In this study, we used an epi-illuminated multiphoton microscope to image three main components of ex vivo human tooth. In particular, we obtained two-photon excited autofluorescence (AF) and second-harmonic generation (SHG) images of the enamel, dentin, and periodontal ligaments (PLs) and constructed three-dimensional projections of sequentially and axially acquired images. We found that the enamel has a strong two-photon AF signal, clearly revealing the structures of the enamel rods. The dentin, on the other hand, has both AF and SHG signals. The contrast provided by the combination of these two imaging modalities can be used to reveal the structure of peritubular dentin and to distinguish the less mineralized circumpulpal dentins. The SHG and multiphoton AF imaging also showed the structure of the PL and the distribution of cells around the PL, respectively. For comparison, we also obtained scanning electron microscopy images of the enamel, dentin, circumpulpal dentin, and the PL. Our results demonstrate the effectiveness of using multiphoton microscopy to visualize the major constituents of teeth, including enamel, dentin, and the PL, and the potential of this minimally invasive technique for monitoring the morphological developments during tooth regeneration.
Detailed morphological and cellular information relating to the human tooth have traditionally been obtained through
histological studies that required decalcification, staining, and fixation. With the recent invention of multiphoton
microscopy, it has become possible to acquire high resolution images without histological procedures. Using an epiilluminated
multiphoton microscope, we obtained two-photon excited autofluorescence and second harmonic generation
(SHG) images of ex vivo human tooth. By combining these two imaging modalities we obtained submicron resolution
images of the enamel, dentin, and the periodontal ligaments. The enamel emits endogenous two-photon
autofluorescence. The structure of the dentin is visible from both the autofluorescence and second harmonic generation
signals. The periodontal ligament composed mostly of collagen can be visualized by SHG imaging. We also
constructed three dimensional images of the enamel, dentin, and periodontal ligament. The effectiveness of using
multiphoton and second harmonic generation microscopy to obtain structural information of teeth suggest its potential
use in dental diagnostics.
The aim of this study is to assess the application of multiphoton autofluorescence and second harmonic generation
(SHG) microscopy for investigating the structural alterations and the pattern of microbial spreading during corneal
infectious process in an in vitro organ culture model. The autofluorescence spectrum derived from pathogens allows us
to monitoring the pattern of microbial spreading within corneal lamellae. In addition, the destruction and regeneration of
second harmonic generating collagen during infectious process can also be monitored in a non-invasive fashion.
Therefore we propose that multiphoton microscopy may potentially be applied as an effective monitoring tool for
corneal infection studies.
The aim of this work is to image the wound healing process of cornea in an in vitro organ culture model with noninvasive
multiphoton imaging modality. Autofluorescence and second harmonic generation (SHG) were respectively
used to monitor the alterations of cellular and collagenous components during wound healing processes. Within
additional developments, this approach may be applied to in vivo visualization of corneal structural destruction and the
subsequent regeneration.
The purpose of this study is to demonstrate the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for the ex-vivo visualization of human corneal morphological alterations due to infectious processes. The structural alterations of both cellular and collagenous components can be respectively demonstrated using fluorescence and SHG imaging. In addition, pathogens with fluorescence may be identified within turbid specimens. Our results show that multiphoton microscopy is effective for identifying structural alterations due to corneal infections without the need of histological processing. With additional developments, multiphoton microscopy has the potential to be developed into an imaging technique effective in the clinical diagnosis and monitoring of corneal infections.
Using the technique of second-harmonic generation (SHG) microscopy we obtained large area image of type I collagen
from rat tail tendon as it is heated from 40°C to 70°C for 0 to 180 minutes. The high resolution images allowed us to
investigate the collagen structural change. We observed that heating the tendon below the temperature of 54°C does not
produce any change in the averaged SHG intensity. At the heating temperature of 54°C and above, we find that
increasing the heating temperature and time leads to decreasing SHG intensity. As the tendon is heated above 54°C, a
decrease in the SHG signal occurs uniformly throughout the tendon, but the regions where the SHG signal vanishes form
a tiger-tail like pattern. By comparing the relative SHG intensities in small and large areas, we found that the
denaturation process responsible for forming the tiger-tail like pattern occurs at a higher rate than the global denaturation
process occurring throughout the tendon. Our results show that second-harmonic generation microscopy is effective in
monitoring the thermal damage to collagen and has potential applications in biomedicine.
