photomultiplier holder 56 is mounted on the 
outer end of another support block 90 which is slidably received in the 
end of housing 24 opposite from support block 82. The wall region of 
holder 56 abutting support block 90 defines another optical stop 92 having 
a central light-transmitting aperture 94. The inner end of aperture 94 
axially aligns with an enlarged aperture 96 which is formed through 
support block 90. Aperture 96 is sufficiently large in diameter to allow 
converging light rays from lens 32 to enter aperture 94 without being 
blocked. Aperture 94 to photomultiplier 28. 
Holder 56 is sufficiently large to cover the aperture 96 in support block 
90. Similarly, optical stop 80 is sufficiently large to cover the aperture 
88 in block 82 except for the opening provided by aperture 84. Housing 24 
is preferably light tight except for apertures 84 and 76. 
As shown in FIGS. 3 and 4, lens 30 and filters 34 and 36 are arranged 
between optical stop 80 and mirror 60 to form an exciting light channel 
100 for system 14. The principal axis or centerline of lens 30 is 
indicated at 102 in FIGS. 3 and 4 and axially aligns with apertures 84 and 
88. Lens 30 is positioned between optical stop 80 and filter 34. Lens 30 
lies closely adjacent to filter 34 so that the spacing between lens 30 and 
filter 34 is considerably smaller than the spacing between lens 30 and 
optical stop 80. Filter 36 is positioned between filter 34 and mirror 60 
and is spaced from mirror 60 as shown. Filter 36 may abut against filter 
34 as shown. 
Still referring to FIGS. 3 and 4, lens 32 and filters 38 and 40 are 
arranged between mirror 62 and optical stop 92 to form an emitted light 
channel 103 for system 14. The principal axis or centerline of lens 32 is 
indicated at 104 and axially aligns with the axes of apertures 94 and 96. 
In the illustrated embodiment, the principal axis 104 of lens 32 also 
axially aligns with the principal axis 102 of lens 30. 
Lens 32 is positioned between filter 40 and aperture 94 and lies closely 
adjacent to filter 40 so that the distance between lens 32 and filter 40 
is considerably smaller than the distance between lens 32 and aperture 94. 
As shown in FIG. 4, light emitted by lamp 26 passes through the optical 
stop's aperture 84. From there, the rays of the exciting light diverge to 
lens 30. These light rays are refracted by lens 30 so that the light rays 
passing beyond 34 and 36. In this embodiment, filters 34 and 36 pass just 
ultraviolet light, while rejecting all other wave lengths. 
The apex 110 of mirror assembly 60, 62 lies on the aligned principal axes 
102 and 104 of lenses 30 and 32. Mirrors 60 and 62 and masks 64 and 66 are 
symmetrically arranged about a vertical plane passing through the mirrors' 
apex 110 and containing the longitudinal axis of aperture 76. Mask 64 is 
located vertically below the reflecting surface of mirror 60 and has its 
upper edge lying just above the aligned principal axes of the lenses so 
that it lies just above the level of the apex 110. Accordingly, light 
passing through the lower half of lens 30 below the principal axis 102 
will be blocked by mask 64, thus preventing the exciting light from 
passing into the system's emitted light channel 103. 
Because of the foregoing arrangement of mirror 60 and mask 64, only the 
light passing through the upper half of lens 30 will strike and be 
reflected by mirror 60. The converging column of light striking mirror 60 
will be reflected downwardly at a small acute angle to a vertical plane 
because of the 45.degree. angle which the reflecting surface of mirror 60 
makes with the principal axis of lens 30. 
The column of excited light reflected by mirror 60 passes downwardly 
through aperture 76 and through the open top of one of the sample-holding 
wells 20 which is selectively positioned to lie vertically below aperture 
76 in alignment with the longitudinal axis of aperture 76. The column of 
exciting light entering well 20 strikes the test sample in the well. As a 
result, the fluorescently excitable substance or substances in the test 
sample will be fluorescently excited to emit light which passes upwardly 
through the open top of well 20 and through aperture 76 to strike the 
reflecting surface of mirror 62. The reflecting surface of mirror 62 
intersects the lenses' principal axes 102, 104 at a 45.degree. angle. 
Because of the angulation of the reflecting surface of mirror 62, the rays 
of the sample's emitted light striking mirror 62 will be reflected towards 
lens 32 and will diverge in the direction of lens 32 as shown in FIG. 4. 
The diverging rays of light reflected from mirror 62 pass through filters 
38 and 40 before arriving at lens 32. 
