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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5528_Библиотеки_им_академика_М_И_Перельмана.pdf
290 E. Rubin
(a) (b) (c)
(d) (e) (f )
(g) (h) (i)
et al.
Figure 2. In vivo temporospatial patterns of hepatocyte necrosis. (Top row) Alterations of
cell shape and fluorescence intensity: (a) Initial swelling and vesicle formation (arrows), followed by shrinking and reduced fluorescence (b, c). (Middle row) Alterations of the cytoplasm.
(d) Cell membrane blebbing, followed by formation of intracellular vesicles (e) and eventual loss of
cell integrity (f). (Bottom row) Nuclear changes. Nuclear blebbing (g), followed by karyorrhexis (h),
and pyknosis (i). Adapted from Ref. [21].
The study also observed single-cell necrosis of hepatocytes as a sign
of inflammation. Inflammation was characterized by bright, dense cellular
material surrounded by a black halo, potentially correlating with cellular
debris. Bile canaliculi were also identified in four patients. However, due
to limited imaging penetration depth, blue laser-CLE could not resolve the
Hepatobiliary System 291
Figure 3. Confocal views of basic non-neoplastic liver pathologies. (Left panel) Steatotic droplets are
seen as rounded black structures. (Middle panel) Bands of fibrosis are seen as strands of increased
fluorescence signal. (Right panel) Necrotic hepatocytes areidentified by a targetoid structure. Adapted
from Ref. [18].
Figure 4. Subcellular resolution with indocyanine green contrast agen. (Left panel) ICG accumulates
in the cytoplasm of hepatocytes, allowing individual cells to be easily visualized.(Middle panel) Hepatic
lobules are separated by non-fluorescing septae that appear as dark bands. (Right panel) Hepatocyte
nuclei are visualized indirectly as non-fluorescing dark round structures within hepatocytes. Adapted
from Ref. [19].
liverparenchyma 250 µm below the capsule. In a follow-up human trial,19a
near-infrared laser-CLE combined with a long-wavelength fluorescent dye
indocyanine green was used to obtain a deeper imaging depth and better
imaging quality. In this study, subcellular details including nuclear details
or intracellularinclusions could be visualized in vivo through the intact liver
capsule (Figure 4). Liver fibrosis and steatosis could be reliably imaged and
scored in vivo. Accuracy of CLE to predict the presence of steatosis and
fibrosis was 81% and 90%, respectively. No liver damage or complications
occurred.
292 E. Rubin
Figure 5. In vivo pCLE imaging of colorectal cancer metastases. (Left panel) A pCLE image of
a healthy liver displaying fluorescent hepatocytes with non-fluorescent nuclei. (Middle panel) pCLE
images of metastatic liver nodules after chemotherapy treatment, without residual cancer. A very
dense and strongly fluorescent scar fibrosis replaced the line-structured hepatocytes. (Right panel)
pCLE images of metastatic liver nodules showing irregular, fluorescent cancerous tubes on a dark
extracellular matrix. Adapted from Ref. [20].
et al.
A more recent human study investigated CLE in ex vivo diagnosis
of metastatic liver nodules in fresh surgical specimens from 30 patients,
with indocyanine green as a contrast agent.
20
The study showed that CLE
can reliably differentiate between colorectal liver metastasis and normal
liver parenchyma, with high specificity and positive predictive value. CLE
of malignant nodules revealed the presence of irregular clusters or tubular
structures of cells surrounded by a relative ly dense extracellular matrix in
contrast to normal liver parenchyma (Figure 5).
Although both intrahepatic biliary and liver CLE imaging feasibility
has been demonstrated in human trials, the application of CLE in routine clinical diagnosis and treatment of hepatobiliary diseases is still in
the process of refinement. Larger size clinical trials are needed to improve
CLE systems and algorithms as well as establish criteria for CLE in vivo
diagnosis in different hepatobiliary diseases. In addition, future studies on
in vivo CLE molecularimaging with specific fluorescence-labeled,diagnostic markers or therapeutic targets will make CLE a valuable clinical adjunct
technology to provide faster and more specific microscopic diagnoses.
