Note: Descriptions are shown in the official language in which they were submitted.
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This application is a division of our Canadian Patent Application
Serial No. 289,221 filed October 21, 1977.
This invention relates to fluorometric systems for the detection
of sample substances derived from biological fluid or tissue tagged with
fluorochromes and to fluorometers adapted for more accurate measurements of
surface mounted fluorescent samples. It is particularly useEul in the de-
tection of hormones, enzymes~ drugs and ot:her substances.
Most infectious diseases of bacterial or viral nature produce anti-
bodies in the blood serwn of the subject. This provides a degree of immunity
against future assaults by the identical infectious agent or antigen. Gne
method for detecting the presence of a particular antigen is to add to it a
specific antibody which binds to the antigen. If the antibody has been pre-
viously tagged with a radioactive element ~RIA technique) or a fluorescent
dye, which does not interfere with its immunological properties, the coupled
comple can be detected by an appropriate detector; and~ in the case of the
fluorescent additive, can be at best semiquantitatively measured~ as is done
in cases in the prior art on a microscope slide for visual inspection.
The~e are many reasons why RIA is not completely satisfactory.
For example, in the presence of small quantities of antigen, only few counts
per second Carl be detected. Since the "noise" of the system is proportional
to the square root of the signal count, large errors in accuracy are made at
low signal levels. Furthermore, radioisotopes have a limited shelf life due
to half-life decay, and require special licensing, handling and disposal.
Testing which relies on fluorescence tagging techniques, as hereto-
~oxe kno~n, has been qualitative, or at best, semiquantitative as an assay.
~n the area of the lar~est diagnostic use of fluorometry (i.e., immunofluor-
escence microscopy in which saDples are typically mounted on a microscope
sta~e and illuminated with an exciting wavelength~ the fluorescence is
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observed through the stage with appropriate filters inte-rposed to select the
wavelength to be observed. Typically, the resultant observation i5 recorded
by a laboratory technician as a comparative degree of fluorescence, for ex-
ample 0, ~ 3, or ~ by comparison to known reference concentrations.
In some instances, where blood ~itre or concentration of antibodies is the
desired unknown, the technician prepares a number of slides; on each is a
differen~ concentration of the test material. Thus, the technician may est-
imate a +4 reaction in the microscope when the blood seruTn or the bacteria
broth medium was diluted 1:4 in distilled water, or 1:16, or 1:128, etc. It
would be of great advantage to medical and clinical authorities if a fluoro-
meter could automatically and quantitatively read titre or concentration
quickly and accurately, without the necessity of making serial dilutions.
In liquid scanning fluorometers, a cuvette usually holds a liquid
containing the substance to be analized and through which excitation ligh~ is
passed and fluorescence observed in a right angle configuration. It has been
found that these systems are unsuitable for the present applications primar-
ily because liquid systems and cuvettes themselves, apart from the sample
being investigated all contribute very subst~ntial background fluorescence,
so that, unless a very high degree of careful chemical separation is utili~ed
together with extremely well-controlled materials selected to have low fluor-
escence in the wavelengths of interest, such systems are unsuitable. Attemp~s
to adapt these instruments for surface measurements have not been particularly
successful. Such systems are typically inef~icient and have not provided for
the discrete handling of individual samples. In general, pas~ fluorome~ers
and RIA syste]Ds have been unduly sensitive to background and non-sample oriented
si~nals. There is, therefore~ a need for a new and improved fluorometric
system.
In general it is an object of the present invention to provide a
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fluorometric system and method which will overcome the above limitations
and disadvantages. Another object of the invention is to provide a fluoro-
metric system of the above character which is quantitative, which is easily
calibrated and which is arranged to e~ficiently operate within acceptable
limits relating to photo-bleaching and fading. Another object of the in-
vention is to provide an improved optical fluorometer particularly adapted
to read samples disposed on a carrier havLng and presenting a free sample
surface thereon, and in which the optical system receives fluorometric data
solely from the sample area of said carrier. Another object of the invent-
ion is to provide both fiber-optic and lens optical fluorometric systems for
carrying out the invention.
The fluorometric system of the present invention measures a sample
substance coated over a surface on a solid substrate. It includes a source
of light filtered to selectively excite fluorescence in the sample and light-
conducting means for conducting light from the source to the sample. An
emitted light detector captures and determines the intensity of fluorescence
emitted from the sample substance by converting the fluorescent light in-
tensity to an electrical signal by a photodetector. Fluorescence is conveyed
from the sample area to the photodetector by suitable light collection optics
terminating proximally adjacent the sample and distally adjacent to the photo-
detector. The optical parameters of the various elements interposed between
the sample and photodetector define a light path which is constructed and
arranged in accordance with this invention to prevent excessive loss of fluor-
escence ~i.e.S cumulative loss is held less than 95%). ThusJ the disclosed
light collection optics Csuch as fiber optical cables or lenses) must be con-
st~ucted ~Yith llmited gap distances at each end and with a large collection
aperture in order to enable efficiency of this level to be obtained over the
e~ission band peak.
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An additional and relate(l criteria is -that the foregoing must be
achieved within the constraint that the input light intensity shall be held
to a value less than causes photo-bleaching or photo-disassociation resulting
in a fading rate of one ~1~) percent per minute. By achieving specifications
that meet the foregoing, a very useful and accurate instrument is disclosed.
The coated substrate to be viewed in the fluorometer may comprise
a single sample coating on a body. Alternatively7 a body adapted to enable
detection and determination of more than one sample substance may include
multiple spaced coating areas (e.g., bands) of different substances. A
single coated area may include diEferent substances in random dispersion
tagged with different fluorochromes.
