U.S. patent number 3,887,812 [Application Number 05/492,501] was granted by the patent office on 1975-06-03 for method and apparatus for detecting and classifying nucleic acid particles.
This patent grant is currently assigned to Block Engineering, Inc.. Invention is credited to Tomas Hirschfeld.
United States Patent |
3,887,812 |
Hirschfeld |
June 3, 1975 |
Method and apparatus for detecting and classifying nucleic acid
particles
Abstract
Free floating viruses are detected and sized by a method which
combines fluorescent staining with the observation of a modulation
of the fluorescence intensity by Brownian motion of the particles
in combination with a particular spatial filter. Intensity
modulation and fluorescence data provides a set of descriptors
useful in distinguishing the type of virus involved, particularly
if also combined with data regarding the scattering of light by the
particles. The method and apparatus are also usable in connection
with detection and classification of other biological particles
having the same order of magnitude of size as viruses.
Inventors: |
Hirschfeld; Tomas (Framingham,
MA) |
Assignee: |
Block Engineering, Inc.
(Cambridge, MA)
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Family
ID: |
27007205 |
Appl.
No.: |
05/492,501 |
Filed: |
July 29, 1974 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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375807 |
Jul 2, 1973 |
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Current U.S.
Class: |
250/458.1 |
Current CPC
Class: |
G01N
21/6428 (20130101); G01N 15/0205 (20130101) |
Current International
Class: |
G01N
15/02 (20060101); G01N 21/64 (20060101); G01t
001/16 () |
Field of
Search: |
;250/304,361,362,363,365,369,432,435,458,461 |
References Cited
[Referenced By]
U.S. Patent Documents
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3497690 |
February 1970 |
Wheeless, Jr. et al. |
3657537 |
April 1972 |
Wheeless, Jr. et al. |
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Primary Examiner: Lawrence; James W.
Assistant Examiner: Willis; Davis L.
Attorney, Agent or Firm: Schiller & Pandiscio
Parent Case Text
This application is a continuation-in-part of copending application
Ser. No. 375,807, filed July 2, 1973.
Claims
What is claimed is:
1. Apparatus for detecting and classifying, in a fluid specimen,
submicron-dimensioned particles containing a nucleic acid and
stained with a fluorescent dye specific to said nucleic acid, said
apparatus comprising,
a radiation source for providing illumination within an excitation
band of said dye,
means for directing said illumination into said specimen,
means for viewing fluorescence emission produced from said dye
responsively to said illumination, and
means for spatially filtering at least one of said illumination and
said fluorescence emission, said means for spatially filtering
comprising aperture means providing, in conjunction with said means
for viewing, a ratio of viewed volume of said specimen to the root
mean square distance across said aperture means of greater than
1000/1.
2. Apparatus as defined in claim 1 wherein said means for spatial
filtering is disposed in the optical path between said source and
said specimen.
3. Apparatus as defined in claim 1 wherein said means for spatial
filtering is disposed in the optical path between said means for
viewing, and said specimen.
4. Apparatus as defined in claim 1 wherein said means for spatial
filtering includes one or more light transmissive portions,
substantially all of which are dimensioned to be as closely matched
to the average dimension of said particles as the wavelengths of
said illumination permits. 0603T0003
Description
The present invention relates to clinical laboratory methods and
apparatus and more particularly to detection and classification of
virus particles.
Virus particles, responsible for a wide variety of plant and animal
pathology, comprise exceedingly small particles sized on the order
of a wavelength of visible light or smaller (3500 to 200 Angstroms)
and consist essentially of either deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA) surrounded by a protein shell. The ratio of
nucleic acid content to protein content varies from 1:100 to
1:2.
Viruses are usually studied in animal, bacterial or plant hosts or
in cell cultures derived from tissues of the hosts. Virus presence
if inferred by observing the development of infectious symptoms in
a patient due to viral infection. The long time involved and the
danger of spread of the infection limit the desirability of this
technique.
Virus-suspect specimens may be subjected to a variety of chemical
reactions using known techniques of immunochemistry to detect and
classify viral species, if any, therein. The reactions involved are
highly specific and a large number of specific antibodies are
necessarily involved. At present however, only a limited number of
such antibodies are known. Culturing suspect specimens and
examination in the electron microscope to observe differences is
limited in its effectiveness by the long time involved in culturing
and sample preparation. It has also been known in the prior art to
produce a suspension of a suspect specimen in liquid, illuminate
the suspension with coherent light and observe back-scattered light
by heterodyne spectometry to obtain data on Brownian motion of
suspended particles correlatable with size of the particles. In
other words, the method measures particle velocity by observation
of the Doppler shift. The use of coherent and essentially
monochromatic radiation permits measurement even of the
comparatively slow Brownian velocities. However, inadequate ability
to descriminate among viral species and between viral particles and
similarly sized cell debris in the specimen limit the utility of
this technique.