Future Directions
In vivo microscopy offers a novel way of visualizing disease processes
that may ultimately yield new insights into disease biology or new criteria
for diagnosis. The development of new endoscopic imaging modalities
Hepatobiliary System 293
with greater penetration depth, wider field of view, a nd three-dimensional
visualization will enable real-time, high-resolution, and more informative
“optical biopsy” with high overall accuracy.
A recent example includes light-sheet microscopy. This technique
offers non-destructive, high-resolution, rapid imaging of intact clinical tissue samples over large 2D and 3D fields of view with the same level of
detail as traditional H&E stains. One study demonstrated the utility and
feasibility of this imaging technology in wide-area surface microscopy to
triage surgicalspecimens, for rapid intraoperative assessment of tumor margins, and for deep volumetric assessment of optically cleared, core needle
biopsies.
22
Three-dimensional (3D) imaging allows real-time assessment of tissue structures and disease distribution by obtaining sequential Z-sections
images. Acquiring Z-sections over time enables the production of 4D rendering modules. Pathophysiology is easily studied using 3D and 4D imaging as the location, shape, volume, and movement of cellular subtypes can
be investigated. An example of applicability is in the pre-transplant donor
setting. This technique could be particularly useful in the determination
of organ viability. The current technique of the frozen section evaluation,
or the cryosection, demonstrates ischemic changes only after 4–12 h after
injury.
Conclusion
In vivo microscopy of the liver, like most solid organs, is at an early stage of
development when compared to the skin and tubular gastrointestinal tract.
However, the availabilityof very small imaging devicessuch as nCLE opens
new possibilities. Much of the groundwork has been laid in the form of animal and ex vivo studies. Large-scale clinical trials are the next logical step.
The possibilities are numerous and include intraoperative margin assessment and biopsy targeting, pre-transplant donor evaluation, as well as more
speculative, but potentially transformative techniques, such as molecular
imaging.
In vivo optical imaging technologies, represented by OCT and CLE,
are powerful approaches that allow physicians to rapidly screen a wide area
294 E. Rubin
et al.
of hepatobiliary pathologyand to reliably identify suspicious lesions in vivo
such as fibrosis, steatosis, and tumors. These imaging modalities hold a
great promise to redefine the clinicians’ approachto diagnose and to manage
hepatobiliary diseases. IVM has the potential to eliminate the need for tissue
biopsy and to facilitate timely application of therapy during the course
of endoscopic procedures. Particularly, with the anticipated development
of new specific fluorescence-labeled biomarkers of diseases (e.g. specific
antibodies for diagnosis, treatment, and monitoring disease progression), it
will be possible to obtain in vivo optical diagnosis with high sensitivity and
specificity. We speculate that in the not too distant future, the new optical
imaging technologieswill open new avenues for diagnosis and management
of hepatobiliary diseases.
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© 2024 World Scientific Publishing Company
https://doi.org/10.1142/9789813206984_0015
Molecular Applications Chapter
15
Satoru Kudose∗and Anne Marie Amacher
Much of routine pathologic examination of specimens still utilizes histochemical stains to distinguish different tissue types, which otherwise have
limited contrast in brightfield microscopy. In general, these stains rely on
differencesin the organization of macromoleculesin tissues, and their interactions with dyes through various modes of chemical bonding.
ular, these dyes are directed to their potential binding sites by electrostatic
forces generated from the manipulation of solvent during the staining process, and actualbinding of the dye totissues (i.e. “staining”)typically occurs
through other forces such as hydrophobic interaction.
non-specific nature of traditional histochemical methods, immunohistochemistry or in situ hybridization are routinely used to obtain more specific
staining for a particular protein or nucleic acid.
†
1
Given the relatively
1
In partic-
∗
Columbia University Irving Medical School, New York, USA.
†
Pathology Department, SSM DePaul Health Center, Bridgeton, MO, USA.