In one embodiment, the flllorometric system includes a branched
fiber optical cable for conducting light from the source to the sample and
for conducting em;tted fluorescent light from the sample to the detector.
One branch conducts light from the source to the sample and the other con-
ducts the fluorescent light to the detector. These branches meet in a
common fiber bundle terminating at the sample end. In this manner, the
area of coincident excitation and emission is maximized at extremely close
gap distances and optical efficiency is obtained.
Another advantageous fiber optical system includes at least two
fiber optical cables for conducting the emitted light to the detector and
means for alternating the input to the detector between the cables. This
system can read at least two coated areas on a single substrate without
movement of the substrate as in a comparison between a standard quantity
of sample and one or more unknown samples. Similarly, both the light con-
ducting means to excite :Eluorescence and to receive fluorescence comprise
branched optical cables for transmitting multiple wavelengths of light to
the sample and receiving different fluorescent signals.
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A thircl preEerred cmbodiment disclosed herein uses lens systems
intersecting a removable stage forming an analysis test station. The stage
is adapted to receive a member having a surface forming a carrier to a fluo-
rometrically active sample substance adhered thereto and presenting a free
surface for examination. The member carrying the sample is constructed and
arranged for insertion and removal from the stage independently thereof and
is adapted to form a light-tight arrangement with the stage so as to exclude
background light from the optical systems. In addition, the stage and carrier
member together with the associated optical components and housing serve also
to form an enclosure for the sample contained therein which enclosure avoids
the circulation of ambient atmosphere (i.e., usually air). In this preferred
embodiment the surface portion is arranged horizontally and includes a slight
depression so that the sample may be disposed thereon in liquid form and so
remain to present a free liquid surface for exposure and examination during
the analysis. By means of the foregoing, the sample is maintained quiescent
in movement and in a stable~ low evaporative environment for analysis. As in
the previous embodiment, the optical systems maintain the excitation light
intensity and collection efficiencies within the limits defined by the present
invention, as will be more clear from the detailed description herein.
In each of the foregoing embodiments filters are utilized for de-
fining the input and excitation wavelength band and for establishing a band
width of sensitivity in the detection system. For optimum performance, these
filters have been found to be very critical and detailed specification for
their selection will be given.
According to the broadest aspect of the invention, there is provi-
ded, in a fluorometric testing apparatus in which a sample is tested by il-
luminating the same with excitation radiation of à~predetermined frequency
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band which 1~ absorbs and re-emits in a second predetermined frequency band,
said f;.rst frequency band being of higher energy than said second band,
emission filter means associated with a ~luorescence collection system for
restricting the sensitivity thereof to a frequency band overlapping the
emission spectra of said fluorometrically active sample and being substantially
non-responsi.ve in the band of excitation radiation, said emission filter
means comprising a multi-cavity interference filter including an emission
frequency pass band filter in combination with a cut-on filter for ellminating
wavelengths shorter than said emission spectra and tr~lsmissive at frequencies
thereabove.
These and other objects and features of the present invention will
become apparent from the following detailed description of the preferred
embodiments when taken in conjunction wi.th the accompanying drawings, of
which:
; Figure 1 is a top, schematic view of one embodiment of a fluoro-
metric system constructed in accordance with the present invention using
fiber bundle optical systems;
Figure 2 is a cross-sectional view, in elevation, taken along the
lines 2-2 of Figure l;
Figure 3 is a schematic diagram of the photo-detector and associated
: circuitry for use in the embodiment of Figure l;
~: Figure 4, on the first sheet of drawings, is a schematic view of
another embodiment of the fluorometric system constructed in accordance with
the present invention utilizing a sample carrier in ~he form of a cylinder;
Figure 5 is a schematic view of an alternate sample member con-
figuration constructed in accordance with the present invention;
Figure 6 is a schematic view of another alte~nate sample member
configuration constructed in accordance with the present invention;
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Figure 7 is a graph relating concentration of fluorescent material
in sample (C) and intensity of detected signal CIo) as a function of effici-
ency of the optical collection system and as a function of input light in-
tensity;
Figure 8 is a schema~ic, isometric view of another preferred em-
bodiment of fluorometer constructed in accordance with the present invention,
shown with external portions in phantom lines;
Figure 9 is a detailed optical diagram of the fluorometer of Fig-
ure 8;
Figure 10 is a perspective view of a test stage assembly including
sample holder constructed in accordance with ~he present invention and part-
icularly adapted for use in the fluorometer of Figure 8;
Figure 11 is a cross-sectional view, in elevation, of the test
stage assembly of Figure 10;
Figure 12 is a cross-sectional view taken along the lines 12-12 of
Figure 11;
Figure 13 is a top plan view of the sample holder of Figure 10;
Figure 14 is a graph depicting performance curves of filters con-
structed in accordance with the present invention for use in the embodiment
of Pigures 8-13;
Pigure 15, on the third sheet of drawings, is a block diagram of
an electronic circuit for use in the fluorometer of Figures 8-14;
Figures 16 and 17 are schematic views similar to that of Figure 1
of modified foxms of fiber-optic fluorometers constructed in accordance with
: the present invention; and
: Pigure 18 is a cross-sectional ~iew of a fiboer-optic cable taken
along the lines 18-18 of ~igure 17.