It is therefore an important object of the present invention to
provide method and/or apparatus for virus detection avoiding the
above cited problems of the prior art.
It is a further object of the invention to provide classification
among viral species present in a suspect-sample consistent with the
preceding object.
It is a further object of the invention to provide a short time of
speciment treatment and data extraction consistent with one or more
of the preceding objects.
It is a further object of the invention to provide inexpensive and
simple apparatus and/or operating steps consistent with one or more
of the preceding objects.
According to the invention, virus-suspect specimens are treated by
the following process:
a. Taking from the patient a body fluid sample containing a
suspension to be examined for virus, and treating the sample with
one or more fluorescent dyes specific to a nucleic acid;
b. Illuminating the sample with light in an absorption band of the
dye or dyes and observing fluorescence, if any, from the stained
sample at spectral peaks associated with deoxyribonucleic or
ribonucleic acid, and substantially simultaneously spatially
filtering either or both the excitation illumination and the
emitted fluorescence, whereby the observed fluorescent intensities
of dyed particles is modulated by the Brownian motion of such dyed
particles.
Scattering behavior and/or polarized light response may also be
observed.
Spatial filtering of observed fluorescence intensities is achieved
in the present invention in one or two basic ways. In the first
method, the radiation used to illuminate the particles is spatially
filtered: For example, a regular grid or a simple aperture is
placed between the illumination source or optics and the suspension
of particles, thereby providing one or more zones of high and low
illumination. In the second method the fluorescence radiation
itself is spatially filtered, for example, by placing a grid
between the suspension of particles and the viewing optics, thereby
providing zones within which fluorescence can or cannot be
observed. In either case, as the particles move from zone to zone
by Brownian motion, the fluorescent emission will be modulated to
produce a repetition rate spectrum which is a function of the
velocities of the fluorescing particles. The particle velocities in
turn are a function of the particle masses. This system can be
readily distinguished from conventional scanning fluorometry in
which either the object or illuminating beam are moved or scanned
in a fixed scanning pattern which creates a signal indicating a
contour of the fluorescent intensity distribution across the
scanned object. The present system depends instead on the
statistical random or Brownian motion of a particle past an edge to
modulate the fluorescent intensity as a function of particle
velocity. By wavelength filtering, one can readily identify the
wavelength of the fluorescent emission and thereby determine the
particular nucleic acid to which the fluorochrome is bound. This,
together with simultaneous observation of the intensity modulation
of the fluorescing particles serves to distinguish viral-like
particles from background and provides useful viral classification
descriptors. The number of useful descriptors can be enhanced
through correlation of simultaneous observations of two-color
fluorescent modulation of instensity by Brownian motion and further
enhanced by correlation of polarized light response and scattering
data with the measurements of intensity modulation by Brownian
motion. The method and apparatus of the invention are also usable
in connection with detection and classification of other biological
particles having the same order of magnitude of size as viruses,
i.e. submicron size.
A conventional laboratory microscope may be modified by the
addition of aperture, filters and a fluid-well specimen holder in
accordance with the present invention to carry out the above
described process. Preferably, the microscope is provided with
multiple detector channels for simultaneous readout of intensity
modulated wavelengths corresponding to each of DNA and RNA.
However, a single channel may also be utilized with changes of
filters for sequential observation for the two colors to be
detected. Scattering data is taken separately or together with the
fluorescence data. Readout is preferably done automatically by
photodetectors coupled to frequency analyzers for each readout
channel. The frequency analysis output for each channel is a
graphical or ditigal representation of intensities of signal at
various frequencies. Whether a signal is generated at all is
determined by coincidence of the color selective properties of the
detection system with the fluorescent emission wavelength of the
fluorochrome attached to the viral particle, which is dependent on
the selection of fluorochrome and whether it is linked to a DNA or
RNA-containing particle.
These and other objects, features and advantages of the invention
will be apparent from the following detailed description with
reference therein to the accompanying drawing in which:
FIG. 1 is a block diagram of apparatus for detection of viral
species and classification by composition of a particular nucleic
acid; and
FIGS. 2-6 are graphical representations of the relationship of
light intensity to intensity modulation frequency in operation of
the invention, all graphs having the same coordinate increments in
abscissa and ordinate.
