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CROSS REFERENCE TO RELATED APPLICATION
This application is related to an application entitled Method, System and
Reagents for DNA Sequencing filed June 12, 1987, Ser. No. 07/057,566 by
Prober et al.
FIELD OF THE INVENTION
This invention relates to a scanning fluorescent detection system and, more
particularly, to apparatus suitable for use with a fluorescence-based DNA
sequencer. This system is capable of distinguishing among similar
fluorophores with relatively low levels of emission. A unique arrangement
of a filter and fiber optic faceplate enables the system to monitor
signals from relatively large detection areas containing multiple sample
regions while still retaining the required optical characteristics of the
combined filters.
BACKGROUND OF THE INVENTION
DNA sequencing, i.e., determining the sequence or order of the nucleotides
or bases comprising the DNA, is one of the cornerstone analytical
techniques of modern molecular biology. The development of reliable
methods of sequencing has led to great advances in the understanding of
the organization of genetic information and has made possible the
manipulation of genetic material, i.e., genetic engineering.
These are currently two general methods for sequencing DNA: the
Maxam-Gilbert chemical degradation method [A. M. Maxam et al., Meth. in
Enzym. 65 499-599 (1980)] and the Sanger dideoxy chain terminatin method
(F. Sanger, et al., Proc. Nat. Acad. Sci. USA 74 5463-5467 (1977)]. A
common feature of these two techniques is the generation of four groups of
labeled DNA fragments, each group having a family of labeled DNA fragments
with each family containing fragments having differing numbers of
nucleotides. The population of fragments within these groups all end with
one of the four nucleotides or bases comprising the DNA. Both techniques
also utilize a radioactive isotope, such as .sup.32 P or .sup.35 S, as the
means for labeling the fragments. The primary difference between the
techniques is in the way the fragments are prepared.
In both methods, base sequence information which generally cannot be
directly determined by physical methods must be converted into
chain-length information which can be determined. This determination can
be accomplished through electrophoretic separation. Under denaturing
conditions (high temperature, urea present, etc.) short DNA fragments
migrate through the electrophoresis medium as stiff rods. If a gel is
employed for the electrophoresis, the DNA fragments will be sorted by size
and result in a DNA sequence determination with single-base resolution up
to several hundred bases.
The Sanger and Maxam-Gilbert methods for DNA sequencing are conceptually
elegant and efficacious but they are operationally difficult,
time-consuming, and often inaccurate. Many of the problems stem from the
fact that the single radioisotopic reporter cannot distinguish between
bases. The use of a single reporter to analyze the sequence of four bases
lends considerable complexity to the overall process. To determine a full
sequence, the four sets of fragments produced by either Maxam-Gilbert or
Sanger methodology are subjected to electrophoresis in four parallel
lanes. This results in the fragments being spacially resolved along the
length of the gel according to their size. The pattern of labeled
fragments is typically read by autoradiography which shows a continum of
bands distributed between four lanes often referred to as a sequencing
ladder. The ladder is read by visually observing the film and determining
the lane in which the next band occurs for each step on the ladder.
Thermally induced distortions in base mobility in the gel (this usually
appears as a "smile effect" across the gel) can lead to difficulties in
comparing the four lanes. These distortions often limit the number of
bases that can be read on a single gel.
Problems relating to use of a single radioisotopic reporter revolve around
its lack of sensitivity and the time required to evaluate a sample. The
long times required for autoradiographic imaging aling with the necessity
of using four parallel lanes force one into a "snapshot" mode of
visualization. Since one needs simultaneous spatial resolution of a large
number of bands one is forced to use large gels. This results in
additional problems. Large gels are difficult to handle and are slow to
run, adding even more time to the overall process.
Once the exposed image of the gel pattern is obtained, there is a problem
of visual interpretation. Conversion of a sequencing ladder into a base
sequence is a time-intensive, error prone process requiring the full
attention of a highly skilled operator. Some mechanical aids do exist but
the process of interpreting a sequence gel is still painstaking and slow.
Finally, the use of radioactive materials has health risks associated with
continued exposure over extended periods. Appropriate use of shielding and
disposal procedures imposes some control of exposure levels, but
elimination of isotope use would be highly desirable.
To solve these problems, efforts are underway to replace autoradiography
with some alternate, non-radiosotopic reporter/detection system using
fluorescence. DNA frequencies labeled with one or more fluorescent tags
(fluorescent dyes) and excited with an appropriate light source give
characteristic emissions from the tags which identify the fragments.