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The present invention relates to a fluorometric sys~em and method
to quantitatively detect and measure a fluorescent sample substance coated
as a layer on a substrate. As defined herein, the term "fluorescent sample
su~stance" is one which includes a material derived from either a biological
fluid or tissue and which, alone or in combination with other materials in
a solid layer form, emits fluorescence upon excitation with a selected wave-
length of light in a solid layer form. Common fluorescent sample substances
include autofluorogenic material derived from a biological flwid (e.g., tet-
racycline), materials derived from such fluids tagged with fluorochrome
before or after isolation, materials derived from such fluids linked in the
layer with homologous fluorochrome-tagged materials ~e.g., antigen or an~i-
body, one of which has been tagged with a fluorochrome). The present descrip-
tion will Make particular reference to the last named substance.
Referring now to the embodiment of the invention as shown in Figures
1 and 2, there is provided a member 10 having a surface portion adapted for
forming a carrier for a fluorometric substance whi'ch is adhered thereto as a
surface coating. In the embodiment sho~n, this member is in the form of a
ball which is prepared, by way of example, in the following manner.
The ball may be about 10 mm in diameter and made of plastic, e.g.
nylon, and bears a dried film or coating 11 of an antibody related to the
antigen to be determined, e.g. to Australian antigen. Since coating of all
balls will be done at substantially the same termperature ~37C), and for
substantially the same incubation period ~30 minutes~, each ball will have
substantially the same amount of antibody on it, which is important for
quantitati~e results. The ball is typically on the order of 5 - 20 mm in
diameter so that an area at least one square mm is viewed by the fluorometer.
The ball is placed into a cuvette 12, in this instance of 12 mm inner diameter.
It is preferred that fluorescence measurements are ~ade in accordance with the
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presen~ inven~ion by viewing the surface to which the fluorometrically active
substance is adhered by optical systems which are very closely positioned
with respect to that surface so as to maintain a predetermined level of op-
tical efficiency. However> if fluorescence is to be detected and measured
with the ball in the cuvette, as shown in Figure 1, the cuvette must be form-
ed of a material, e.g. glass, which is non-fluorescing at the wavelength to
be measured.
Referring now particularly to Figure 2, an example is now given of
the prepa~ation of the ball, it being understood that this description is
specific for the sake of giving a detailed background on o-ne procedure for
such preparation. However, it is to be understood that it is given solely by
way of example and that many other procedures will be fo-~nd useful and in
accordance with the present invention. Examples of such procedures are given
in United States Patent application Serial No. 553,582, now United States
Patent 3,992,631, in greater detail in conjunction with many other illustrat-
ions and examples of the use of the fluorometric system set forth herein, the
content of which application is not deemed necessary for understanding the
invention as presented and claimed herein, and, accordingly, will not be
repeated.
The ball is placed in a cuvette 12. One ml. of serum 13 from a
patient or subject is added to cover the ball and the cuvette is gently rock-
ed for 5 minutes to obtain room temperature incubation. Australian antigen
14, if present, binds to ~he coating 11 of the antibody on the ball. A cap
16 having a hole 17 is placed on the open end 18 of the cuvette 12 and the
cuvette 12 is inverted, permitt m g the serum to run out. A small second
hold 20 tD pe~mit p2ssage of air is also provided in the cap 16. Suitably,
the cuvette 12 has a rounded or generally hemispherical inner surface 21 at
its bacie 22~ whereby the ball 10 is held in position and does not move or roll
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around ~uring the Eluorescence test.
After incubation with the subject serum, the cuvette is rinsed
out, e.g. with aqueous phosphate buffer or distilled water, which is then
also allowed to pour out of hold 17J the ball 10 remaining in the cuvette
and antibody solution tagged with a substance which fluoresces under ultra-
violet light. Such a fluorescent tag or label substance can be, e.g. sodium
fluorescein isothiocyanate or other suitable substance. However, sodium
fluorescein isothiocyanate, with excitation at ~60 nanometers and emission
at 520 nanometers, is advantageous. The ma-terial in the cuvette 12 is again
incubated as described above, the liquid poured off and the cuvette and ball
rinsed as before. The ball now bears the antigen and attached fluorescent
substance 23 where Australian antigen is present. The cuvette 12 and/or
member 10 are now ready for insertion into the fluorometer system.
Referring now to Figures 1 and 3 in the fluorometer system, the
member 10 and/or cuvette 12 are so placed that a fiber optical cable 2~
conducts ultraviolet or visible light from a light source 27, which can be
any desired source which includes the excitation wavelength. The light
then passes through a gelatin filter 28, which ensures that only light of
the exciting wavelength and excluding the emission wavelength reaches the
carrier surface on member 10 to excite fluorescence of the coupled substance
23. A second fiber optic cab]e 25 is disposed preferably at a small angle,
less than 30, from the cable 2~; and the emitted fluorescence passes through
; a gelatin filter 30, which ensures that only emitted fluorescence reaches
photodetector ~e.g., a photomultiplier) and associated circuitry 29 via
the fiber optic cable 25. The photodetector 29 converts the intensity of
the emitted fluorescent light into an electronic signal. This signal is
passed through an electronic filter 31 to a processor 31a (which converts the
AC signal to a DC signal, e.g. through a peak-to-peak detector and linearizes
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the relationship betw~en fluorescent light intensity and DC vvltage, as by a
Eour-step diode linearizer), an amplifier 32, and an analog-to-digital con-
verter 33 and then is displayed on a digital panel meter 34 calibrated direct-
ly in titre.
In the e~nbodiment shown in Figure 4, a nylon cylinder 35 is employed
as a carrier member 10 instead of the balL shape. The upper portion of the
cylinder 35 is coated with a standard fluorescent coating 36, i.e, of the
same fluorescent substance as is used to tag the antibody coating 38 of the
lower portion of the cylinder 35 and has a known titre as measured on the de-
tector devi.ce which is employed in the test or assay, in this instance, the
fluorometer described herein. A blank space 37 is left around the surface
of cylinder 35, separating the upper and lower coating, 36 and 38, respectively.