According to a preferred embodiment of the invention, the known
techniques of tissue culture preparation are used to obtain a
virus-suspect sample. The known techniques are modified to the
extent of being carried out in a time too short to produce the
growth areas of cell destruction associated with plague
preparation. The sample so obtained is suspended in a liquid.
Alternatively, a natural body fluid suspension of virus particles
(e.g., plasma or spinal fluid) may be directly utilized. The
suspension is mixed with fluorochrome dye stains which are soluble
in the fluid and specific to the viral or other small
nucleic-acid-containing particle to be detected.
The apparatus of a preferred embodiment of the invention comprises
a modified microscope with a laser or other high intensity light
source 12 for projecting light through a wavelength filter 14, a
spatial filter 18, and conventional projection optics 16. Filter 14
restricts the illumination to an excitation band of the
fluorochrome dye. Spatial filter 18 may be a single aperture or a
multiaperture filter such as a grid or the like as hereinafter
described. The spatial filter is imaged in the plane of the sample
which is held in a transparent fluid well in sample holder slide
20. It will be understood the spatial filter may precede the slide
or the slide may precede the spatial filter in the light path. In
the latter case, the sample is imaged onto the spatial filter by
viewing optics 22.
Spatial filters generally are well known to those skilled in the
optical arts and may take the form of gratings, grids, annuli, and
the like. Each defines one or more zones or edges between a
relatively light-transmitting element and a relatively
non-transmitting element. It will be apparent that as a particle,
moved by Brownian forces, crosses the edge or zone of a filter to a
light-transmitting area, the detector will see that particle
somewhat as a light burst or scintillation. As the particle crosses
the edge into a non-transmitting area, the burst is extinguished.
Thus, assuming that a particle travels in a straight line across a
grid, the observed intensity of emission from the particle will
fluctuate between maximum and minimum values at a frequency
depending on the grid spacing and the particle velocity.
Theoretically, for optimum modulation and duty cycle, the grid
spacing should, at least in order of magnitude, match the particle
size. For particles of submicron size, such matching is a difficult
task at least. However, spatial filters approaching
diffraction-limited apertures for the wavelengths employed,
although having aperture sizes far greater than viral dimensions,
will nevertheless provide quite useful results. It should be noted
that the present invention is intended to provide viral
classification descriptors which are derived by frequency analysis
of a viral population of statistically valid magnitude. A typical
sample can be expected to contain viral particles in excess of
about 1 .times. 10.sup.6 /ml.sup.3 which thus provides an adequate
population for the proposed analysis. However, if one assumes that
this population of particles is viewed in a volume 1.mu. thick
(e.g. the depth of field of the viewing optics is 1.mu.) through a
spatial filter which is simply a diffraction-limited circular
aperture, the observation time to obtain adequate data for analysis
would be intolerably long because of the very few scintillations
per minute that would occur. Simply increasing the aperture area
would improve the situation by providing a longer edge across which
more particles would be expected to move, but the mean dwell time
of the particles in the aperture would also increase thereby
impairing the signal-to-background ratio, so little gain would be
made toward providing an adequate observational sample in a
reasonable time period.
The criterion used in the present invention to select an adequate
spatial filter is that the ratio of viewed volume to the root mean
square distance across the aperture of the filter, be greater than
1000/1. The root mean square distance (also known as the mean
crossing distance) is defined as the mathematical root mean square
distance to the edges of the aperture (or the several apertures of
a multiple aperture) from the mean of all points in the aperture
(or in each of the apertures in the multiple aperture situation).
In other words, to obtain a statistically large number of
scintillations per unit time it is desirable to increase the viewed
volume (i.e. the product of the aperture times depth of field) but
the dwell time of the individual particles in the viewed volume is
minimized to reduce background.
For example, an aperture in the form of an elongated slit 1.mu.
wide (assuming a depth of field of 1.mu. deep) should then be more
than 1000.mu. long to achieve adequate signals. Such slit need not
be linear but can be in the form of a spiral. Closely related
filters formed of multiple slits are gratings (e.g. an array of
parallel linear slits), a group of concentric ring segments (e.g.
an array of curved slits having a common center of curvature) and
the like. Similarly, one can employ a grid or a plurality of small
(rectangular or otherwise) openings to form the desired filter. All
of the latter are of course multiple volume apertures that can be
employed in the present invention provided that they meet the
criterion hereinbefore set forth.
The light passing through the sample and/or emitted by the sample
is viewed through conventional viewing optics 22, a beam splitter
24, and photoelectric detectors 26, 28, 30. Detectors 26 and 28 may
have wavelength filters 32 and 34 associated therewith to limit
their effective spectral ranges to different fluorescence bands or
may themselves have limited spectral ranges. Detector 30 may also
be provided with a wavelength filter and known means 48 to mask out
direct radiation from light source 12 so that detector 30 sees only
scattered light.