In the illustrated embodiment, filter 38 is designed to reject light in the 
ultraviolet range while passing all other wave lengths above the 
ultraviolet range. Filter 40 is of the band pass type for passing just one 
preselected wave length (or a narrow wave length band) of the emitted 
light passed by filter 38. The light wave length passed by filter 40 is 
selected to measure the fluorescence of light emitted by a particular 
substance of interest in the sample in well 20. 
Mask 66 is positioned vertically below the reflecting surface of mirror 62 
and has its upper edge lying just slightly above the level of the mirror 
apex 110. Mask 66 is positioned between mask 64 and the pack of filters 
38, 40 to block transmission of stray light at and below the aligned 
principal axes 102 and 104 of the lenses. Accordingly, the only light 
transmitted to lens 32 will lie above the aligned principal axes of the 
lenses. Mask 64 blocks the entry of emitted light and any stray light into 
the system's exciting light channel 100. 
The rays of the sample's emitted light entering lens 32 will be refracted 
by lens 32 such that the light rays leaving lens 32 will converge 
virtually to a point in the optical stop's aperture 94 which directs the 
sample's emitted light to photomultiplier 28 for measurement. 
Photomultiplier 28 measures the intensity of the sample's emitted light. 
The measured intensity of the emitted light in turn is a measure of the 
quantity of the fluorescently excited substance which produced the emitted 
light at the wave length passed by filter 40. 
Preferably, lenses 30 and 32 are the same and have equal focal lengths. 
In the illustrated embodiment, the object "seen" by lens 30 is the exciting 
light passing through the optical stop's aperture 84. The exciting light 
passing through aperture 84 represents the light source as viewed from 
lens 30. 
In accordance with this invention, the length of the path followed by the 
exciting light from aperture 84 to a desired image point or location in 
the sample-holding well 20 (as measured along the principal axis from 
aperture 84 to mirror 60 and from mirror 60 to well 20) is set to equal or 
at least substantially equal four times the design focal length of lens 
30. Lens 30 is positioned at the midpoint of this path. Because of this 
arrangement, the length of the foregoing path between aperture 84 and lens 
30 will be equal to twice the design focal length of lens 30. Likewise, 
the length of the foregoing path between lens 30 and desired image 
location in well 20 is also equal to twice the design focal length of lens 
30. This image location in well 20 is selected so that it lies at or at 
least closely at the surface of the sample in well 20. 
Accordingly, where the object (the light source) lies at spot f.sub.1 in 
aperture 84, the sharply focused image will appear at spot f.sub.2 
centrally in well 20 as shown in FIG. 4. 
Because of the optical system thus far described, the image of the exciting 
light will be sharply focused centrally in well 20 on the sample in well 
20 without causing the downwardly reflected exciting light column to 
strike the side wall of the well or the surface region of plate 18 around 
the open top of the targeted well. Fluorescent excitation of the sample 
will therefore be maximized for a given intensity of the exicting light to 
enhance or strengthen the light emitted by the fluorescently excited 
sample. In addition, fluorescent excitation of the microtest plate will be 
reduced by directing the downwardly reflected exciting column into well 20 
without striking the well's side wall or the top wall of plate 18. 
The length of the path travelled by the exciting light from the optical 
stop's aperture 84 to the image location in well 20 is selectively 
adjustable to compensate for imperfections in lens 30. Such imperfections 
cause small deviates in the lens' focal length from the design or ideal 
length. 
In the illustrated embodiment the foregoing adjustment is accomplished by 
selectively adjusting the position of optical stop 80 along the principal 
axis of lens 30. Any suitable means may be employed for adjusting the 
optical stop 80. 
For example, support block 82 may be releasably fixed in place by screws 
120 extending through horizontally elongated slots 122 in housing 24 and 
threaded into tapped bores in block 82 as shown in FIG. 2. If the image of 
the exciting light is not precisely focused on the desired location in 
well 20 after system 14 is assembled, screws 120 may be loosened to allow 
the assembly of block 82 and optical stop 80 to be shifted to a new 
position along the lens' principal axis where the image of the exciting 
light focuses more sharply at the desired location in well 20. 
The same focusing techniques used for the exciting channel 100 are applied 
to the emitted light channel 103. In particular, the length of the path 
followed by the emitted light from the sample in well 20 to a desired 
image location at the optical stop's aperture 94 (as measured along the 
principal axis from aperture 94 to mirror 62 and from mirror 62 to well 
20) is set to equal or at least substantially equal four times the design 
focal length of lens 32. Lens 32 is positioned at the midpoint of this 
path. Because of this arrangement, the length of the path between aperture 
94 and lens 32 will be equal to twice the design focal length of lens 32. 
