U.S. patent application number 12/367194 was filed with the patent office on 2009-08-06 for systems and methods of identifying biomarkers for subsequent screening and monitoring of diseases.
This patent application is currently assigned to Vitrimark, Inc.. Invention is credited to Nazneen Aziz, Arijit Bose.
Application Number | 20090196484 12/367194 |
Document ID | / |
Family ID | 36119445 |
Filed Date | 2009-08-06 |
United States Patent
Application |
20090196484 |
Kind Code |
A1 |
Bose; Arijit ; et
al. |
August 6, 2009 |
Systems and Methods of Identifying Biomarkers for Subsequent
Screening and Monitoring of Diseases
Abstract
A system for generating an image of ultrastructural biomarkers
from a biological sample is provided. The system includes a grid
onto which a sample to be imaged may be placed and a cryogenic
reservoir into which the grid and sample may be immersed for
vitrification of the sample. The system also includes a stage onto
which the grid and sample may be situated for subsequent imaging in
a high contrast imager to permit identification of ultrastructural
biomarkers therein. A method for generating an image of
ultrastructural biomarkers from a biological sample is also
provided. The generated image of ultrastructural biomarkers may be
used subsequently for screening and monitoring diseases, evaluating
drug and therapeutic efficacy, and assessing risks associated with
a drug or therapeutic candidate, among other things.
Inventors: |
Bose; Arijit; (Lexington,
MA) ; Aziz; Nazneen; (Lexington, MA) |
Correspondence
Address: |
GREENBERG TRAURIG, LLP
ONE INTERNATIONAL PLACE, 20th FL, ATTN: PATENT ADMINISTRATOR
BOSTON
MA
02110
US
|
Assignee: |
Vitrimark, Inc.
|
Family ID: |
36119445 |
Appl. No.: |
12/367194 |
Filed: |
February 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11232597 |
Sep 22, 2005 |
7507533 |
|
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12367194 |
|
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|
60612713 |
Sep 24, 2004 |
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Current U.S.
Class: |
382/133 |
Current CPC
Class: |
H01J 2237/204 20130101;
G01N 21/6456 20130101; B01L 3/022 20130101; G01N 33/68 20130101;
G01N 1/42 20130101; G01N 21/6428 20130101; H01J 2237/2001 20130101;
G01N 2001/2873 20130101 |
Class at
Publication: |
382/133 |
International
Class: |
G06K 9/00 20060101
G06K009/00 |
Claims
1. A system for identifying ultrastructural biomarkers from a
biological sample, the system comprising: a grid onto which a
biological sample to be imaged may be placed; a cryogenic reservoir
into which the perforated grid and sample may be immersed for
vitrification of the sample; a stage, provided with a temperature
substantially similar to the cryogenic reservoir, and onto which
the grid and sample may be situated for subsequent imaging; and a
high contrast imager designed to receive the stage with the grid
for imaging a region of the thin sample for subsequent
identification of ultrastructural biomarkers.
2. A system as set forth in claim 1, wherein the grid includes a
plurality of holes across which the sample may extend, so as to
enhance generation of a thin film thereacross.
3. A system as set forth in claim 1, wherein the grid further
includes a first plate onto which the grid may be positioned and a
second plate for placement onto the grid.
4. A system as set forth in claim 3, wherein the first and second
plates act to spread the sample across the grid, so as to generate
certain portions that can be substantially thinner in thickness
than others across the grid.
5. A system as set forth in claim 3, further including
cryogenically cooled forceps to permit separation of the grid from
the first and second plates.
6. A system as set forth in claim 1, wherein the cryogenic
reservoir includes an inner chamber for accommodating a first
cryogenic fluid, and an outer chamber situated about the inner
chamber for accommodating a second cryogenic fluid.
7. A system as set forth in claim 6, wherein the first and second
cryogenic fluids are different and the presence of the second
cryogenic fluid in the outer chamber helps to maintain the first
cryogenic fluid substantially close to its melting point.
8. A system as set forth in claim 6, wherein the cryogenic
reservoir further includes a grid holder positioned within the
outer chamber for placement of the grid thereon prior to
transference onto the stage.
9. A system as set forth in claim 1, wherein the stage includes a
container for accommodating a cryogenic fluid, and an arm extending
from the container for placement of the grid thereon, the arm
having a channel along which cryogenic fluid from the container may
flow toward the grid, so as to maintain the temperature of the grid
substantially similar to that of the cryogenic reservoir.