The use of a fluorescent tag as opposed to a radioisotopic level allows one
to specify a DNA fragment detection system that responds to the optical
parameters characterizing tag fluorescence. For example, the use of four
different fluorescent tags, distinguishable on the basis of some emission
characteristic (e.g., spectral distribution, life-time, polarization),
allows one to uniquely link a given tag with the sequencing fragments
associated with a given base. Once such a linkage is established, one can
then combine and resolve the fragments from a single sample and make the
base assignment directly on the basis of the chosen emission
characteristic. When electrophoresis is chosen as a separation means, for
example, a single sample containing DNA fragments with base-specific
fluorescent tags can be separated in a single gel lane.
The "real-time" nature of fluorescence detection allows one either to scan
in the electrophoresis direction a gel containing spatially resolved bands
(resolution in space) or to monitor at a single point on the gel and
detect bands of separated fragments as they pass in sequence through the
detection zone (resolution in time). Large gels are not necessarily
required to discriminate between the fragments when time resolution is
selected. Furthermore, a "real-time", single lane detection mode is very
amenable to fully automated base assignment and data transfer.
A known "real time" fluoresence-based DNA sequencing system developed by
the California Institute of Technology is disclosed in at least one
published patent application and at least two journal articles: L. M.
Smith, West German Pat. Appl. DE No. 3446635 Al (1985); L. M. Smith et
al., Nucleic Acids Research, 13 2399-2412 (1985) and L. M. Smith et al.,
Nature, 321: 674-679 (1986). This system employs four sets of DNA
sequencing fragments, each labeled with one of four specified fluorescent
dyes. Unfortunately, the fluorescence (emission) maxima are spread over a
large wavelength range (approximately 100 nm) to facilitate discrimination
among the four dyes, but, the absoption (excitation) maxima for the dyes
are comparably spread. This makes it difficult to efficiently excite all
four dyes with a single monochromatic source and adequately detect the
resulting emissions.
It would be preferable to use dyes with closely spaced absorption (and
corresponding emission) spectra, selected to enhance the excitation
efficiency. But such closely spaced spectra cause other difficulties.
Recalls that a real time detection system for DNA sequencing must be able
to distinguish between four different dye emission spectra in order to
identify the individual labeled fragments. The emissions are typically of
relatively low intensity. The detection system must have a high degree of
selectivity and sensitivity (better than 10 (-16) moles DNA per band), and
a means to minimize stray light and background noise, in order to meet
desired performance characteristics. The system must also be able to
monitor the detection area frequently enough to avoid missing any
fragments that may migrate past the detection window between scans.
In order to effectively utilize an electrophoresis gel, a typical DNA
sequencing experiment involves running multiple samples simultaneously in
parallel lanes of a slab gel. Therefore, an excitation/detection system
must also be able to monitor each lane of such a gel at essentially the
same time. A system must be capable of monitoring a detection zone which
spans the majority of the usable gel width. Typical sequencing gels have
lanes that are 4-5 mm wide with 1-2 mm spacing between lanes. Therefore,
in order to monitor a 10 lane gel, a detection system must excite and
detect emissions from a region typically as wide as 70 mm.
Another fluorescence detection system developed for similar applications,
is disclosed in a U.S. patent application Ser. No. 07/057,566 filed June
12, 1987 by Prober et al. This application discloses a system for
detecting the presence of fluorescent energy from different species,
typically dye-labeled DNA, following separation in time and/or space, and
identifying the species. A set of four labels are chosen such that all
four are efficently excited by a single source, yet have emission spectra
that are similar but distinguishable in wavelength. Since differential
perturbations in electrophoretic mobility of the attached DNA fragments
are small, any disturbance to this behavior is minimized by using four
tags that have similar molecular weights, shape and charge.
The scheme of Prober et al. provides for modulating and ratioing the
signals corresponding to the fluorescent energies in two different
wavelength ranges to obtain a resultant signal that determines the
identity of the species. A dichoroic filter, with a
transmission/reflection characteristic that various as a function of
wavelength, or two filters with passbands that vary as a function of
wavelength, effect the modulation. Two detectors are positioned
respectively to receive the transmitted and reflected emissions and
generate first and second signals that vary in different senses
corresponding to the intensities of each. Preferably the dichroic filter
characteristic has a relatively sharp transition from transmission to
reflection which occurs near the center of the species emission spectra.