Likewise, the length of the path between lens 32 and the sample in well 20 
is also equal to twice the design focal length of lens 32. Accordingly, 
where the object (the sample) lies at spot f.sub.3 in well 20 the sharply 
focused image of the sample will appear at spot f.sub.4 in the aperture 
94, all as shown in FIG. 4. 
Because of the foregoing arrangement, substantially the full image of the 
fluorescently excited sample in well 20 will be sharply focused in 
aperture 94 and thus on photomultiplier 28 to maximize the intensity of 
the emitted light detected by the photomultiplier. 
Similar to support block 82, support block 90 also is mounted for selective 
adjustment along the principal axis of lens 32 by screws 124 extending 
through horizontally elongated slots 126 in housing 24 and threaded into 
tapped bores in block 90. If the emitted light lens 32 is not precisely 
focused on the desired location in well 20, screws 124 may be loosened to 
allow the assembly of block 90, photomultiplier holder 92 and 
photomultiplier 28 to be shifted as a unit along the principal axis of 
lens 32 to a new position where the image of the emitted light focuses 
more sharply on photomultiplier tube 28. In checking for the focus for 
lens 32, the photomultiplier tube 28 may be replaced by a lamp, thus 
becoming a light source type of object for lens 32 to provide for the 
focusing of the lamp's image in well 20. 
Although the focus adjustments described for channels 100 and 103 are 
advantageous for obtaining optimum focusing they are optional in the sense 
that satisfactory focusing can be achieved in the initial assembly of the 
component parts of the fluorometer. 
In summary, it will be appreciated that the downwardly reflected exciting 
light beam is directed and confined to strike the sample in well 20 
without striking the well's side wall or the top surface of plate 18. It 
also will be appreciated that photomultiplier 28 detects just those rays 
of the sample's emitted light passing upwardly through the open top of 
well 20. 
The fluorometer of this invention is therefore particularly suitable for 
measuring the fluorescence of substances in microtest wells making it 
unnecessary to transfer samples prepared in microtest plates or strips to 
special cuvettes or tubes for holding the samples during the fluorometric 
measurements. 
Where the sample-holding microtest plate or strip exhibits a substantial 
level of fluorescence when exposed to the exciting light in the 
fluorometer, it will be appreciated that a reference reading may be taken 
of the plate's native fluorescence to adjust the fluorometric measurements 
of the samples. Alternatively or additionally, the operator may use 
non-fluorescent or low-fluorescent microtest plates or strips of the type 
described in copending application Ser. No. 433,826 filed on even date 
herewith for Non-Fluorescent Vessels For Holding Test Samples In 
Fluorescent Assays and assigned to the assignee of the subject 
application. 
In view of the foregoing, it will be appreciated that a reference reading 
may be taken of the plate's native that the samples may be prepared in 
microtest plate 18 and that the plate may then be placed in the 
fluorometer of this invention for individually measuring the fluorescence 
of the samples in wells 20. Any suitable, conventional mechanism may be 
utilized for shifting carriage 16 in an X-Y plane to individually and 
sequentially target the wells 20 for fluorometric measurement to obtain 
separate fluorometric measurements of the samples in plate 18. 
Alternatively, it is evident that carriage 16 could be shifted manually to 
individually target the samples in plate 18. 
From the foregoing description it will be appreciated that lens 30 produces 
a spot image of the exciting light (as seen at perture 84) in well 20 at 
spot f.sub.2. The diameter of aperture 84 is such that the diameter of 
spot image produced in well 20 is nearly equal to or approaches the 
diameter of well 20 to excite a maximum area of the sample without causing 
the downwardly reflected, image-producing exciting light beam to strike 
the well's side wall of plate's top wall 22 before striking the sample in 
well 20. For a nonmagnifying lens and a 1/4 inch diameter well, the 
diameter of aperture may be lightly less than 1/4 inch. 
The diameter of aperture 94 is lightly smaller than the diameter of well 20 
or larger if lens 32 is of the type which magnifies the object. 
Finally, it will be appreciated that the downwardly reflected, converging 
beam or column of exciting light enters the open circular top of well 20 
at a small acute angle with well's vertically positioned longitudinal 
axis. 
The invention may be embodied in other specific forms without departing 
from the spirit or essential characteristics thereof. The present 
embodiment is therefore to be considered in all respects as illustrative 
and not restrictive, the scope of the invention being indicated by the 
appended claims rather than by the foregoing description, and all changes 
which come within the meaning and range of equivalency of the claims are 
therefore intended to be embraced therein.