The research in our laboratory focuses on developing optical techniques and the application of these techniques in elucidating fundamental biophysical processes and improving human healthcare.

1.  Developing optical technologies in bioimaging

Second order susceptibility microscopy                              

       Nonlinear phenomena such as multiphoton fluorescence excitation and second harmonic generation have been successfully applied to the imaging of biological structures. Unlike one-photon processes, two-photon excitation (lowest order of multiphoton excitation process) involves the simultaneous absorption of two less energetic photons in molecular excitation (Fig. 1). In this manner, molecular excitation is limited to the focal volume, minimizing photodamage to biological specimens. Furthermore, limited excitation focal volume also allows axial image sectioning to be achieved. Finally, the near-infrared photons used in two-photon excitation can penetrate deeper into biological specimens, enabling in-depth imaging to be achieved. These advantages have enabled multiphoton fluorescence excitation microscopy to be the preferred technique in a wide arry of bioimaging applications. Second harmonic generation, the nonlinear polarization response of non-centrosymmetric materials, share the common advantages as multiphoton fluorescence excitation and is also widely used in studying biological systems.



Fig. 1 Principles of two-photon fluorescence excitation. In one-photon excitation (left), the molecule is excited by the absorption of one UV or visible photon. In two-photon excitation (right), two near-infrared photons are simultaneous absorbed by the molecule in reaching the excited state.

      In addition to conduct research with multiphoton excitation fluorescence and second harmonic generation microscopy, we found that the second order susceptibility of can also be used as a contrast mechanism for imaging non-centrosymmetric tissues such as collagen. Since collagen is the most abundant mammalian protein responsible for key roles in maintain the structure and function in human physiology, the development of an imaging technique capable of resolving different collagen types is important in collagen biophysics and may be applied to tissue engineering in constructing tissues for organ repair. We found that second order susceptibility microscopy (SOSM) can be used to discriminate types I and II collagen. Specifically, the second order susceptibility ratios of (cxzx/czxx)  and (czzz/czxx) for the two types of collagen are different. These differences can be converted to images to identify the presence of each molecular species (Fig. 2).



Fig. 2 Second order susceptibility images of engineered cartilage from human mesenchymal stem cells. (A) and (B) are the (cxzx/czxx)  and (czzz/czxx) resolved images respectively. Yellow arrows indicate location of type I collagen while the green arrows show the presence of type II collagen.

Femtosecond laser ablation

      In addition to microscopic imaging, focused laser beam can also be used to modify biological structures on a microscopic scale. As shown in Fig. 3, after scanning a focused beam of femtosecond, titanium-sapphire laser across a collagen fiber, the fiber can be severed. The precise control of biological structure at the micron-scale enable studying the effects of opto-mechanical perturbation on the evolution of different constituents of organisms and may provide a way to create tissues of desired spatial organization.



Fig. 3 Femtosecond laser ablation can be used to alter the structure of collagen fiber from rat tail tendon. (A) and (B) are the respective second harmonic generation images of the tendon before and after light induced excision (along the blue line shown in (B)). Scale bar: 20 μm.

2.  Elucidation of biophysical processes

Nanoparticle transport in biological systems

      There has been considerable interest in the use of nanoparticles as vehicles for delivering drug molecules or in other therapeutic processes. Therefore, understanding the transport of nanoparticles into and out of biological tissues may help to design improved delivery strategies. We have studied nanoparticle transport into the skin and liver. Shown in Fig. 4 are images we acquired of fluorescent nanoparticles in these two organs. In the skin, the nanoparticles are primarily confined to the intercellular domain while in the liver, negatively charged fluorescent nanoparticles are processed by the hepatic Kupffer cells.


Fig. 4 (A) Upon delivery, fluorescence nanoparticles (blue) are located primarily in the intercellular region. (B) Uptake of negatively charged fluorescet nanoparticles (yellow arrow) by hepatic Kupffer cells (red). Scale bar: 50 mm.

Intravital hepatobiliary metabolism

      The liver is a vital organ responsible for a plethora of important physiological functions such as the excretion of albumin and detoxification of waste molecules. Through the use of intravital multiphoton microscopy, we can observe the uptake, processing, and excretion of fluorescent molecules. Shown in Fig. 5 are the time-lapse, multiphoton images of the probe molecule 6-carboxyfluorescein diacetate (6-CFDA) by the liver. Upon enzymatic processing by intracellular esterases of the hepatocytes, 6-CFDA is converted into the fluorescent form of 6-carboxyfluorescein (6-CF) which is eventually excreted into the bile canaliculi. In these images, the entire metabolic process can be followed as the appearance of intracellular fluorescence (green) indicate locations of the processed 6-CF molecules. With time, the 6-CF molecules are excreted into the bile canaliculi, resulting in a decrease of fluorescence within the hepatocytes. This approach enables us to study dynamic transport process in the liver and yield insights how different disease can affect this biophysical process.


 Fig. 5 Time-lapse, multiphoton imaging of the uptake, processing, and excretion of the 6-carboxyfluorescein diacetate (6-CFDA) by the liver. Red arrow: sinusoid. Green arrow: processed 6-CF molecule. Yellow arrow: 6-CF excretion into the bile canaliculi. The stars indicate positions of the nuelci. Scale bar: 20 mm.


3.  Development of diagnostic techniques in the clinics

Skin photoaging

      Changes in skin dermis occurs when a person ages. Specifically, the elastic fibers increase relative to collagen fibers. We have performed multiphoton autofluorescence and second harmonic generation imaging of human skin dermis from patients of different ages. As shown in Fig. 6, since elastic and collagen fibers can generate spectrally distinct autofluorescence and second harmonic generation signals respectively, our approach can be used to identify changes in skin dermis as a person ages. This approach may be applied in the clinical setting for an accurate assessment of skin photoaging.


Fig. 6 Multiphoton autofluorescence and second harmonic generation imaging of human skin dermis from patients of different ages (left to right: 20, 40, and 70 years old). As a person ages, the elastic fiber (green: autofluorescence) increases relative to collagen fibers (blue: second harmonic generation). Scale bar: 50 mm.

Corneal disease

      Cornea, the main focusing element of human vision, is transparent to visible photons. As a result, optical diagnostics of corneal diseases is a challenge to ophthalmologists. However, since the cornea is composed primarily of type I collagen, non-centrosymmetric connective tissues capable of generating second harmonic signals, corneal disease can be effectively characterized by a combination of multiphoton fluorescence excitation and second harmonic generation microscopy. Shown in Fig. 7 are the images of keratoconus and a cornea co-infected with Acanthamoeba castellinii and Pseudomonas aeruginosa.


Fig. 7 Multiphoton fluorescenc excitation (green) and second harmonic generation (blue) imaging of (A) keratoconus and (B) cornea co-infected with Acanthamoeba castellinii and Pseudomonas aeruginosa.