10. A system as set forth in claim 1, wherein the high contrast
imager can generate substantially artifact-free images in the
absence of contrasting agents.
11. A system as set forth in claim 1, further including a positive
pressure environment within which the grid may be transferred onto
the stage and into the high contrast imager, so as to maintain the
integrity of the sample and to minimize risks of contamination of
the sample.
12. A system as set forth in claim 1, wherein the biomarkers
include components from one of intracellular organelles or
components, extracellular organelles or components, tissue
components, and biological fluids.
13. A system as set forth in claim 12, wherein the identified
ultrastructural organelles or components, extracellular organelles
or components can be used to evaluate, determine, or predict drug
or therapeutic efficacy, patient response or response rate, or
clinical trial participant response or response rate.
14. A system as set forth in claim 12, wherein the identified
ultrastructural biomarkers, when compared to ultrastructural
biomarkers from healthy or control intracellular organelles or
components, extracellular organelles or components, tissue
components, and biological fluid can act to assess risks for
adverse events, toxicity, or serious adverse events associated with
drug or therapeutic candidates in preclinical development, in
animal models, or in clinical development, as well as risks for
drug attrition in preclinical development, animal models, clinical
development or in marketed drugs.
15-41. (canceled)
Description
RELATED U.S. APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Application Ser. Nos. 60/612,713 filed Sep. 24, 2004, which
application is hereby incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention relates generally to imaging systems and
methods, and more particularly to imaging systems and methods for
identifying ultrastructural biomarkers for subsequent screening and
monitoring of diseases.
BACKGROUND ART
[0003] The deepening productivity crisis in the pharmaceutical
industry, the high cost to the pharmaceutical industry of
introducing new drugs to the market, partly because of expenses
related to Phase I, II and III clinical trials, as well as late
stage failures for many drug candidates have spurred intense
across-the-board activity around biomarker discovery and
validation. Biomarkers, defined by the FDA as a characteristic that
is objectively measured and evaluated as an indicator of normal
biologic or pathogenic processes or pharmacological responses to a
therapeutic intervention, are being sought actively to help make
early and cost-effective "go/no-go" decisions on drugs, for patient
stratification, clinical trial analysis, and finding niche markets
(e.g., sub-population of patients who respond to drugs or in whom
no drug-related toxicity is seen) for new drugs under development.
In addition, the FDA has recently recommended that validated or
investigational biomarker data be included in IND and NDA packages.
These are powerful drivers for the biomarker market, whose size is
estimated at $428 millions in 2005, and is growing at 20 percent
per year.
[0004] The use of biomarkers is rapidly gaining momentum in the
pharmaceutical industry and in the medical management of patients.
Current methods for identifying biomarkers involve the use of
biochemical assays for identifying "functional" biomarkers, such as
genes or protein arrays or metabolite analysis. The use of
biochemical assays in this context requires probing for functional
alterations in genes and proteins, the need for a priori knowledge
of their function, as well as extensive assay development and
optimization.
[0005] While there has been an explosion of biomarker discovery
efforts utilizing genomics, proteomics and metabolomics, these
technologies also focus only on functional biomarkers. With many
diseases, the presence of observable functional biomarkers often
occurs late in the disease state. As such, preventive measures for
these diseases may be ineffective when developed in connection with
the management of the disease, or in early evaluation of drug
efficacy.
[0006] Contributions towards understanding ultrastructural
morphology have been made in recent years. Such an approach focuses
on the ultra-structural differences in the biological samples that
can occur much earlier in the diseased state, even before
functional differences are observable. Since these target
structures typically range from between about 5 nanometers (nm) and
1 micrometer, one approach to visualize them is through the use of
conventional transmission electron microscopy (TEM). However, the
use of conventional TEM has some critical limitations. For example
(i) the high vacuum used in TEM removes solvent, leaving behind
structures that are quite different from those present in the
original solution, (ii) adequate contrast between the sample
features and background is usually not available, necessitating the
use of stains (the addition of stains, which usually are heavy
metal salts, can cause dramatic changes in aggregate morphology),
and (iii) the exposure of the sample to the electron beam often
damages the sample.
[0007] Accordingly, it would be desirable to provide an approach
that can generate substantially artifact-free images of structural
biomarkers of a cell or biological sample without compromising the
integrity of the biomarkers in the sample.
