KEYWORDS: Luminescence, Phase only filters, Signal detection, Calcium, Tissue optics, Tissues, Microscopes, In vivo imaging, Objectives, Polymer optical fibers
The fiber-coupled microscope (FCM) enables in vivo imaging at deep sites in the tissues or organs that other optical techniques are unable to reach. To develop FCM-based intravital imaging, we employed a plastic optical fiber (POF) bundle that included more than 10,000-units of polystyrene core and polymethyl methacrylate cladding. Each POF had a diameter of less than 5 μm; the tip of the bundle was less than 0.5 mm wide, and the flexible wire had a length of 1,000 mm. The optical performance of the plastic FCM was sufficient for detection of significant signal changes in an acinus of rat pancreas labeled with a calcium ion–sensitive fluorescent dye. In the future, the potential power of plastic FCM is expected to increase, enabling analysis of structure and organization of specific functions in live cells within vulnerable organs.
To study neuronal functions in brain, we developed a higher resolution type fiber-coupled microscope (FCM), and
measured the activity-dependent fluorescence intensity of the excitable cells over time. FCM was constructed by
combining a fluorescence microscope with the high density type of fiber bundle, which consisted of 1.5 x 104 unit fiber in the assemble less than 0.5 mm tip. The spatial resolution was calculated to be 2.4 mm with the 5 mm focal depth. The
activity-dependent Ca signals were detectable in each cell of either the pancreatic spheroids or the brain slices. The
present FCM is very promising for detailed studies with the live imaging of signal molecules in the body at a single cell
level.
Cell membrane motions of living cells are quantitatively measured in nanometer resolution by low-coherent full-field
quantitative phase microscopy. Our setup is based on a full-field phase shifting interference microscope with a very lowcoherent
light source. The reflection mode configuration and the low-coherent illumination make it possible to
differentiate the weak reflection light from the cell membrane from the strong reflection from the glass substrate. Two
cell populations are quantitatively assessed by the power spectral density of the cell surface motion and show different
trends.
In the PDT practice for tumor patients, the dose and irradiation time for the treatment are chosen by experience and not
by real need. To establish advanced PDD-PDT model system for patients, we developed a method for monitoring the
cell-death based on a spectrophotometric real-time change in fluorescence in HeLa-tumors during Photofrin®-PDT and
ALA-PDT. Here, we describe the results of application of the new PDD-PDT system to human tumors. The fluorescence
spectra obtained from human tumors were analyzed by the differential spectral analysis. The mass-spectral changes of
tumor tissues during PDD-PDT were also examined by MALDI-TOF-MS/MS. The first author's seborrheic keratosis was
monitored with this system during the PDD-PDT with a topically applied ALA-ointment. The changes in fluorescence
spectrum were successfully detected, and the tumor regressed completely within 5 months. The differential spectral
analysis of PDD-PDT-fluorescence monitoring spectra of tumors and isolated mitochondria showed a marked decrease of
three peaks in the red region indicative of the PDD (600 - 720 nm), and a transient rise followed by a decline of peaks in
the green region indicative of the PDT (450 - 580 nm). The MALDI-TOF-MS analysis of PDD-PDT HeLa-tumors
showed a consumption of Photofrin-deuteroporphyrin and ALA-PpIX, and decreases in protein mass in the range of
4,000 - 16,000 Da, m/z 4929, 8564, 10089, 15000, and an increase in m/z 7002 in a Photofrin® PDD-PDT monitoring
tumor.
The photodynamic therapy (PDT) on tumors is quite effective and widely applied but usually carried out without an immediate evaluation of results. We measured the tumor fluorescence in mice with a fiber probe connected to a linear array spectral analyzer (PMA-11, Hamamatsu Photonics). The spectrum showed a transient change in fluorescence color from red to green during Photofrin-mediated PDT. In order to examine the source of green fluorescence, the mitochondria were accessed under a Nipkow disk-scanning confocal microscope in the HeLa cell in culture after labeling them with a red fluorescent protein (DsRed1-mito) and staining the cell with Photofrin (Axcan Scandipharm). Changes in fluorescence color from red to green were observed in the area of mitochondria upon their swelling during irradiation. This finding in vitro provided clear evidence that the change in fluorescence color from red to green observed in vivo was due to the mitochondrial destruction associated with the cell-death by PDT. This technique of spectral monitoring in tumor may be useful for detection of the cell-death signal during PDT in patients.
In order to study the dynamic change in the cell, we modified the evanescence microscope with an ultra high NA objective lens so as to modulate the penetration depth of the evanescent wave. We employed a galvanomirror to aim and switch the laser beam rapidly at the back focal plane near the periphery of 1.45 or 1.65 NA objectives. Under this microscope equipped with a 1.45 NA objective, images of the fluorescent bead were clearly distinguishable by the modulation of the penetration depth of the evanescent wave. Thus, translocation dynamics of protein kinase Cα (PKCα) upon cell activation were compared every 0.5 s between two modes using HeLa cells expressing PKCα fused with the green fluorescent protein (GFP). Stimulation of the cell with phorbol ester induced a transient increase in GFP fluorescence images illuminated by the thin evanescent field, but not in the image illuminated by the thick evanescent field. Later, a persistent increase in fluorescence appeared at cell borders in the both images. Using a 1.65 NA objective, trafficking of secretory vesicles was studied in MIN6 cells expressing insulin-GFP. Occasionally, the change in fluorescence of a vesicle observed under one illumination mode appeared very different from the other, allowing unique assignments of the fluorescence change to a certain combination of vesicle movement and a chemical response of fluorescent molecules. The ultra high NA lens provides a large window for evanescent illumination with a wide range of penetration depth, thus is useful for analyzing 3D events in the cell.
KEYWORDS: Confocal microscopy, Microscopes, In vivo imaging, Luminescence, Objectives, Signal detection, Real time imaging, Photodynamic therapy, Tumors, Clinical research
To study cellular morphology and functions in vivo in realtime, we developed a fiber-coupled confocal microscope (FCM), and observed fluorescently-labeled cells inside the body of anesthetized rat. We developed an imaging fiber bundle (IFB), which consisted of an objective lens and a multi-fiber assembly (unit fiber: NA > 0.4, 3 micron in diameter). By combining the IFB with a real-time confocal scanner, we detected intracellular signals of the molecular messenger, and the death signals in the form of fluorescence changes even from cells located deep (> 2 mm) inside the solid organs. The FCM we developed is very promising for detailed studies in both the cell-based researches and clinical researches.
By employing the total internal reflection fluorescence (TIRF) microscope with an ultra high NA (1.65) objective lens, we demonstrated detailed dynamics of exocytosis in various types of secretory vesicles. However, the TIRF microscopy could be applied to observations only on the plasma membrane and its immediate vicinity. To observe the vesicles in the deeper region of cytoplasm, we modified the TIRF optics to project a slit beam thinner than 1 μm in width to the cell. The slit beam illumination spotted single secretory vesicles inside the cell better and their movement and exocytosis easier. By scanning the slit beam, a fluorescence microscopy was possible at a high signal-to-noise ratio useful for
measurement and analysis of single exocytosis in neurons and endocrine cells.
Optical imaging with a high detecting power is very instrumental to dynamic observations of bio-molecular objects in water. To pioneer this field of imaging researches further, we used novel objective lenses of 1.65 and 1.45 in NA comparatively with 1.35 NA lens. The lenses of ultra high NA have a high resolving power and very useful properties for observation of high contrast images of fluorescently labeled molecules and subcellular organelles including DNA and membrane fusion proteins.
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