This system overcomes many of the problems of Smith et al. and has the
ability to distinguish in real time between relatively small wavelength
differences in emission spectra, while maintaining a relatively high
degree of sensitivity. Further, the system delivers a high portion of the
usable light onto the two photometric detectors to maintain continuous
monitoring of the gel containing the fluorescent species.
Both of the systems described above operate a fixed light beam and fixed
detectors which together can monitor only a single point within the
monitoring region. In order to monitor more than one spacial position
(lane or lane position) of a gel, either the light beam must be scanned
while providing a means to detect the emissions from the dyes, or the gel
must by physically shifted while holding the beam fixed. The latter of the
two alternatives, moving the gel, is not always practical since a large
electrophoresis gel along with its associated buffer reservoirs are
physically cumbersome. The other alternative, moving the beam while the
gel is stationary, has its own problems since the detectors must remain
closely coupled to sources of emission to prevent the entry of stray light
and maximize collection of the emitted light.
One method of accomplishing this task is to physically move either of the
two previously discussed detection systems and their associated optics and
light beam so that several lanes in the gel are effectively scanned. This
type of system has the disadvantage of being mechanically complex while
introducing additional noise into the system. Reliability and the high
costs associated with this type of system would also be a concern.
Another known "scanning" detection system is discussed in U.S. Pat. No.
3,764,512 issued to Green et al. This system discloses a laser scanning
electrophoresis instrument and system for determining the electrokinetic
or zeta potential of dispersed particles in an aqueous solution. This
system utilizes a galvanometer mirror to scan a laser light beam across an
electrophoresis medium. The system is not capable of detecting multiple
samples moving perpendicular to the scanning motion of the beam.
Another scanning system is disclosed in U.S. Pat. No. 4,162,405, Chance et
al. which describes an apparatus for measuring the heterogeneity of oxygen
delivery to perfused and in situ organs. A laser is employed as a flying
spot scanning excitation source and uses two photodetectors to monitor the
emission signal and excitation wavelength light. Although an x-y scanner
is used to move the laser beam over the sample area, the total scanned
area is only 1 cm by 1 cm. (As mentioned earlier for a multiple sample DNA
sequencer, a sample area 7 times wider is needed).
To implement the schemes of Chance et al. for a large area, the detectors
must be either larger in size, or located further from the sample thus
diminishing the collection efficiency. Furthermore, the detection of
closely spaced emission spectra of relatively low light intensities in the
presence of a much more intense excitation source requires the selective
transmission properties offered by interference filters. In order to
monitor a relatively large spacial area, both large detectors and large
filters must be used. Unfortunately, large interference filters that
collect light even a large solid angle are subject to transmission
properties which vary with the angle of incidence of the light. Thus, when
placed close to the emission source, light impinging on the filter with an
angle of incidence greater than about 22 degrees can experience
significantly less rejection of the excitation light than light at normal
incidence. Consequently, if the filter subtends a relatively large solid
angle with respect to the source of emission, the overall excitation
wavelength rejection properties of the filter will be compromised due to
leakage of excitation light entering at the higher angles of incidence.
SUMMARY OF THE INVENTION
Many of the above noted problems of the prior art radiant energy detecting
systems are overcome by this invention which has particular application to
a DNA sequencing system. This invention finds use in a system for
detecting the presence of radiant energy from different species, typically
dye-labeled DNA, following separation in time and/or space, and
identifying the species, the system having first detection means
responsive to the radiant energy emitted by the species of generating a
first signal that varies in amplitude in a first sense as a function of
the nature of the species, second detection means responsive to the
radiant energy for generating a second signal that varies in amplitude in
a second sense different than the first sense as a function of the nature
of the species, and third means responsive to the first and second signals
for obtaining a third signal corresponding to the ratio of functions of
the first and second signals, the amplitude of the third signal being
indicative of the identity of each of the species.
The invention is an improvement of such system wherein the first and second
means each include: a detector having a large solid entrance angle
positioned adjacent to the species to receive radiant energy emitted from
the species, and a wavelength selective filter means positioned between
each respective detector and the species, each wavelength filter means
having transmission vs. wavelength characteristics that are complementary,
and wherein one of the first and second detection means includes a
transmission filter means for rejecting radiant energy incident on a
detector at an angle greater than a predetermined value.