SUMMARY OF THE INVENTION
[0008] The present invention provides, in one embodiment, an
approach through the use of cryogenic transmission electron
microscopy (cryo-TEM), as well as modified freeze fracture direct
imaging (M-FFDI) to identify ultra-structural biomarkers, which may
subsequently be used for screening and monitoring a range of
diseases. The use of cryo-TEM and M-FFDI can generate substantially
artifact-free images, unlike images obtained from conventional
TEM.
[0009] In accordance with one embodiment of the present invention,
a system for generating an image of ultrastructural biomarkers from
a biological sample is provided. The system includes a grid onto
which a sample to be imaged may be placed. The grid may be
perforated so that a thin film of the sample may be generated
across a hole. The system also includes a cryogenic reservoir into
which the perforated grid and sample may be immersed for
vitrification of the sample. In an embodiment, the reservoir
includes an inner chamber for accommodating a first cryogenic fluid
and into which the grid and sample may be immersed, and an outer
chamber situated about the first chamber for accommodating a second
cryogenic fluid. The system further includes a stage, provided with
a temperature substantially similar to the cryogenic reservoir, and
onto which the grid and sample may be situated for subsequent
imaging. The system may also be provided with a high contrast
imager, such as an electron microscope, designed to receive the
stage with the grid for imaging a relatively thin film region of
the sample to permit identification of ultrastructural biomarkers
therein.
[0010] The present invention also provides a method for generating
an image of ultrastructural biomarkers from a biological sample.
The method includes, in one embodiment, providing a substantially
thin film of a sample to be imaged. The thin film may be generated
from blotting or alternatively from sandwiching the sample between
two plates. Next, the sample may be immersed in a cryogenic fluid
so as to cause the sample to vitrify. This rapid vitrification
allows the objects present in the sample to substantially maintain
their original morphology. Once vitrified, the sample may be
transferred onto a stage for placement in a high contrast imager,
such as a transmission electron microscope, under positive dry
pressure to minimize the risks of contamination of the sample. The
transfer to the high contrast imager also includes keeping the
sample at a temperature range of from about -170.degree. C. to
about -150.degree. C. in the imager to maintain the integrity of
the sample. Thereafter, an image of the thin film sample may be
generated for subsequent identification of ultrastructural
biomarkers. The generation of the image, in one embodiment,
includes producing a substantially artifact-free image in the
absence of contrasting agents.
[0011] The method for generating an image of ultrastructural
biomarkers from a biological sample may be used subsequently for
screening and monitoring diseases or disease susceptibility,
evaluating drug or therapeutic efficacy, and assessing risks
associated with a drug or therapeutic candidate, among other
things. In one embodiment, a vitrified biological sample from a
test subject may initially be provided. Next, an image from the
vitrified sample may be generated, in a high contrast imager, for
subsequent identification of ultrastructural biomarkers.
Thereafter, the biomarkers from the vitrified sample may be
compared to those biomarkers from a healthy subject or control
population, for structural or morphological variations
Subsequently, the presence of structural or morphological
variations may be analyze and used as determinants or predictors
for a disease, for evaluating drug or therapeutic efficacy, or
assessing risks associated with a drug or therapeutic
candidate.
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIGS. 1A-C illustrate a system for use in preparing samples
for subsequent imaging and identification of ultrastructural
biomarkers.
[0013] FIG. 2 illustrates a cryotransfer station onto which a
sample may be transferred for subsequent placement into a high
contrast imaging device for in imaging and identifying
ultrastructural biomarkers.
[0014] FIG. 3 illustrates schematically another method for
preparing samples for subsequent imaging and identification of
ultrastructural biomarkers, in accordance with an embodiment of the
present invention.
[0015] FIGS. 4A-D illustrate cryogenic TEM images of a human blood
serum sample.
[0016] FIGS. 5A-B illustrate a change comparison in morphology and
aggregate state between a control sample and a diseased sample.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0017] The present invention provides, in one embodiment, a method
for identifying ultrastructural biomarkers through the use of
cryogenic transmission electron microscopy (cryo-TEM), or modified
freeze fracture direct imaging (M-FFDI). In particular, the
combination of cryogenic vitrification of biological samples and
subsequent high contrast imaging of the samples, can preferably
generate substantially artifact-free images, unlike those images
obtained from the use conventional TEM alone. As such, the ability
to visualize `soft` structures that range, for instance, from
between about 5 nanometers (nm) and about 500 nm, makes these
artifact-free imaging techniques ideally suited for high resolution
imaging of biomolecular aggregates, such as proteins, viruses and
cellular organelles in their native hydrated states for
ultrastructural analysis. Moreover, the data obtained by cryo-TEM
or M-FFDI can complement atomic level information from, for
instance, X-ray diffraction (where crystals of the sample have to
be produced before identification--these crystals do not represent
the true hydrated configuration in solution) and NMR, as well as
micron level information from light microscopy for biomarker
identification.