The lower coating 38 contains, for example, streptococcal fluorescent-tagged
antibody, being prepared in the same manner as described above with respect to
the member 10, except that only the lower portion is immersed in the body of
liquid serum to determine if any of the suspected antigen or antibody is
present in the serum being tested. In this embodiment, the ultraviolet or
visible light source 27, the fiber ~ptical cable 24, and the filter 23 are
again provided. Two fiber optic cables 39 and 47 are provided with respective
filters 39a and 47a. Cne such cable 39 conducts fluorescent light from the
standard fluorescent coating to the photomultiplier tube 29, and the other
- such cable 47 conducts emitted fluorescence from the lower coating 38 to the
photomultiplier tu~e 29. A chopper wheel 46 operated by a motor 45 rotates
and alternates the flow of light from each coating 36 and 38 to the tube 29.
In this manner, a direct co3~parison is obtained between the standard refer-
ence and the test poxtions.
In the em~odi~ent of Figure 5, the carrier member 10 comprises a
paddle-shaped body 40 having a handIe 41 at one end extending out of the
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cuvette 12, a stem ~2, and a wide~ El~-t head 43 at ~he other end, the head
~3 bearing a coating ~4 of sample. In this embodiment, the two fiber optic
cables 24 ~for excitation light) and 25 (for emitted fluorescent light) are
parallel to each other, or at an angle of 0 with respect to each other.
Conveniently, the two cables 24 and 25 can also be arranged as a coaxial
cable. The other elemen-ts of the device and system are as previously des-
cribed and shown.
Another embodiment of multiple test sample carrier body is shown
in ~igure 6. ~lere, the body is a cube 50 at the end of a handle 51. The
cube S0 can present four faces, two faces 52 and 53 being visible. Each face
has a different sample. Four different fluorescent tags can be provided, and
the fluorometer may have a filter wheel with four selected wavelength regions
to isolate energy going to the photodetector 29.
~he following description of Figures 7-14 relates to a commercially
developed fluorometer developed in accordance with the present inven~ion.
Before proceeding furkher, it will be helpful to consider certain limitations
and restraints which are placed upon the fluorometer of the present invention
in order to achieve the required performance levels.
In general, fluorescence from a sample may be expressed as follows:
~here I~. is fluorescent intensity; I~ is excitatio~ intensity;
is quantum efficiency; and E, B are geometric factors
IF = I~Cl-e~EBC)
For small concentration of C on a surface
-EBC = l-EBC
then
I~ = Ix~EBC
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I.et K = ~EB, a phys.ical c~nd geometrical constant for the substance and sys-tem,
then
Cl~ Ip = IXKC
Define IX = EiIS and Io = EoIF where Is is source intensity;
ID is intensity at detector; Ei is light transmissive efficiency from source
to sample; and Eo is light trc~nsmissive efficiency from sample to detector,
then
C2) D i o sC
Fluorescence fading occurs at high intensities
. IF (t) = KIxCe kt
where k = koIX and ko is a rate constant for decomposition
then
ID Ct~ = KEiEOIsCe o i 5k
For s~all ~alues of t ~short times)
-k E.I t
o 1 s
e = l-koEiIst
then
~ D Ct) EiEoI~CCl koEiEst~
: For a practical linut, fading should be less th~n 1% per minute and this
Yalue ls given predominantly;in that desirable laboratory procedure accomp-
lishment ti~e in one minute and that the largest amount of fading that is
acceptable is of the order of 1% per minute. Given these constraints, the
following equations hold:
~ iEOIsC = KE~iEoI5C- E o(EiEoIs)2C
and
O.01EiEoI = ko CE )2
O
0.01 q KoEiI
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~) O-O Is< 0.01
koEi
Substituting ~4~ into ~2~
~5~ ID = O.OlEo
ko
The variables of Equation 5 are illustrated in the graph of Pigure
7 wherein the line 100 represents the one (1%~ percent fading ra*e at 100%
efficiency ~Eo =1.0). Above and to the left of -this line, high powers of
input light intensity are required and are found to cause photo-bleaching
and fading at an unacceptable level and, therefore, cannot be used in this
invention.
~ith the optical systems disclosed herein, it is possible to achieve
efficiencies of the order of 5%, the 5% line being indicated at 101 for ill-
; ustration. The claimed operating domain 103 of the present invention liesbetween lines 100 and lOl. As shown, this domain extends down to the limit
where the signal and background become comparable in strength (S/N = 2.0 being
indicated at 102) at least to which satisfactory performance is provided by
the present invention. Below about 5% efficiency, it becomes impossible to
detect low concentrations of sample. It is also seen that the fading rate
limit and efficiency requirement are mutually interdependent and must both be
satisfied. Accordingly~ the use of the optical systems disclosed herein
achieves an efficiency of greater than 5% in the collection and detection
systems and the input light intensities are held to values such that the fad-
ing rate is less than one ~1%) percent per minute.
Referring now more particularly to Figure ~, there is sho~n such a
len~ type fluoro~etex, the overall out~ard appearance of which is shown in the
phantom lines. The optical system of the fluorometer is developed within c~n
opaque block 104 of plastic, as for example Delrin~selected because of its
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dimensional and thermal stability The block is machined to accept the var-
ious components as shown in the drawings. The details of the machining are
not believed essential to understanding the invention and accordingly, they
have been omitted For the sake of clarity.