Preferably, the species are excited by a beam of radiant energy from a
laser and the system includes means to separate molecules (typically
fragments of DNA) labelled with emitting species of materials. The
detection means are positioned on opposite sides of the region propagating
the laser beam of energy in which beam is swept across the separation
means to excite the species in sequence. The wavelength selective filters
have a transition in their transmission vs. wavelength characteristics
centered at about the middle of the species' radiant energy spectra. The
transmission filter has an extra mural absorber among plural optical
fibers positioned to have parallel generatrices transverse to the first
and second detectors.
This system is optically efficient and does not require the use of lenses
or other collection optics. It is capable of and does, by the use of
detectors having a wide entrance angle, view large areas capable of
accommodating plural electrophoresis lanes. These plural lanes are
sequentially and repetitively scanned. Because of these efficiencies, the
system can operate using low levels of emitted radiant energy. The only
moving part required in the system is an optical element which effects the
laser scanning. The use of the transmission filters and associated extra
mural absorbers substantially reduce extraneous light impinging on the
detector. The system is capable of detecting and distinguishing the
radiant energy emitted from plural sources that emit energy at different
but closely spaced wavelengths.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention may be more fully understood from the following detailed
description thereof taken in connection with accompanying drawings which
form a part of this application and in which:
FIG. 1 is an isometric view of the electrophoresis gel slab showing plural
sample wells and lanes;
FIG. 2 is a partial diagrammatic layout of a system constructed in
accordance with this invention for detecting the presence of radiant
energy from different sources that each emit energy at different but
closely spaced wavelengths;
FIG. 3 is a side elevation view of the detector/filter arrangement for
detecting the presence of radiant energy;
FIG. 4 is a graph depicting the complementary filter transmission
characteristics as a function of wavelength;
FIGS. 5A, 5B, and 5C are flow diagrams describing the routines and
subroutines used to obtain sequence of DNA fragments using the systems of
this invention; and
FIG. 6 is a pictorial view of a transmission filter utilizing optical
fibers.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The radiation from very closely spaced emission bands may be detected using
the system of this invention. These closely spaced emissions are produced
from preselected species which typically act as reporters and are
irreversibly bound to materials that are to be analyzed. Acceptable
reporters are generally one or more species chosen for their ability to
emit radiation over a narrow range of wavelengths, typically between a 50
and 100 nm range, preferably over a 20 to 50 nm range. Preferably, the
peak maxima should be spaced no closer than 2 nm. One reporter species may
be capable of emitting energy at more than one wavelength, depending upon
the manner of attachment to the materials of interest and the conditions
of analysis in the system. However, individual reporters with unique
emission characteristics in the system are more conventionally chosen to
emit radiation in the wavelength range to be detected. Since the preferred
form of the invention is directed to detecting reporter-labeled DNA
sequencing fragments, it will be described in that context. It is to be
understood, however, that the invention may be used to detect any light
emitting labelled samples and is particularly advantageous where the
emission radiation has closely spaced wavelengths. The invention may be
used to detect, for example, fluorescense, chemiluminescence, and the
like. Thus dye labelled DNA sequencing fragments are passed through an
electrophoresis apparatus for separation. For this purpose, as is
illustrated in FIGS. 1 and 2, the electrophoresis may be carried out by a
suitable slab 10 arrangement typically having a thickness of about 0.3 mm
and about 40 centimeters long and 15 centimeters wide. Other sizes may be
used as appropriate. This slab 10 has a suitable gel 11, typically 6%
polyacrylamide; sandwiched between glass or low fluorescing plastic
supports 12.
The slab 10 is typically placed in an upright position in a holder with the
upper end of the slab 10 extending through and into an upper container 16
holding a buffer 24 and downwardly into a second container 14 also holding
a buffer 18. The buffer solution could be any suitable buffer such as that
obtained from a solution consisting of 0.1M tris, 0.1M boric acid, and
0.05M Na.sub.2 EDTA, with a final pH of approximately 8.3. In this manner,
the buffer contacts the gel at either end of the slab in order to make
electrical contact therewith.
With this arrangement, a sample containing reporter dye-labeled DNA
fragments can be pipetted into cavities 15 that are created at the top of
the gel 11 and define separation lanes. The reservoir containers 14 and 16
are filled with buffer solutions 18 and 24. An electrical circuit is then
completed through the terminals 20 in reservoir containers 14 and 16. A
suitable electrical field (typically 50 volts/cm) is needed to obtain
separations for gels of this particular length and thickness. The positive
electrode is located at the lower end of the slab to cause the DNA
fragments to migrate downwardly. Under these conditions, as the fragments
migrate through the gel they are separated spatially into bands (not
shown).