Cryo-TEM Technique:
[0018] 1. The Controlled Environmental Vitrification System
[0019] Referring now to FIGS. 1A-C, there is illustrated a
Controlled Environmental Vitrification System (CEVS)10, such as
that available from The Department of Chemical Engineering &
Material Sciences at the University of Minnesota, for use in the
preparation of samples for subsequent ultrastructural biomarker
identification. In general, the CEVS 10 includes a housing H
equipped with temperature and humidity control. The CEVS 10 also
includes a vial 11, within which a volume of sample may be stored.
The sample within the vial 11, in one embodiment, may be thermally
equilibrated relative to the interior of housing H. To extract an
amount of the sample from the vial 11, a pipette 12 may be provided
adjacent to the vial 11. The CEVS 10 may also include a grid 13
onto which the extracted sample may be deposited from the pipette
12. The grid 13, in one embodiment, may be a perforated disc with
holes 131 that are sufficiently spaced to support the deposited
sample. In an embodiment, the grid 13 may be a specially prepared
holey carbon grid that is approximately 3 millimeters (mm) in
diameter, if circular in shape. Of course, the grid may be designed
with other geographic shapes if necessary. Such a holey carbon grid
is well known in the art and includes, among other things, a
perforated copper disc having a carbon layer, approximately 200 nm
in thickness, extending across holes 131. The carbon layer, in an
embodiment, is provided to decrease the diameter of the holes 131
on the grid 13, and does not completely cover these holes 131. In
accordance with an embodiment, the holes 131 may be provided with a
diameter ranging from about 1 micrometer to about 10 micrometers.
In this manner, when the sample is deposited onto the grid 13, a
thin film of the sample may be generated across the reduced-size
holes 131. To facilitate the generation of the thin film, the CEVS
10 may be provided with a blotter 14 adjacent the grid 13 to blot
excess amount of the deposited sample from the surface of the grid
13, such that an amount sufficient for generating the thin film
remains on the grid 13. In addition, to maintain the grid 13 in
position during the deposition process and blotting process, a
plunging mechanism 15 may be provided to which the grid 13 may be
affixed.
[0020] The CEVS 10 may further include a portal 16 through which
the plunging mechanism 15 may extend, so as to push the grid 13
from within the CVES 10, as illustrated in FIG. 1B. In this manner
the grid 13 may be immersed within a cryogenic reservoir 17
situated in proximity to the portal 16. The reservoir 17, in one
embodiment, includes an inner chamber 171 within which a volume of
a first cryogenic fluid may be accommodated and into which the grid
13 along with the sample may be immersed. In one embodiment, the
first cryogenic fluid may be liquid ethane, or similarly cold
cryogenic fluid, such as liquid propane. For ethane, the normal
melting point is about -183.degree. C., while the normal boiling
point is about -89.degree. C. The reservoir 17 may also include an
outer chamber 172 situated about the inner chamber 171 for
accommodating a volume of a second cryogenic fluid, such as liquid
nitrogen, or a similarly cold cryogenic fluid. By positioning outer
chamber 172 about inner chamber 171, the volume of liquid ethane in
inner chamber 171 may be kept sufficiently cold and substantially
close to its melting point by the presence of the liquid nitrogen
in the outer chamber 172 to maximize heat transfer away from the
sample and to allow the sample to vitrify rather than crystallize.
The reservoir 17 may further include a grid holder 18 submerged
within the liquid nitrogen in the outer chamber 172 for subsequent
placement of the grid 13 thereon.
[0021] 2. Preparation of the Sample
[0022] Still looking at FIGS. 1A-C, approximately 1-10 microliters
of the liquid sample may be withdrawn from vial 11 using pipette
12, and subsequently deposited onto perforated grid 13. In one
embodiment, the sample may be about 2-5 microliters in volume, and
preferably, about 3 microliters in volume. The sample may
thereafter be blotted by blotter 14, so as to leave on grid 13 a
thin film having a thickness ranging from about 50 nm to about 200
nm spanning the holes 131. It should be noted that thickness of the
thin film may not be substantially uniform throughout. To that end,
areas of the film that is relatively thin can provide an ideal
location for imaging.