~ eferring simultaneously to Figures 8 and 9, the system generally
consists of three major optical systems: an illumination optical system 105
for supplying excitation light to the sample; a collection optical system 106
for receiving fluorescent output from the sample; and a reterence level optic-
al system 107 for periodically establishing and checking zero and preset in-
tensity levels.
The illumination optical system 105 consists of lamp 110~ condens-
ing lens 111, excitation filter 112, chopper 113 and its lens system 114a
and 114b, as driven by an electric synchronous motor 115 and focusing lens
116 for imaging excitation light onto a test stage 150 including sample 118.
The collection optical system 106 consists of a collecting lens 120,
emission filter 121, and photodetector lens 123 which images the sample
member surface onto a photodetector 124.
The reference level optical system 107 ccnsists of a beam-splitter
130~ a turning mirror 131, chopper 113 and associated reference lens system
133, a diffusing screen 134, a portion of which is developed by lens 135 and
reinserted at beam-splitter 136 into the collection optical system 106. Ro-
tation of the chopper 113 causes light to pass alternately through the ill-
umination optical system 105 or through the reference optical system 107.
Thus, the output at the detector is an alternating signal during one period
of which the intensity of the excitation source is ~easured while the other
` period measure~ the ~luorescent output.
~any of the optical components shown and described herein can be
selected from a wide v~riety of a~ailable designs, but the following have
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333'7
been folmd to be particularly satisfactory for use in the
present inventionO By way of example, the lens 114a, 114b,
133a, 133b, 120 and 135 may be selected from the aspheric
type produced as standard products of Mells Griot~ product
Serial No. 01 LAG 005. Alternatively, these same lenses
may be aspheric lenses having a 24mm diameter and an 18mm
focal length produced under the designation as an aspheric
condensing lens, stock No. 17.1050 and available from Rolyn
Optical Co. of Arcadia, California. Lens 116 is plano-convex
with a 25mm diameter and 50nlm focal length; lenses 1]1 and
123 are plano-convex lenses with a 25mm diameter and a 25mm
focal length. Lens 120 is located approximately 0.60 inch
from the sample surface.
Figure 9 contains ray tracing lines thereon which
serve to illustrate the functions of the lenses and optical
components. Thus, lens 111 collimates the light from the
light source and passes it in parallel rays through the
first filter at 112 after which the beam splitter 130 divi-
des off a certain portion of the light to be delivered to
the reference optical system 107. The remainder passes to
the chopper lens pair 114 in the excita-tion optical system
105. The first lens of this pair 114a focuses the light
down to a small spot so that the cut-o~f and cut-on times
and general level of intensity of light exiting from the
chopper is well-defined in value over an appropriate period
of time. The second lens 114b o that pair collimates the
light for delivel~ to a focusing lens 116 whîch reduces
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an image of the filament down into a small focused spot on a central area of
the sample carrier at 118.
The outp~lt from the s~mple is collec-ted by planoaspheric lens 120
over a wide solid angle and delivered in parallel rays through the emission
filter 121, through a beam insertion device 136, and thence focused by de-
tector lens 123 onto the active element of the photodetector 12~.
The portion of the beam which is removed for -reference purposes is
turned by turning mirror 131 to follow a path parallel to c~ld alongside the
excitation path. This light is passed through lens pair 133 which also brings
the light source to a ocus in the plane of the chopper so as to also provide
for rapid turn-on, turn-offl and well-defined value of intensity when the
reference beam is on. The reference beam is then passed in parallel rays to
a diffusing screen which can consist of a film of polyester, such as that
made by DuPont ~Type A), having a dull matte finish on the side facing the
detection system 106. The matte finish serves as a light scattering function
and thus con~erts the film into a secondary or reference sourcs of high uni-
formity. A portion of the central area of the film is taken by lens 135 and
the rays made parallel for being passed to insertion mirror (partial) 136
lying in the collection stream path between the emission filter 121 and the
photodetector focusing lens 123.
Referring now more particularly to Figures 10-13, the sample carrier
stage 15Q is shown in detail. This stage is removable in its entirety to
facilitate cleaning. As will be noted from Figures 10-12, the stage is in-
serted into a cylindrical passageway in block 10~ at a lowermost positiGn
such that the carrier 118 containing the sample thereon faces upwardly and
in a suhstantially hori7ontal plane within the assembly as a whole. By mak-
ing this provision, together with certain structural features of the sample
car~ier, it is possible to insert and measure a sample ha~ing a substantially
:
3.333~
free liquid containing surface while simultaneously maintaining that surface
free of changes duTing analysis (such as by evaporation). The stage is rota-
tionally oriented for insertion into the optical block 104 by virtue o an
elongate axial slot 151 formed along one side of the stage and adapted to
accommodate a locating pin 152 mounted in the optical block. A similar loca-
ting pin 152 projecting in the elongate slot 151 indicates when the stage
has been brought to a fully inserted position within the block.