These hands are detected by the system and apparatus of this invention as
they migrate downwardly in a detection zone 19 located near the bottom of
the slab 10. In this zone 19, the DNA fragments are irradiated by a laser
beam 32 of appropriate excitation wavelength and the different reporters
attached to the several fragments emit detectable radiation. Since the
reporters and their attachment to the DNA fragments are not the subject of
this invention, such will not be described in detail. However, an example
of appropriate reporters is described in the copending Prober et al patent
application.
Four fluorescent dyes were selected with emission maxima at 505, 512, 519,
and 526 nm. These maxima may tend to shift somewhat when in the
environment of gel electrophoresis. These emission characteristics were
created by the appropriate chemical group substitutions, such as methyl
groups, at specified loci in the parent compound
(9-carboxyethyl-6-hydroxy-3-oxo-3H-xanthene). Each of the four dyes
prepared have reactive carboxy groups provided by a sarcosinyl moiety
covalently bound to the 9-position of the parent compound, which are
capable of forming covalent attachments with amine groups in linking
moieties that join the dyes with selected nucleotides. Useful linking
moieties found are a group of alkynylamine derivatives which contain a
terminal amino group that can form covalent attachments with the dye
carboxy groups. A preferred linker is a 3-aminopropynyl derivative which
is covalently attached to the 5-position of uracil (T) or cytosine (C), or
to the 7-position of deazaguanine (d-G) or deazaadenine (d-A).
Appropriate linker-nucleotide derivatives for use in the system of this
invention were prepared with 2',3'-deoxyribonucleotides, which are known
to serve as DNA chain terminating substrates for DNA polymerases. It was
found that covalent attachment of the
aminopropynyl-2',3'-dideoxynucleotides to the fluorescent dyes in
appropriate combinations, did not substantially diminish the chain
terminating properties of the unsubstituted 2',3'-dideoxynucleotides. The
four dye-linker-dideoxynucleotides A,G,C,T selected are illustrated by the
structures:
##STR1##
They were found to serve as useful chain terminating substrates for reverse
transcriptase (avian myeloblastosis virus) in a modification of the
well-known Sanger DNA sequencing method. The classical Sanger method uses
a primer, DNA template, DNA polymerase I (Klenow fragment), three
unlabelled deoxynucleotides and one radiolabeled deoxynucleotide in each
of four reaction vessels that each contain one of four
2',3'-dideoxynucleotides, which correspond to the four DNA bases
(A,C,T,G).
Appropriate reaction conditions are created which allow the polymerase to
copy the template by adding nucleotides to the 3' end of the primer. A
multitude of reactions occur simultaneously on many primer copies to
produce DNA fragments of varying length which all contain the radiolabel
at appropriate nucleotides in each fragment, and which also irreversibly
terminate in one of the four dideoynucleotides. This set of fragments is
typically separated on a polyacrylamide slab electrophoresis gel in four
lanes, one lane corresponding to each of the four dideoxynucleotide
reaction mixtures. After the fragments have been separated, a photographic
film is placed on the gel, exposed under approprate conditions, and a DNA
sequence is inferred from reading the pattern of bands created by the
radiolabel on the film in order of their appearance in the four lanes from
the bottom of the gel.
The modifications permitted by using these dye-labelled terminators include
omitting the radiolabeled nucleotide and substituting the dye-labelled
chain terminators for the unlabeled 2',3'-dideoxynucleotides. Reaction
mixtures (actually a single reaction member can be used) will now contain
fragments which are labeled on their 3' ends with a fluorophore that
corresponds to each of four DNA bases. The reaction mixture(s) are
combined and electrophoretically separated. Sequence is inferred by the
order of appearance of bands being resolved in time or space that are
revealed by the presence of fluorescent radiation. Therefore, the
fluorescent dye-labelled dideoxynucleotides previously described are the
preferred sources of closely spaced emitted radiation to be detected in
the system and method of this invention. An alternative source of emitted
radiation which can also be useful in the system and method of this
invention are the fluorophores described in the Smith et al. application.
Their use would require selection of the appropriate laser frequencies and
wavelengths of the preselected filters.