[0023] The sample on grid 13 may then be immersed by plunging
mechanism 15 into reservoir 17, as shown in FIG. 1B, and in
particular, into chamber 171 of liquid ethane having a temperature
range of from about -170.degree. C. to about -150.degree. C. As
noted above, the liquid ethane may be kept close to its melting
point by the liquid nitrogen in the outer chamber 172 to maximize
heat transfer from the grid. Contact of the sample on the grid 13
with the liquid ethane in chamber 171 can induce rapid
vitrification of the sample on grid 13 within a few milliseconds.
It should be appreciated that during vitrification, liquid, such as
water, in the solution solidifies without crystallization. In this
manner, substantially all of the microstructures in the sample may
be preserved in their original state. The grid 13 may next be
transferred from the inner chamber 171 of the reservoir 17 to the
outer chamber 172 and placed onto the grid holder 18 in liquid
nitrogen.
[0024] Referring now to FIG. 2, the grid 13 may thereafter be
transferred from the holder 18 onto a cold stage 20 in the liquid
nitrogen environment of the outer chamber 172 of reservoir 17.
Transferring the grid 13 in a liquid nitrogen environment can help
to maintain the integrity of the sample. As illustrated, cold stage
20 may include a container 21 within which a volume of, for
instance, liquid nitrogen or any other similarly cold substance may
be stored, and an arm 22 extending from the container 21. Arm 22,
in an embodiment, may be designed for placement within the liquid
nitrogen environment of the outer chamber 172, and may be tubular
in shape. As such, arm 22 may be made from a material that can
withstand immersion in liquid nitrogen. Arm 22 may also include a
channel 23, so as to permit liquid nitrogen from container 21 to
advance to tip 24 and maintain the temperature of the arm 22
thereat from about -170.degree. C. to about -160.degree. C.,
substantially well below the amorphous to crystalline phase
transition temperature of about -155.degree. C. in ice, to minimize
any compromise to the integrity of the sample. As shown in FIG. 2,
arm 22 may include a depression 25 towards tip 24 to provide an
area onto which grid 13 may be placed for subsequent imaging. Cold
stage 20, in one embodiment, can be a commercially available cold
stage, such as the Cryotransfer System--CT3500J from Oxford
Instruments.
[0025] Once the grid 13 has been transferred onto arm 22 of cold
stage 20, the cold stage 20 may be inserted into, for instance, a
high contrast imager (not shown), such as a TEM, under positive dry
pressure to minimize the risks of contamination of the sample by,
for example, atmospheric contaminants, including moisture. The
positive dry pressure may be generated from any gas, such as
nitrogen or oxygen. During imaging, such as phase contrast imaging,
the tip 24 of arm 22 continues to be maintained at a temperature
range well below the amorphous to crystalline phase transition
temperature of about -155.degree. C. in ice, in the electron
microscope to maintain the integrity of the sample being
imaged.
M-FFDI Technique:
[0026] In samples having a viscosity that may be relatively high,
i.e., greater than about 100 centipoise, for blotting to
effectively thin down the samples, the use of cryo-TEM may not be
sufficient. As such, the present invention contemplates the use of
M-FFDI.
[0027] Looking now at FIG. 3, in one embodiment, approximately 100
nanoliters (nL) of the sample 30 may placed onto a plate or
planchette 31. Planchette 31, in an embodiment, may be a copper
planchette, or may be made from a similar material of approximately
3 mm.times.3 mm in size. Next, a grid 32, either standard electron
microscope grid or a holey carbon grid, such as that described
above, may be placed onto the planchette 31 over the sample 30, so
that the sample 30 may permeate across the perforations of the grid
32. In an alternate embodiment, the sample 30 may initially be
placed on the grid 32 and the grid 32 subsequently placed onto the
planchette 31. The planchettes 31, in accordance with an
embodiment, may be relatively larger in size than the grid 32, so
as to accommodate the grid 32 thereon.
[0028] Thereafter, a second planchette 33 may be gently lowered
onto the grid 32 to sandwich the grid 32 between the two
planchettes 31 and 33. It should be appreciated that gentle
placement of the second planchette 33 onto the grid 32 allows the
sample 30 to be squeezed between the planchettes 31 and 33, and
spread out over the surface of the grid 32 into previously
unoccupied areas. Moreover, the spreading of the sample 30 across
of the grid 32 into previously unoccupied areas generates certain
thin film portions that can be substantially thinner in thickness
than others across the perforations of the grid 32. The presence of
the relatively thin portions can facilitate imaging of the
structures within these thin portions of the sample 30.