The sample carrier 118 is mounted on a removable sample carrie:r
member in the form of a spatula 154 ~shown inserted in Figures lO and 11 and
separately in Pigure 13) consist.ing of an end portion 155 having a subs-tanti-
ally planar surface 156 and an area of slight depression 157 formed at -that
end of the member on which carrier 118 is disposed to form a support for a
film or coating of sample. The spatula 154 further consists of an elongate
blade 158 having a registry projection 159 thereon and terminates at its other
end in a paddle 160 suitable for being easily gripped by the operator. The
spatula is easily removed and inserted into the stage, the latter normally re-
maining in place within the instrument. Thus, an elongate stop in the form of
slot 162 is provided within the stage for receiving the carrier, together with
an upwardly extending groove 163 which accommodates projection 159. When
fully home, the parts appear as shown in Figure 11. It will be further noted
that the outward extremity of the mounting recess 162 ~at 16~) is provided with
a converging wedge-shaped configuration so that when the spatu]a is fully seated,
: the end 166 thereof is urged downwardly against the floor of slot 162 into
precisely positioned contact within the stage. With the utilization of plas-
tic parts for many of the components, it is found preferable that these com-
ponents be restrained laterally and in every other dimension so that any
: : slight warpage of the plastic parts of which the carrier member is made is
comp~ns-ted for, the membel being urged into exact position ag~inst the bottom
.
.
33'~
of slot 162 within the analysis stage.
Consideration of the optical diagram with respect to thc ;Focusing
of light onto the stage and to the carrier spatula and the collection of
light therefrom as shown in ~igure 9 will explain the purpose for the relieved
portions 168, 169 provided by the removed portions o:F the end of stage 150
and laterally located adj acent the sample 118. Once positioned, me-mber 154
is maintained in position by virtue of urging contact made by a resiliently
mounted setting member, including a spring-loaded ball 170 carried in the
body 104 of the optical system and adapted to engage and urge the back in-
clined surface of projection 159 to urge the carrier member inwardly against
slot 162 for precise positioning. In additionl ball 170 falls behind that
back surface and assumes a position serving together with the projection it-
self to block light from passing through the channel 162. Such light is cap-
able, if not blocked, of causing non-dark background readings.
An aperture 172 is provided through the stage and passes light from
a small light-emitting diode 174 to a small light detector 173. This aperture
is closed by the passing of the member 154 as the same is pushed into the unit
and when closed provides a "ready" signal for the associated electronics. An
additional aperture 176 e~ctends from the region immediately below the sample
carrying end of member 154 and in general alignment with the excitation light
beam so that a photodetector 178 positioned immediately b~hind this second
aperture indicates when the sample is actually in position and blocking light
from the beam.
The carrier stage 150, even though removable together with its in-
dependently removable sample carrier member 154, nevertheless when assembled,
fo~s both a li,ghtti~ht enclosure of quiescent air within the optical assembly.
The former eli~ninates background light; the latter retards evaporation from
an aclueous fil~ oYer the free sa~ple surface at 118 during the measurement
- 19 --
3'~
period. The latter generally quiescent enclosure is defined and formed
b~ the carrier member 15~ itself which closes aperture 176, by the stage and
its close fitting relationship with the adjacent block, and by lenses 116 and
128, both of which are sealed into contact within bores supporting the respec-
tive excitation and collection optical systems.
In constructing the fluorometer in accordance with the present in-
vention, it is important that the emission filter design be carefully select-
ed, and that the excitation filter design compliment that of the emission
filter design. In the following discussion, excitation and emission filters
will be disclosed which are of substantially identical ~although compliment-
ary) structure and which are produced by interference layering techniques in
accordance with applicants' specifications and with particular reference to
application in the present invention. The examples will be given in connect-
ion with filters designed for use with fluoroscein dye which has an absorp-
tion frequency band at approximately 480 nm (blue) and an emission frequency
band at approximately 530nm ~green). This dye is widely used because of its
high quantum efficiency, i.e. about 80-90% of the input radiation that is
absorbed is re-emitted.
Referring now to Flgure 14 there is shown a series of curves which
illustrate the absorption and emission bands 180, 181 as well as the contours
of the characteristics of the fil~ers which are constructed in accordance with
the present invention. In general, it is desired to obtain a compromise where-
in the maximum aYailable input radiation is applied to the sample while ob-
taining the maximum output fluorescence emission without overlap or cross-
talk between the illumination and collection channels. Since the absorption
band 180 o fluo~escence and the emission band 181 oYerlap each other, there
are certain techniques which must be used to ob~ain optimum performance. In
general,~the present filters are characteri~ed as follows: a combination pass
- 20 -
,; ' ' ' ' ' ' ' ,
:
.
33'~
band~ plus cut-on Cor cut-ofQ , filters developed by 1nterEerence coatings
laid upon a single substrate. By dolng so, it is possible to obtain a high
transmission filter having very high rejection oE the adjacent channel.
Thus, the emission collection filter 121 includes a three-cavit~
pass band type having a wavelength of highest transmission at 540nm and
half-width of 16nm for 50% transmission. This is combined with a cut-on
filter having a through transmission of 80% at 550nm. 'L'he pass band curve
is shown at 182 while the cut-on curve of this filter is shown at 183 in
Figure 14 while the combination filter ta~en together is shown as curve 184.
The illumination filter 112 is similarly constructed with a pass band curve
having a center wavelength of 475nm with half-band width of 16nm, combined
with a cut-off filter having its 80% transmission at 465nm and incorporated
with the pass band filter on a single substrate to form a combination filter
of very satisfactory performance. The excitation pass band curve is shown
at 185, the cut-off curve is shown at 186 and the combination illumination
filter at curve 187. The following are the specific specifications of inter-
ference filters which were made to applicants' specifications and which are
available from Ditric Optics, Inc. of Marlboro, Massachusetts.
Illumination Interference Filter
CWL: 475nm ~ 3nm
HBW: 12 to 17nm
%T: > 50%
BLOCKING: > 4 O.D. outside of passband to
1200nm >6 O.D. from 520nm to
540nm
Collection (Emission) Interference Filter
C~L: 54Gnm + 3nm
HB~: 12 to 17nm
%T > 50%
BLOCK~NG: > ~ O.D. outside of passband to
1200nm > 6 O.D. from 460nm to
490nm
_ 21 -
- ~ ' '
,
~3~3'~
It will be noted that the crossband rejection is 6 O.D. or .0001%
crosstalk between the channels.
