Chen-Yuan Dong

Innovations in two-photon deep tissue microscopy

The goal of this article is to provide a review of deep tissue studies based on two-photon microscopy. We will further explore three new developments that have significantly enhanced the power of two-photon imaging: (1) simultaneous two-photon fluorescence and confocal reflected-light imaging, (2) two-photon video-rate imaging, and (3) fluorescent spectroscopic measurements in deep tissue.

Implementation of intensity-modulated laser diodes in time-resolved, pump–probe fluorescence microscopy

We present the implementation of intensity-modulated laser diodes for applications in frequency-domain pump-probe fluorescence microscopy. Our technique, which is based on the stimulated-emission approach, uses two sinusoidally modulated laser diodes. One laser (635 nm) excites the chromophores under study, and the other laser (680 nm) is responsible for inducing stimulated emission from excited-state molecules. Both light sources are modulated in the 80-MHz range but with an offset of 5 kHz between them. The result of the interaction of the pump and the probe beams is that a cross-correlation fluorescence signal at 5 kHz is generated primarily at the focal volume. Microscope imaging at the cross-correlation signal results in images with high contrast, and time-resolved high-frequency information can be acquired without high-speed detection. A detailed experimental arrangement of our methodology is presented along with images acquired from a 4.0-mum-diameter fluorescent sphere and TOTO-3-labeled mouse STO cells. (TOTO-3 is a nucleic acid stain.) Our results demonstrate the feasibility of using sinusoidally modulated laser diodes for pump-probe imaging, creating the exciting possibility of high-contrast time-resolved imaging with low-cost laser-diode systems.

Basic Principles of Multiphoton Excitation Microscopy

Multiphoton microscopy is one of the fastest growing areas in biomedical imaging. The potential of multiphoton excitation was first theorized by Maria GoppertMayer in 1931. Generating three-dimensionally resolved microscopic images based on nonlinear optical excitation was postulated in the 1970s (Gannaway and Sheppard, 1978; Wilson and Sheppard, 1984). The definitive experiment was done by Denk, Webb, and co-workers (1990), who accomplished two-photon, three-dimensional (3D) imaging of biological specimens. Furthermore, they demonstrated 3D localized uncaging and photobleaching using two-photon excitation to trigger a photochemical reaction in a subfemtoliter volume.

Tissue imaging using two-photon video rate microscopy

Non-invasive optical diagnosis of cellular and extracellular structure and biochemistry in thick tissue is becoming a reality with the maturation of the two-photon imaging. Today, the slow imaging speed of typical two-photon microscopes is a major hurdle in realizing their clinical potential. We have developed a high-speed two-photon microscope optimized for acquiring 3-D tissue images in real time. The scanning speed improvement of this system is obtained by the use of an air bearing polygonal mirror. The maximum achievable scanning rate is 40 microseconds per line, which is about 100 times faster than conventional scanning microscopes. High-resolution fluorescence images were recorded in real-time by an intensified CCD camera. Using this instrument, we have monitored the movements of protozoas and mapped the collagen/elastin fiber structures in excised human skin.

Application of two-photon microscopy to elucidate oleic-acid-induced changes in microscale transdermal transport processes

In a novel application of two-photon scanning fluorescence microscopy (TPM), three-dimensional spatial distributions of the hydrophilic and hydrophobic fluorescent probes, sulforhodamine B (SRB) and rhodamine B hexyl ester (RBHE), in excised full-thickness human cadaver skin were visualized and quantified. These findings utilizing TPM demonstrate that, in addition to providing three-dimensional images that clearly delineate probe distributions in the direction of increasing slun depth, the subsequent quantification of these images provides additional important insight into the mechanistic changes in transdermal transport underlying the visualized changes in probe distributions across the slun.

Applications of two-photon fluorescence microscopy in deep-tissue imaging

Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

Frequency-domain pump-probe microscopic imaging using intensity-modulated laser diodes

We report the implementation of intensity modulated diode lasers in frequency-domain pump-probe studies, diode lasers are compact, stable, and economical units that require little maintenance. In our study, a 365 nm diode laser is used as the excitation source and the output of a 680 nm unit induces stimulated emission from excited state fluorophores. By modulating the intensities of the two diode lasers at slightly different frequencies, and detecting the fluorescence signal at the cross-correlation frequency, both time-resolved and high spatial resolution imaging can be achieved. The laser diodes are modulated in the 100 MHz cross-correlation signal has been used for time-resolved imaging of fluorescent microspheres and mouse fibroblasts labeled with nucleic acid stains TOTO-3. These results demonstrate and feasibility of using intensity modulated diode lasers for frequency-domain, pump-probe studies.

Characterization of two-photon point-spread function in turbid medium by direct measurements, multicolor imaging, and blind deconvolution

Over the past decade, scanning fluorescence microscopy based on two-photon excitation has become an important branch of microscopic bio-imaging. Compared to traditional scanning techniques, two-photon microscopy offers a number of distinct advantages. First, scanning of the point-like excitation spot used for imaging results in images with excellent axial depth discrimination. In addition, the limited extent of the excitation volume also limits specimen photo-damage to the focal volume. Finally, the long, near-infixed wavelengths used for sample excitation allow in-depth, non-invasive imaging of optically turbid biological samples. For in-depth imaging, the microscopic objective and often optically heterogeneous biological specimen forms a complex system. To optimize imaging quality in two-photon microscopy, an understanding of the point-spread-function (PSF) is essential. In this work, we attempted to characterize the two-photon PSF by two methods: direct imaging of 0.1 ym fluorescent microspheres and multicolor imaging of 2 ym green fluorescent microspheres in a uniform blue fluorescent background. In both measurements, the turbidity of the surrounding medium was varied by changing the concentration of Liposyn III, a scattering component in the specimen. We found that at discrete Liposyn III concentrations between 0 and 2%, the PSF widths were not affected by the amount of scatterers present. However, the imaged contrast continued to degrade as a function of the amount of scatter. This suggests that the broadening of the tail region of the PSF can be the cause of image contrast loss. We will also discuss the possibility of using blind- deconvolution as a method to obtain PSF information in complex biological specimen.

Magnetic tweezers microscope for cellular manipulation

We present the design of a magnetic tweezers microscope for cellular manipulation. Our design allows versatile and significant 3D stress application over a large sample region. For linear force application, forces up to 250 pN per 4.5 micrometers magnetic bead can be applied. Finite element analysis shows that variance in force level is around 10 percent within an area of 300 X 300 micrometers 2. Our eight-pole design potentially allows 3D liner force application and exertion of torsional stress. Furthermore, our design allows high resolution imaging using high numerical aperture objective. Both finite element analysis of magnetic field distribution and force calibration of our design are presented. As a feasibility study, we incubated fibronectin coated 4.5 micrometers polystyrene beads with Swiss 3T3 mouse fibroblast cells. Under application around 250 pN of force per magnetic particle, we observed relative movement between attached magnetic and polystyrene beads to be on the order of 1 micrometers . Elastic, viscoelastic, and creeping responses of cell surfaces were observed. Our results are consistent with previous observations using similar magnetic techniques.