[0029] The planchette-grid-planchette sandwich may then be immersed
into, for instance, liquid ethane that is maintained near its
melting point with a temperature range of from about -170.degree.
C. to about -160.degree. C. Once vitrification of the sample 30 has
taken place, the copper planchettes 31 and 33 may be separated
(e.g., peeled apart) while they remain immersed in the liquid
ethane to remove the grid 32 therebetween. In an embodiment, a
cryogenically cooled forceps (not shown) may be used to separate
the grid 32 from the two planchettes. Next, the grid 32 may be
withdrawn and stored under liquid nitrogen, for instance, on a grid
holder, such as that shown in FIG. 1B, until it is ready to be
transferred to cold stage 34 for direct imaging of the
ultrastructural biomarkers within the sample 30.
[0030] This technique can be suited for preparation of highly
viscous samples and gels, where blotting may not be feasible, for
subsequent high resolution imaging. In other words, those samples
that cannot be prepared for imaging using cryo-TEM can be prepared
using this M-FFDI technique. This technique can also be employed to
prepare samples that have a predominant organic phase that tend to
dissolve if exposed directly to ethane. Accordingly, by providing
these two approaches for imaging, a substantially complete range of
solutions or biological fluids that can be imaged.
[0031] The combination of cryogenic vitrification for sample
preparation and the high contrast microscopy for imaging of the
sample can produce reliable, substantially artifact-free direct
images of ultrastructural biomarkers, for instance, nanoscale
aggregates in solution, or of soft tissue sections in their native
states. It should be appreciated that neither cryo-TEM nor M-FFDI
requires the use heavy metal salts to create contrast, thus
avoiding salt-related phase transitions. In addition, the
structural information obtained from cryo-TEM or M-FFDI, when
applied to, for instance, computer based reconstruction of images
obtained at different angles or stage tilts, can provide three
dimensional (3-D) structural information on macromolecular
assemblies. Such 3-D reconstruction is well known in the art, for
example, 3-D images created from CAT scans.
[0032] Although the use of cryogenic vitrification is described
above in connection with cryo-TEM and M-FFDI, it should be
appreciated that such can be employed with other imaging
approaches. For instance, cryogenic vitrification may be used in
connection with Cryotoming to image and examine ultrastructural
biomarkers in various tissue samples. As an example, a sample of
about 1 mm square may be vitrified by high pressure freezing. The
vitrified sample may then be positioned and secured on, for
instance, a cold aluminum pin. Thereafter, a section of
approximately 50 nm-100 nm may be microtomed (i.e., sliced) using a
cold diamond knife. This sample may subsequently be placed on a
carbon-coated electron microscope grid, and imaged at from about
-170.degree. C. to about -150.degree. C. on a cold stage.
EXAMPLES
[0033] In an experiment, a serum sample from a test subject was
prepared using cryogenic vitrification in the manner set forth
above and subsequently imaged through the use of cryo-TEM. The
images of the serum sample are illustrated in FIG. 4. In
particular, the images are taken from different regions of the same
holey carbon grid (i.e. different relatively thin regions of the
thin film on the grid). As can be seen, the images show a
relatively rich range of structures, including (a) multilamellar
vesicles, (b) single and (d) compound discs, and (c) compound
vesicles, all at nanoscale resolution of approximately 500 nm or
less.
[0034] It should be noted that serum typically contains
macromolecules, such as metabolites, lipids, hormones, peptides,
and proteins. Certain of these biological macromolecules can also
organize into 3-D complexes, which can be biochemically homogeneous
or heterogenous in nature. For examples, serum can contain many
glycoproteins, glycopeptides, lipoproteins, and hormones and
metabolites complexed with proteins, and lipids.
[0035] In FIGS. 5A-B, there is illustrated a comparison of the
structural and physical changes between a control sample (FIG. 5A)
and a diseased sample (FIG. 5B). Images of both can, of course, be
obtained using the protocol set forth above. It is noted that
protein structures 51 in the control sample can become more
aggregated in the diseased sample, whereas vesicles 52 in the
control sample can change shape, becoming more elliptical or larger
in size. These structural or morphological changes can act as
determinants, among other things, screening and monitoring
diseases.