Collagen shrinkage associated with denaturation from thermal treatment has a number of important clinical applications. However, individualized treatment is hindered by the lack of reliable noninvasive methods to monitor the process of collagen denaturation. We investigate the serial changes of collagen denaturation from thermal treatment of rat tail tendons at 58 °C by use of second harmonic generation (SHG) microscopy. We find that rat tail tendon shrinks progressively from 0 to 9 min of thermal treatment, and remains unchanged in length upon further thermal treatment. The SHG intensity also decreases from 0 to 9 min of thermal treatment and becomes barely detectable from further thermal treatment. Collagen shrinkage and the SHG intensity are well correlated in a linear model. In addition, SHG imaging reveals a tiger-tail-like pattern of collagen denaturation. The bands of denatured collagen progressively widen from increased thermal treatment and completely replace the adjacent bands of normal collagen after 9 min of thermal treatment. Our results show that collagen denaturation in rat tail tendon from thermal treatment is inhomogeneous, and that SHG intensity can be used to predict the degree of thermally induced collagen shrinkage. With additional development, this approach has the potential to be used in biomedical applications.
The purpose of this investigation is to characterize corneal wound healing under in vitro conditions. Multiphoton
autofluorescence and second harmonic generation (SHG) microscopy will be used to visualize cells and collagen fibers
associated with corneal wound healing. Using the near-infrared excitation source from a titanium-sapphire laser pumped
by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton
autofluorescence and SHG signals from the cornea specimens. A home-modified commercial microscope system with
specified optical components is used for optimal signal detection. To acquire both high resolution and tissue-level
information of the specimen, a sample positioning stage is used in conjunction with the beam scanning system. Finally,
the organ level image can be assembled from individual area scans. The in vitro samples we used are cornea buttons
acquired from porcine eyes. Localized wounds will be induced by #11 blade and imaged using multiphoton microscopy.
Based on these results, we envision the in vitro imaging chamber to be able to follow the wound healing process without
damaging histological procedures. We envision this approach will enable us to further understand wound healing process
associated with corneal scar and can lead to in vivo methodology for diagnosing cornea damage.
The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic
generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as
being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser
pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton
autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial
microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button
from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The
specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of
lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In
addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the
cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover,
increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area.
In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological
features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future
the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.
The purpose of this work is to demonstrate the combination of reflective confocal microscopy and multiphoton
microscopy and its application in imaging cornea. The difficulty of optically imaging the highly translucent cornea has
prevented the development of an effective non-invasive system for the clinical monitoring of the physiological or
pathological states of corneas. In this work, we combine reflective confocal microscopy with multiphoton microscopy
to demonstrate the potential of our methodology in the minimally invasive imaging of the cornea. The visible reflection
signals from cornea can provide structural information of interfaces of different refractive indices while the multiphoton
signals generated from the use of near infrared excitation allows deep tissue penetration and reduced photo-damage. In
multiphoton imaging, the second harmonic generation (SHG) signal is used to detect collagen in the stroma of the cornea,
and the reflective confocal imaging allows detection of the cellular components located in the epithelium. The
combination of reflective and multiphoton imaging can be used to reveal complementary structural information of the
corneal architecture.. The system is first tested on porcine eye cornea. Assessment of the result on the porcine eye will be
used to evaluate the potential of the system as a technique for in vivo clinical applications.
The purpose of this study is to assess the application of multiphoton fluorescence and second harmonic generation (SHG)
microscopy for imaging and monitoring the disease progress of infectious keratitis in an experimental model, and to
investigate the possible correlation of tissue architecture with spreading patterns of pathogens in an experimental model.
Porcine eyes are to be obtained from slaughter house and processed and placed in organ culture system. Fungal
infections by common pathogens of infectious keratitis are to be induced in porcine cornea buttons. Multiphoton
fluorescence and SHG microscopy will be used for imaging and for monitoring the progression and extension of tissue
destruction and possibly the pattern of pathogen spreading. We found that SHG imaging is useful in identifying
alterations to collagen architecture while autofluorescence microscopy can be used to visualize the fungi and cells within
the stroma. In summary, multiphoton fluorescence and second harmonic generation microscopy can non-invasively
demonstrate and monitor tissue destruction associated with infectious keratitis. The pattern of pathogen spreading and its
correlation with the tissue architecture can also be shown, which can be useful for future studies of the tissue-microbial
interactions for infectious keratitis.