The foregoing filters are generally manufactured by a technique
known in interference filter manufacture clS a three-cavity technique and
may, of course, employ more cavities as tlle need may require. In general,
it is found that the foregoing design in displacing the spectra maxima of
the filters slightly away Erom each other and slightly oEf of the response
characteristics of the dye, nevertheless provides very satisfactory perfor-
mance. In addition~ the use of cut-on and cut-off filters on the substrate
with maximum transmissions of about 80% yields overall transmission of the
desired band of the order of 40-50%.
Referring now to Figure 15 there is shown electronic control cir-
~ cuitry which processes the output fluorometer of Figure 8 for providing
; an output reading therefrom.
In general, the control circuitry serves to time demultiplex the
output of the photo-multiplier tube by suitable circuitry in demultiplexer
190 driven by a synchronizing pulse from a light emitting diode/detector pair
192 positioned across the path of the chopper 113 to derive a sample channel
signal and a reference channel signal, the respective ones of which are pro-
cessed in a sample channel and reference channel respectively. The channels
include AC amplifiers 193a, 193b, demodulators 194a, 194b, and integrators
195a, 195b for developing DC s;gnals S and R, the magnitude of which is pro-
portional to the magnitude of signal strength as seen by the photodetector
124 when viewing the respective sample or reference channel. The DC signal
outputs are passed through respective DC amplifiers 195a, 195b for isolation
and applîed to a ratLo circuit, the outputs S~R of which is displayed on a
digital displa~ unit 198, and also is displayed digitally on the instrument's
front panel at 198 shown in Figure 8. ~y using this ratio technique, the
- 22 -
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: . . . . .
',. ",
, '' ' ' , ' : ' ' , .
: : ' - . .,
- , .
~ ,' ' .
333'~
stability of the unit is increased and electronic, photomultiplier and illu-
mination lamp output drift is minimized.
Figure 16 shows a modified form of fluorometer 260 of the fiber
optic type in which a single branched fiber optic cable 261 replaces the two
separate cables 224 and 225. A single-bundle portion 262 of the cable 261
leads to and away from a solid base 263 having a fluorescent surface 264.
One branch 265 of the cable 261 transmits light from a lamp 266 or other light
source and a suitable ("blue") filter 267 to the fluorescent surface 264. A
second branch 270 of the same cable 261 conducts the emitted fluorescence
from the surface 264 to a suitable ("green") filter 271 and thence through a
lens 272 to a solid state or photomultiplier type of detector 273. Operation
is basically the same as in Figure 1 with readily apparent differences.
Figure 17 shows another modified form of fluorometer of the fiber
optic type 280 in which branches 265 and 270 of fiber optic cable 261 replaced
with b-ranch fiber optic cables 281, 282, 283~ and 284, respectively. A single
bundled portion 286 of the cable leads to and away from a single base 287
having a fluorescent surface 288. Branches 281 and 282 transmit light from
lamps 289 and 290, respectively, or other light sources, to fluorescent sur-
faces 288. ~ranches 283 and 284 of the same cable 286 conduct the emitted
fluorescent from the surface 288 through suitable lenses 291, 292, respectively,
to solid state or photomultiplier type of detector 293 and 294, respectively.
Gne method for employing *he device of Figure 17 which is highly
advant~geous is to view surface 288 which includes a plurality of biologically
derived substances in random dispersion. Each of the substances is tagged
with a fluorochrome which emits fluorescence responsive to a different wave-
length of light. Thu5, lamps 289 and 290 emit the different fluorescence
exciting wavelengths while the multiple fluorescence is received simultaneously
by detectors 293 and 294 through light conducting branches 283 and 284,
~; - 23 -
~:
, .
- -
-: ~,. .', - .
.
', - :
',
33'i~
respectively. The multiple fluorochrome tagged substances in random dis-
persion may also be read using the single branched fiber optical cable of
Figure 16. In this instance, a single lamp or other lamp source replaces
lamps 289 and 290 and a plurality of filters are employed to provide the
proper wavelengths to excite the respective fluorochromes in the samples.
Similarly, the light-conducting cables 283 and 284 may be replaced with a
single cable and detectors 293 and 294 may be replaced with a single detector
so long as the wavelengths to which the detector is responsive is synchron-
ized to the selected fluorochrome to be excited.
Referring to Figure 18, a cross-sectional view of the common fiber
bundle 286 of the branched cable 280 is illustrated schematically in which the
fibers of the various branches are enlarged for viewing clarity. Such fibers
are schematically represented by a solid circle, an open circle, a circle
containing "x" and a circle containing "y". It is apparent that the four
different types of fibers in this particular arrangement are randomly disper-
sed. It may be desirable to accomplish a specific optical effect to arrange
them schematically as in concentric circles, not shown, or to use fibers of
different diameters. An important feature of the present fluorometric system
is the maximization of fluorescent light which is received from the sample in
accordance with the general criteria discussed in connection with Figure 7.
This is particularly important when the fluorescent substance is present at
very low concentrations and illumination is held to low values in order to
limit fading. In the fiber optic systems of Figures 16 and 17, this objec-
tive is accomplished by avoiding gap distances in the light path between the
fluorescent substance and the means for converting the light intensity into
an electrical signal for quantitative measurement. ~ith a fiber optic cable
conducting light from the light input end adjacent the sample to the con-
version means, such gaps include the distance between the light input end
- 24 -
.