Other Applications
[0036] In accordance with an embodiment of the present invention,
ultrastructural biomarkers identified through the use of cryo-TEM
or M-FFDI may be used for a wide variety of applications, for
example, to make early disease screening (i.e., prediction,
susceptibility), disease monitoring, early markers of drug related
toxicity, and drug efficacy, among others. Other applications that
can be imagined include those for diagnostic, therapeutic,
prophylactic, drug discovery, and patient stratification
purposes.
[0037] A biomarker or biological marker is defined by the FDA as a
characteristic that is objectively measured and evaluated as an
indicator of normal biologic or pathogenic processes or
pharmacological responses to a therapeutic intervention.
[0038] In diseased individuals, the composition of, for instance,
serum components can be altered due to cell proliferation,
metabolic, hormonal, inflammatory or secretory changes, thus
impacting the structure and morphology of the resulting imaged
components. As there are numerous diseases where ultrastructural
abnormalities occur in cell organelles, tissue structures, and
biological fluids, the utilization of ultrastructural analysis of
components therein can reveal critical biomarkers associated with
these diseases. Moreover, by screening and comparing biomarkers
from a sample of a test subject to those biomarkers from a healthy
subject or control population for structural or morphological
variations, the presence of variations in the ultrastructural
biomarkers from the test subject sample, in one embodiment, can act
as determinants or predictors of disease, disease predisposition,
and disease susceptibility.
[0039] Examples of biological fluids from which ultrastructural
abnormalities can be observed include, but are not limited to
blood, mucosa, plasma, serum, cerebral spinal fluid, spinal fluid,
joint fluid, urine, saliva, bile, pancreatic fluid, peritoneal
fluid, lung fluid, alveolar sac fluid, sinus fluid, lachrymal
fluid, nasal mucous and fluid, intrathoracic fluid, gastric fluid,
gastrointestinal fluid, ovarian fluid, testicular, prostrate fluid,
uterine fluid, cystic fluid, renal fluid, brain fluid, opthalmic
fluid, tear, ear fluid, auditory canal fluid, subcutaneous or
muscular fluid.
[0040] Examples of cell organelles and tissue structures within
which ultrastructural abnormalities can occur include plasma
membrane, organelle membranes, basement membrane, extracellular
matrix, intercellular organelles, intercellular structures,
intracellular membranes, intracellular organelles, cell-cell
junctions, cell-cell adhesion, gap junctions, tight junctions,
nucleus, nucleolus, nuclear membrane, nuclear pore, chromosomes,
chromatin, ribosomes, polyribosomes, monosomes, cellular proteins,
cellular protein complexes, cellular protein subunits,
extracellular proteins, extracellular protein complexes,
extracellular protein subunits, secretory proteins, secreted
protein complexes, secretory protein subunits, secreted
intracellular or extracellular protein aggregates, golgi,
lysosomes, mitochondria, endosomes, mitochondrial membranes,
peroxisomes, endoplasmic reticulum, mRNA, DNA, tRNA, rRNA, small
RNA, proteosomes, vacuoles, intracellular and extracellular
vesicles, cavity, and droplets, cellular lipids or carbohydrates,
cellular lipid or carbohydrate complexes, cellular lipoproteins,
cellular glycoproteins, intracellular and extracellular lipids,
extracellular lipoprotein complexes, extracellular lipoprotein
subunits, secreted proteins, secreted protein subunits, lipoprotein
or glycoprotein aggregates can reveal critical biomarkers
associated with these diseases.
[0041] Moreover, it is well established that structural and
morphological variations in secreted components of biological
fluids, such as serum, precede or occur simultaneously with
functional changes. Accordingly, the ability to monitor the
structural or morphological changes in aggregates present in
biological fluids in their native, hydrated states at nanoscale
resolution, and which can be correlated to functional and
phenotypic changes has the potential for early and simple detection
of disease, classification of disease sub-categories, and
monitoring of disease progression. In one embodiment, to
effectively monitor the progress of a disease via an image-based
platform, such as that employed in the present invention, an
accurate, precise and temporally contiguous picture of the progress
of the disease is needed. The method of the present invention can
provide an accurate and precise image of the ultrastructural
biomarkers from samples taken from a subject over a period of time.
As a result, these images may be compared against one another for
any structural or morphological changes in the biomarkers being
observed to determine and monitor the progression of the
disease.
