Scanningless depth-resolved microscopy is achieved through
spatial-temporal focusing and has been demonstrated
previously. The advantage of this method is that a large area may be imaged without scanning resulting in higher
throughput of the imaging system. Because it is a widefield technique, the optical sectioning effect is considerably
poorer than with conventional spatial focusing two-photon microscopy. Here we propose wide-field two-photon
microscopy based on spatio-temporal focusing and employing background rejection based on the HiLo microscope
principle. We demonstrate the effects of applying HiLo microscopy to widefield temporally focused two-photon
Two-photon excitation microfabrication has been shown to be useful in the field of photonics and biomedicine. It
generates 3D microstructures and provides sub-diffraction fabrication resolution. Nevertheless, laser direct writing, the
most popular two-photon fabrication technique, has slow fabrication speed, and its applications are limited to
prototyping. In this proceeding, we propose high-throughput 3D lithographic microfabrication system based on depthresolved
wide-field illumination and build several 3D microstructures with
SU-8. Through these fabrications, 3D
lithographic microfabrication has scalable function and high-throughput capability. It also has the potential for
fabricating 3D microstructure in biomedical applications, such as intertwining channels in 3D microfluidic devices for
biomedical analysis and 3D cell patterning in the tissue scaffolds.
Second harmonic generation (SHG) microscopy has become an important tool for minimally invasive biomedical
imaging. However, differentiation of different second harmonic generating species is mainly provided by morphological
features. Using excitation polarization-resolved SHG microscopy we determined the ratios of the second-order
susceptibility tensor elements at single pixel resolution. Mapping the resultant ratios for each pixel onto an image
provides additional contrast for the differentiation of different sources of SHG. We demonstrate this technique by
imaging collagen-muscle junction of chicken wing.
Both reflected confocal and multiphoton microscopy can have clinical diagnostic applications. The successful combination of both modalities in tissue imaging enables unique image contrast to be achieved, especially if a single laser excitation wavelength is used. We apply this approach for skin and corneal imaging using the 780-nm output of a femtosecond, titanium-sapphire laser. We find that the near-IR, reflected confocal (RC) signal is useful in characterizing refractive index varying boundaries in bovine cornea and porcine skin, while the multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) intensities can be used to image cytoplasm and connective tissues (collagen), respectively. In addition, quantitative analysis shows that we are able to detect MAF from greater imaging depths than with the near-IR RC signal. Furthermore, by performing RC imaging at 488, 543, and 633 nm, we find that a longer wavelength leads to better image contrast for deeper imaging of the bovine cornea and porcine skin tissue. Finally, by varying power of the 780-nm source, we find that comparable RC image quality was achieved in the 2.7 to 10.7-mW range.
Laser scanning systems for two-photon microscopy and fabrication have been proven to be excellent in depth-resolving
capability for years. However, their applications have been limited to laboratory use due to their intrinsic slow nature.
The recently introduced temporal focusing concept enables wide-field optical sectioning and thus has potential in both
high-speed 3D imaging and 3D mass-production fields. In this paper, we use the ultrafast optical pulse manipulation to
generate two-photon excitation depth-resolved wide-field illumination (TPEDRWFI). The design parameters for the
illumination were chosen based on numerical simulation of the temporal focusing. The imaging system was
implemented, and the optical sectioning performance was compared with experimental result.
For the last two decades, multiphoton excitation microscopy/microfabrication based on laser scanning/writing techniques
has been popular in the life science as well as photonics. Due to the slow scanning/writing nature, these applications are
very limited to the production of prototypes, although its submicron optical resolution and intrinsic 3D optical sectioning
capability are very attractive for creating 3D structures. In this proceeding, we introduced multiphoton excitation
microscopy and microfabrication based on wide-field illumination. We derived mathematical model for wide-field
illumination in the microscopy and microfabrication, and identified the design parameters that affect axial resolution for
the proposed system. The future work of developing optical model combined with photopolymerization is also discussed.
Histological analysis is the clinical standard for assessing tissue health and the identification of pathological states. Its invasive nature dictates that its use should be minimized without compromising diagnostic accuracy. A promising method for minimally invasive histological analysis is optical biopsy, which provides cross sectional or 3D images without any physical sectionings. Optical biopsy method based on multiphoton excitation microscopy can image cross-sectional image for deep tissue structures with subcellular resolution based on tissue endogenous fluorescence molecules. Despite its suitability for tissue imaging, multiphoton microscopy has not been used for in vivo clinical applications due to both compactness and imaging speed problems. We are developing a high-speed, handheld, miniaturized multifocal multiphoton microscope (H<sup>2</sup>M<sup>4</sup>) as an optical biopsy probe to enable optical biopsy with subcellular resolution. We incorporate a compact raster scanning actuator based on optimizing a piezo-driven tip tilt mirror by increasing its bandwidth, and reducing its nonlinearity. For flexible light delivery, we choose a photonic bandgap crystal fiber, which transmits ultrashort pulsed laser delivery with reduced spectral distortion and pulse width broadening. We further demonstrate that this fiber produces minimal spatial mode distortion and can achieve comparable image point spread function (PSF) as free space delivery. We further investigate the applicability of multiphoton microscopy for clinical dermal investigation by imaging ex vivo human skins with both normal and abnormal physiologies. This demonstrates the performance of H<sup>2</sup>M<sup>4</sup> and the possibility of optical biopsy for diagnosing skin diseases.
For the last decade, multiphoton excitation fluorescence microscopy has found numerous applications in biology. Multiphoton microscopy provides several advantages over conventional fluorescence microscopy, including increased penetration depth, improved signal-to-background ratio, and reduced photodamage. Despite its suitability for tissue imaging, multiphoton microscopy has not been used for in-vivo clinical applications due to its lack of portability and its slow imaging speed. Multiphoton microscopy has recently been improved with the development of high speed imaging systems and handheld devices. High speed imaging has been achieved by simultaneously exciting multiple foci in the specimen, known as multiphoton multifocal microscopy (MMM). Compact devices have been developed by combining fiber optic delivery and miniaturized scanning devices. We have developed a handheld device for high speed multiphoton microscopy based on optical fiber delivery, multifoci excitation/detection and a novel scanner. Our system is designed to be sufficiently compact such that it can be used for in vivo clinical imaging, or optical biopsy with potential applications in dermal, cervical and colorectal cancer diagnosis. The power available from a typical Ti:sapphire laser is fully utilized by using multifoci excitation; this results in reduced image acquisition time. Femto-second pulses from a Ti:sapphire laser are delivered to our system through conventional optical fiber. We realize multifoci excitation with a microlens array, and multifoci detection with a multi-anode PMT. A high bandwidth tip tilt mirror is further used as the scanning element for high speed imaging. The feasibility of this handheld MMM is demonstrated by measuring the performance of major components individually. This work is supported by NIH R33 CA091354.