Thermal treatment induced collagen shrinkage has a great number of applications in medical practice. Clinically, the there is lack of reliable non-invasive methods to quantify the shrinkage. Overt treatment by heat application can lead to devastating results. We investigate the serial changes of collagen shrinkage by thermal treatment of rat tail tendons. The change in length is correlated with the finding in second harmonic generation microscopy and histology. Rat tail tendon shortens progressively during initial thermal treatment. After a certain point in time, the length then remains almost constant despite further thermal treatment. The intensity of second harmonic generation signals also progressively decreases initially and then remains merely detectable upon further thermal treatment. It prompts us to develop a mathematic model to quantify the dependence of collagen shrinkage on changes of SHG intensity. Our results show that SHG intensity can be used to predict the degree of collagen shrinkage during thermal treatment for biomedical applications.
The ability to image tissue engineering products without damaging histological procedures is important for the understanding of the dynamics of tissue reorganization and formation. In this work, we test the ability of multiphoton autofluorescence and second harmonic generation microscopy to image engineered tissues following chrondrogenic induction. The system we used is human bone marrow stem cells seeded in the scaffold polyglycolic acid (PGA). Our results show that autofluorescence can be used to image cells while second harmonic generation signal can be used to visualize the synthesis
of extracellular matrix. This approach demonstrates the ability of multiphoton imaging in the study of tissue engineering products.
Multiphoton microscopy has evolved into a powerful bioimaging tool in three dimensions. However, the ability to image biological specimens in-depth can be hindered by sample spherical aberration and scattering. These two phenomena can result in the degradation of image resolution and the loss of detected multiphoton signal. In this work, we use the correction collar (for cover glass thickness) associated with a water immersion objective in an attempt to improve multiphoton imaging. In the two samples we examined (human skin and rat tail tendon), we found that while the improvement in image resolution was not visible qualitatively, the measured axial fluorescence or second harmonic generation signal profiles indicate that the use of the correction collar can help to improve the detected multiphoton signals. The maximum increases are 36% and 57% for the skin (sulforhodamine B fluorescence) and tendon (second harmonic generation) specimens, respectively. Our result shows that for in-depth multiphoton imaging, the correction collar may be used to correct for spherical aberration. However, each tissue type needs to be examined to determine the optimal correction collar setting to be used.
Physical and chemical enhancement techniques have proven to be useful in promoting transdermal delivery of therapeutic agents. However, it has been difficult to follow the changes to the skin during these processes microscopically. In this work, we use multiphoton polarization (P) and generalized polarization (GP) imaging of the membrane probe Laurdan to monitor changes to the stratum corneum under conditions of chemically enhanced delivery by oleic acid. We show that the combination of P and GP Laurdan imaging helps to reveal both the physical and chemical changes to the stratum corneum under enhanced delivery. Our work can help to develop more efficient transdermal delivery strategies.
In recent years, non-linear imaging techniques such as multiphoton fluorescence and harmonic generation microscopy have been successfully applied to dermatological imaging. Confocal-like image quality, enhanced depth penetration, and non-linear spectral signatures are among the main advantages of this family of techniques. In this presentation, we will focus on the applications of multiphoton microscopy to skin specimens in different physiological states. Images of normal and diseased tissue specimens will be presented and spectrally characterized. Our work has potential applications in developing multiphoton microscopy into a clinically applicable diagnostic tool.
As a major component of the connective tissues, collagen fibers are responsible for various physiological functions inside the body. They provide support for the skin, partial focusing through the cornea, and coordinate movements via tendons, ligaments, and cartilages. In many medical procedures, thermal reorganization of the collagen structure is inevitable or desired. Therefore, the optimization of the therapeutic values of these procedures requires the characterization of thermal changes to collagen fibers. In this presentation, we use multiphoton microscopy to achieve this task. We will show that second harmonic generation (SHG) microscopy can characterize the thermally altered states of collagen and that they have potentials to be used in imaging applications in vivo.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.