The optical arrangement for irradiating the lanes of the electrophoresis
slab 10 is shown in FIG. 2. The system and apparatus of FIG. 2 may be used
with any fluorescent or other type system to distinguish between and
measure the intensity of closely spaced emission radiation bands. However,
it will be described by way of example of detecting the emissions from DNA
fragments labeled with the particular reporters (dyes) set forth in the
Prober et al. application, which application is incorporated herein by
reference. The dyes described in Prober et al. have peak emission
wavelengths of about 505, 512, 519, and 526 nm. It includes a laser 30
which is selected to provide an exciting beam of radiation 32, with a
specific wavelength determined as a function of the excitation wavelengths
of the fluorophores used. The specific source used with the dye
fluorophores disclosed in Prober et al. is an argon ion laser with a
wavelength of 488 nm and a 0.8 mm diameter light beam 32 operated at about
50 mW. The light beam 32 passes through an excitation filter 34 and is
then directed into scanning optics 36. The filter 34 is selected block out
any undesired excitation wavelengths that could otherwise interfere with
the detection process. However, for sufficiently spectrally pure lasers
this filter may be omitted.
The scanning optics 36 include a prism or mirror 38 mounted on a fixed
support (not shown), an astigmatic focusing lens 40, a second prism or
mirror 43, and a cylindrical optic support 44 all mounted to the shaft of
a stepping motor 46. The beam 32, upon entering the scanning optics 36 is
first directed downward by the prism 38 into the cylindrical opening of
the optical support 44 and through the focusing lens 40. Prism 38 serves
to direct the beam from the laser into the scanning optics 36 thus
facilitating convenient placement of the laser 30. The light beam, passing
through the focusing lens 40 is concentrated into an elliptical spot, in a
preferred case of about 0.2 mm.times.1--2 mm in cross-section. The focused
light beam 32 is directed through an exit aperture 42 by the second prism
43. The optic support 44 is mounted to the shaft of the stepping motor 46
such that by actuating the stepping motor 46, the lens 40 and the prism 43
are rotated to cause the light beam 32 to angularly scan, in a horizontal
plane perpendicular to the shaft axis and to the plane of the gel 11. This
light beam 32 is directed at the detection zone 19 of the electrophoresis
slab 10.
The light beam 32, upon entering the slab 10 excites the reporter material,
here fluorescent dye labelled DNA fragments, as they migrate through the
detection zone 19, causing them to fluoresce at wavelengths shifted from
the excitation wavelength. The peak emission wavelengths for the dyes
described in Prober et al. are about 505, 512,519, and 526 nm; however the
system is adaptable to discriminate wavelengths associated with other sets
of dyes with closely spaced emission bands. Furthermore, while a laser
source is preferred since it allows a minimum of extraneous filtering and
optics, other sources including a non-coherent source such as a xenon arc
lamp could be used.
To increase the total radiant energy emitted by the fluorescent species, a
reflective surface 50 (FIG. 3) can be positioned opposite from the
excitation source. In the preferred apparatus, a mirrored surface is
deposited directly onto the outside of the outer plate 12 which supports
or contains the gel. The excitation light 32 which is not absorbed by the
emitting species continues through the plate 12 and is reflected back
towards the species by surface 50 to provide essentially twice the amount
of excitation light. Additionally, the light given off by the fluorescent
fragments is emitted in all directions so that light directed towards the
reflective surface 50 is reflected also. The net increase in fluorescent
signal available for detection is approximately 4 times the amount
available without the reflective surface. The preferred method of
providing a reflective surface is accomplished by coating the outside of
the plate which supports the gel 10. Alternatively, a mirror could be
external to the glass but the increased number of interfaces that the
light passes through would cause additional undesirable scattered
excitation light. The radiant energy or light emitted by the fluorescent
species is collected by two suitably positioned upper and lower
photodetector modules 52 and 54, respectively. These detector modules can
be seen most clearly in FIG. 3 in which the details of their construction
is shown.
In accordance with this invention, the modules 52 and 54 are positioned
above and below the plane of scanning of the light beam. The modules are
light tight in such a way as to eliminate stray light not directly coming
from the excitation region. Each module comprises a photomultiplier tube
(PMT) 56 of conventional type having a wide entrance area. A suitable
photomultiplier tube is the Hamamatsu R1612. Each module 52, 54 also has a
separate wavelength selective filter 58 positioned between its PMT 56 and
the fluorescent species in the gel slab 10. The filters 58 preferably are
custom interference filters which may be obtained from Barr Associates in
Westford, MA, which have complementary transmission band characteristics
as shown in FIG. 4 and are positioned to be transverse (preferably
perpendicular on average) to the light 60 emitted from the species. | | |