~333~
and the s~n~lle substance and any distance bctween the optical cable and con-
version means.
It has been set forth for average fluorescence intensity, at a
minimum, the cumulative fluorescence loss across the above light path from
sample to detector should not be greater than 95% of the fluorescence avail-
able for transmisslon along that path. Such losses do not include losses
due to viewing only a portion of a fluorescent sample surface. To take this
into account, such loss is related only to the fluorescent light emitted
from the sample within an area defined by the light input end perimeter pro-
jected onto the sample surface. Although limiting the loss in fluorescenceto 95% across the total light path is a significant improvement over con-
ventional fluorometers, it is preferred to limit such loss to below 50 to
90%. At such loss levels, even minute quantities of sample may be detected
and determined quantitatively.
The above considerations deal primarily with fiber optic cables
and light pipes and the importance of their close proximity~ as expressed
by gap distance, to surfaces from which they receive and to which they de-
liver light. Light conducting systems may also contain such components as
lenses to collect and focus light, mirrors to reflect and redirect it, and
apertures through which light passes after dispersion. When components
such as these receive light from a surface that is radiating it into a hemis-
phere, the amount of such light they capture is approximately proportional
to that portion of pi steradians delined by the circumference of the area
they project on the hemispherical surface generated by a radius equal to the
gap distance between the light emitting surface and the component receiving
it.
Based upon the above relationship, the circumference which permits
loss of no greater than 95% of emitted fluorescence corresponds to one that
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33~7
will genera-te a solid angle no less than approxilnately 0.3 steradian. ~he
solid angle of non-c;rcular cross-section is defined as one generated by an
equivalent circular area.
A study of the fluorometric systems of the present invention
illustrate the percentage loss can be directly related to the ratio of the
gap distance between the sample and the effective diameter of the light
input end of the emitted light collection optical system. ~le term "effec-
tive diameter" means either the diameter of a light input end of circular
cross-section or the equivalent diameter of a non-circular cross-section.
0 This latter term may be approximated by reference to the formula:
area: ~d2
The effective diameter, d', of non-circular cross-section is defined as
4 x area
~ . Reference to the relationship of gap distance to effective dia-
meter is based upon the approximate relationship that intensity of fluor-
escence is inversely proportional to the square of the distance Erom the
fluorescent substance. This approximation does not take into account an
increase in capture accomplished by minimizing the angle of reflectance,
i.e. the angle between the light conducted to the fluorescent substance to
excite fluorescence and that received by the light input end of the detector
means. It has been found that this value is not as significant as the gap
distance. It is apparent that the effective diameter of the light input
end is significant since an increase in the area of that surface causes a
corresponding increase in light captured.
Using the above calculations, a gap distance adjacent the sample
which permits loss of no greater than approximately 95% of emitted fluor-
escence corresponds to a ratio of gap distance to effective diameter of the
light input end of no greater than about 5:1. Similar calculations may be
made to determine the theoretical ratio of other :Eluorescent loss percen-
- 26 -
~q~;~33i7
tages. It should be understood that this ratio is only an approximation.
The same Eormula applies to oti)er gap distances in the light path such as
between the fiber optical cable and the portion of the detector which con-
verts the light to an electrical signal and between any lenses and mirrors
which may be employed in the light path.
The branched fiber optical system oE F;gures 10-17 is particularly
effective in reducing to a minimum the gap distance which can be obtained
to minimize loss of emitted fluorescence. This is based upon the principle
that the only area of the fluorescent substance which can be received by the
detector is where the light transmitted to the substance for exciting fluor-
escence coincides with the viewing area of ~he light input end of the
detector. This can be accomplished with separate fiber optical cables as
in FIGURE 1 until a gap distance is reduced to relatively small values. As
this reduction occurs, the area of coincidence of totally separate light
exciting and light emitting cables continuously reduces. It is apparent
that this may be a limiting factor on the gap distance and consequently may
cause excessive fluorescence loss for a sample substance in extremely small
quantities. On the other hand~ the use of branched cables each including
a plurality of light transmitting fibers which terminate in a common fiber
bundle at the light input end enable the fluorometer to be disposed extremely
close to the fluorescent sample without lack of coincidence. The only limit
on this is when the gap distance approaches zero at which point the fine
fibers of the fiber bundle act like independent cables.
The common fiber bundle is particularly effective in embodiments
such as multiple branching of Figure 17. Cables with separate light input
and output ends for each of the branches of cable 280 would require a fairly
substantial gap distance to assure a sufficient area of coincidence.
Another technique to avoid loss of fluorescence is to maintain the
- 27 -
- . ' '
-
.
3~3'7
gap between the sample coating and light input end of the detector free ofsolid medium which prevents transmission of excessive quantities of fluor-
escent light. It has been found that glass or certain plastics (e.g.,
polystyrene) at moderate thicknesses of less than 0.05 inch causes a loss
of fluorescence less than 30%. Although it is preferable to avoid the
interposition of such a solid medium, such losses are acceptable if necessary
or convenient to the system. For example, in the embodiment schematically
illustrated in Figure 1, it may be convenient to employ a thin-walled
cuvette to retain a coated substrate of a spherical shape. If so, the
cuvette should be formed of a material which does not cause the loss of
the fluorescence in excess of 30%.
The above description makes reference to fiber optical cables
as one preferred light conducting means. It should be understood that
other optical conduits such as light pipes may also be employed in those
instances where the distance does not require the use of common fiber
optical bundles.
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