[0042] The ultrastructural biomarkers identified can also be
employed for drug or biological therapeutics screening. For
example, in cell-based or in vitro drug screening, any
intracellular or extracellular markers of change can be detected
and utilized as a marker of drug or therapeutic efficacy or an
indicator that the drug target is being hit. In particular, in one
embodiment, different drugs, candidate drugs or therapeutics may be
administered to test subjects, and the side effects, including
desired effects, toxicity, adverse effects or serious adverse
effects, may be documented. Any conventional metrics of side effect
severity can be used. In addition, before and after drug
administration, the biomarkers may be identified and analyzed to
determine which of the biomarkers has changed. In this way, the
biomarkers affected by each drug can be correlated with the
particular desirable and undesirable effects of the drug.
[0043] It is anticipated that new drugs being developed will have
fewer adverse effects due to extensive use of biomarkers to
identify adverse events in preclinical animal models or in clinical
trial patients. As additional generations of drugs continues to be
developed, the list of relevant biomarkers and their changes can be
refined further. In addition, as it becomes clear whether each
biomarker is indicative of desired or undesired effects, more
information about the mechanisms of drug action are learned,
helping to direct development of next generation drugs.
Accordingly, these ultrastructural biomarkers can allow for the
monitoring and evaluating of drug or therapeutic safety and
efficacy during discovery, preclinical and all levels of clinical
trials, as well as post sales monitoring and testing. Furthermore,
a similar approach may be used to determine and evaluate patient
response or response rate, as well as clinical trial participant
response or response rate.
[0044] Similarly, the above protocol can be employed to generate
information that can lead to the understanding of the risks of
adverse events, toxicity or serious adverse events associated with
marketed drugs or therapeutics, drug or therapeutic candidates, as
well as risks for drug attrition. Such an understanding can assist
in a decision making process during clinical development, thereby
driving informed stop/go decisions early in, or prior to clinical
development. The information may also be used, in an embodiment, in
designing and developing drugs or therapeutics that can be tailored
to address only relevant disease mechanisms while causing fewer
adverse effects.
[0045] The present invention, in addition to being able to resolve
ultra-structural features in the morphology of cells, cellular
organelles, and extracellular matrix, can also employ Cryo-TEM and
M-FFDI to detect macromolecular structural differences in lipid
droplets, vesicles, and other structural components in biological
fluids.
[0046] Furthermore, the present invention permits, in an
embodiment, identification of the spatial positions of proteins in
a larger assembly or changes of protein complex morphology. This is
important because proper assembly can be critical to the
functioning of the protein complexes and cell organelles. In
particular, there are potential changes in morphology and
aggregates in different stages of disease which can change size or
size distribution. Since these changes are physical, the
identification process employed by a method of the present
invention does not require any a priori knowledge of specific
biological targets. Accordingly, sole reliance on biochemical
assays can be eliminated.
[0047] The high resolution images of these ultrastructures, ranging
from nanometers to micrometers in size, thus provide clear
indications that cryogenic vitrification and high contrast imaging
achieved through cryo-TEM or M-FFDI can generate a powerful tool
for analyzing these nanostructures and changes to these
nanostructures in biological samples. Whether, the sample is fluid
or viscous, the provision of the either cryo-TEM or M-FFDI, as
disclosed herein, can create broad capability to examine relevant
bio-samples under conditions that most closely resemble their
native states. For instance, biomarkers that are from any of parts
of the human body, including any viruses, bacteria or other
pathogens residing in any part of the human body can be identified
employing the methods of the present invention.
[0048] Moreover, since the present invention involves utilization
of resolutions relatively far beyond those traditionally used, the
potential for discovery of early changes of structural markers can
be substantially high. Thus, the use of cryo-TEM and M-FFDI coupled
with image analysis can provide a novel and high resolution
approach for the identification of ultrastructural biomarkers.
Furthermore, such an approach has the potential to change the
paradigm and dramatically reduce the cost associated with biomarker
discovery and validation, by providing a robust and relatively
sensitive approach to diagnosing and monitoring diseases, while
simultaneously reducing drug development costs.
[0049] Although the above description has been provided in the
context of human subjects, it can be equally well applied to animal
models, particularly those with immune systems similar to the human
immune systems. For instance, suitable animals include mice, rats,
and rabbits.
[0050] While the invention has been described in connection with
the specific embodiments thereof, it will be understood that it is
capable of further modification. Furthermore, this application is
intended to cover any variations, uses, or adaptations of the
invention, including such departures from the present disclosure as
come within known or customary practice in the art to which the
invention pertains, and as fall within the scope of the appended
claims.
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