The electrical output signals from the detectors are amplified by
preamplifiers 36, 38 and 40 and applied to known frequency
analyzers 42, 44, 46 which are synchronized by mechanical or
electronic means such as a common clock circuit associated
therewith. The outputs of the frequency analyzers are applied to
data reduction apparatus which may be a strip chart recorder and/or
a computer.
To obtain desired specificity of nucleic acid staining, solubility,
and the property of fluorescence at desired wavelengths under
excitation by light of desired wavelenghts, one may use the
cationic dyes: acridine orange or yellow GR, quinacrine mustard,
ethydium bromide, pyronine B, aurophosphine, euchrysine 2GNX and
3R, vesuvin, rhodamine S, B and 6G, coriphosphine 0, civanol,
acrifalvine, atabrine, phosphine, benzoflavine, rheonine A,
thioflavine T and berberine. Many other dyes can be used with
appropriate adaptation of sample chemistry or the light source to
suit such dyes.
With an appropriate spatial filter, the apparatus of FIG. 1 can be
operated with a 0.05 cc. sample having a particle concentration of
about 10.sup.8 particles (of approximately 200 to 3500 Angstrom
size) per cubic mm., to make classification measurements in one
minute or less. Sample handling can be accomplished at a rate of
five minutes or less per sample. Accuracy of size classification
can be within .+-. 3 percent.
The outputs of the frequency analyzer for a single sample are shown
schematically in FIGS. 2-5 which are intensity vs. modulation
frequency graphs (in Hertz). The sample was studied with acridine
orange and its fluorescence was measured through different channels
or in a single channel with changes of detection filtering between
observations.
FIG. 2 is a graph taken with a filter passing an appropriate
wavelength (5500 A in this example) to detect the presence of DNA
particles. The location of each of peaks A, B, C on the resulting
curve are proportional to the equivalent diffusional diameters of
various DNA-containing particles. Peak heights and areas are
proportional to quantities of the DNA-containing particles of the
sizes indicated by peak location.
The curve in the graph of FIG. 3 is created by the frequency
analyzer and its printout device when using a wavelength filter
(5500 A in this case) chosen to detect RNA-containing
particles.
Clearly peak B of the curve in the graph of FIG. 2 corresponds to a
DNA-containing virus because there is no corresponding peak in FIG.
3. Similarly peak D of FIG. 3 corresponds to an RNA-containing
virus. As viruses have to belong in one of these types, the
continued presence of peaks A and C indicates a slightly shifted
DNA spectral behavior which indicates a structural change in the
DNA'S arrangement. These flucturations in the overlap between both
channels have recognition value.
FIG. 4 shows the result of a repeat run with polarized filter
specific for scattering due to the total particle mass. The
illuminating source is an argon laser. This filter is chosen to
correspond to the illumination, and is 4880 Angstroms in this
case.
It is clear from comparison of FIG. 4 with FIGS. 2 and 3 that the
peaks E, F, G, H, and I of FIG. 4 are due to the presence of
particles that do not contain nucleic acid and may be
disregarded.
FIG. 5 shows the result of a repeat run with the 4880 Angstrom
polarized filter with a different orientation.
The ratio between the readings of FIGS. 4 and 5 describes the
polarization in the particle scattering and thus the particle's
elongation.
Tha average of the curves of FIGS. 4 and 5 gives the total particle
mass, whose ratio to the FIG. 2 or 3 curves gives the nucleic
acid/total weight ratio in the particle.
FIG. 6 is a similar plot, for apparatus calibration purposes, of
intensity vs. modulation frequency, using the apparatus of FIG. 1
with a single tiny aperture as a spatial filter to detect suspended
particles of 880 Angstrom polystyrene spheres, a known phage virus,
and 2340 Angstrom polystyrene spheres. The polystyrene spheres were
observed in scattered light.
The results are shown, respectively, on the curves marked 880, V,
and 2340. The half-width of the half-heights of the respective
curves occur at 34.5, 13.2 and 27.5 Hertz indicate the frequencies
relatable to known diameters of the particles.
It is evident that those skilled in the art, once given the benefit
of the foregoing disclosure, may now make numerous other uses and
modifications of, and departures from the specific embodiments
described herein without departing from the inventive concepts.
Consequently, the invention is to be construed as embracing each
and every novel feature and novel combination of features present
in, or possessed by, the apparatus and techniques herein disclosed
and limited solely by the scope and spirit of the appended
claims.
* * * * *