297
298 S. Kudose & A. M. Amacher
Similarly, many in vivo microscopy modalities provide contrast by
exploiting differences in physical and chemical properties of the tissues
that affect how these tissues interact with electromagnetic waves. Consequently, when these modalities are used without the use of dye to differentially stain various tissue components, the contrasts they provide can be
limited and are typically not specific for a particular protein or DNA. To
overcome this problem, multiple approaches have been developed, which
can be categorized broadly into those with and without the use of exogenous
agents.
Exogenous agents are typically composed of probes attached to
reporters, and conceptually are similar to immunohistochemistry or in situ
hybridization. Probes are ideally specific to cells and tissues of interest and
can be proteins, such as peptide fragments or antibodies, or other small
molecules. The use of probes in vivo is more challenging than immunohistochemistry on ex vivo tissue for multiple reasons. These probes must
be soluble, stable, and non-toxic to the tissues of interest and preferably
2
should not interfere with downstreamsignaling.
Also, theyshould not elicit
immune response. Furthermore,these probes must be able to reach their target without manipulation of tissues and the surrounding environment, since
these surrounding environment cannot be manipulated to optimize staining
behavior of these probes unlike in ex vivo applications. Lastly, they must
remain excitable in tissues long enough to be detected but cleared promptly
afterward.In contrast to antibody-based probes, which can be immunogenic
and do not cross the plasma membrane, which limits their use for surface
antigen only, smaller peptides and nucleic acids have been found to have
similar specificity with reduced immunogenicity.
3
These probes are conjugated to a reporter which can be relatively
familiar molecules, like fluorescein, or nanoparticles that contain metals,
like gold. To serve as a useful reporter for the study of in vivo processes,
these reporters must be soluble, stable, and non-toxic to the tissues of interest similar to probes. Furthermore, these reporters must be conveniently
excitable and emit light differently from surrounding tissues to provide a
2
high signal-to-noise ratio.
Reporters that fluoresces at near-infrared region
(NIR, 650–900 nm) have been found to have relatively deep tissue penetration with reduced autofluorescence.
4
Molecular Applications 299
Various nanoparticle-based reporters have been studied in recent
years, and they can be divided into plasmonic and non-plasmonic types.
Plasmonic nanoparticles are made of metals such as gold in varying shapes
and sizes, like cages and rods, and utilize the surface plasmon resonance
effect. These are typically used in Raman imaging and photoacoustic imaging, fluoresces at NIR region with good photostability, and havebiocompat-
6,7
ibility.
Non-plasmonic types depend on other means to generate light,
examples of which include carbon nanotubes and quantum dots. Quantum dots are nano-scale s emiconductors with narrow and tunable emission
spectra and broad absorption spectra with good photostability. While these
properties make quantum dots promising for in vivo microscopy applications, their utilizationhas been limited by their toxicity in cases of Cd-based
8
quantum dots.
These nanoparticles can be made specific by conjugating
them to other molecules, such as antibodies, however some nanoparticles
have been observed to preferentially accumulate in tumors due to enhanced
permeability and retention (so-called EPR effect), believed to be caused by
tumor-associated microvascular proliferation and poor lymphatic drainage
within the tumor.
9
A study by Burggraafet al.
illustratessome of the practical issues that
must be considered to design these probes. They designed a water-soluble
peptide probe using a phage library and conjugated it to a fluorescent dye
to improve the detection of tubular adenoma by colonoscopy. The probe
was directed against c-Met, which is known to be upregulated in colorectal
adenoma and adenocarcinoma, and was tested to ensure little binding to
plasma protein. A fluorescent reporter with spectral characteristics that
do not overlap with colonic autofluorescence was chosen. The probe was
intravenously injected and was tested for safety in rats and monkeys at
various doses, including investigation specifically for genotoxicity. Then,
the probes were tested in healthy volunteers and in 15 patients high-risk for
colorectal neoplasia. Although a total of 101 lesions were detected using
conventional colonoscopy only, additional 22 lesions were found using the
custom colonoscopy, of which 17 were not visible grossly (Figure 1). These
lesions were small and had non-polypoid morphology. A similar approach
has been used to visualize Barrett’s esophagus using probes sprayed over
a region of interest instead of intravenous agents.
10
5