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For deep imaging depth and least invasiveness, people commonly use 1100-1300 nm femtosecond laser sources to perform label-free in vivo microscopy. The modalities include reflectance confocal, two & three photon fluorescence, and second & third harmonic generation microscopy. However, most of the laser sources are typically based on bulky oscillators, which are sensitive to environment conditions and less stable for routine clinical use. In contrast, fiber-based lasers have simpler cavity design and potentially compact size for movable use. In this presentation, we demonstrate a fiber-based 1150 nm femtosecond laser source, with 6.5 nJ pulse energy, 86 fs pulse-width, and 11.25 MHz pulse repetition rate. It was achieved by a Bismuth Borate (BIBO) or Magnesium-doped periodically poled Lithium Niobate (MgO:PPLN) mediated frequency doubling of the 2300 nm solitons, generated from an excitation of 1550 nm Er:fiber femtosecond laser pulses on a large mode area photonic crystal fiber. Combined with a laser scanned microscope and a home-build data acquisition card, we achieve a pulse-per-pixel harmonic generation microscopy in vivo at a 30 Hz frame rate. In the future, this solution is potential to be used for label-free clinical virtual optical biopsy.

In recent years, it has been suggested that epicardial adipose tissue (EAT) plays an important role in development of coronary artery disease (CAD) and diabetes mellitus (DM). In this article, we used two-photon fluoresce microscope to measure the fluorescence metabolic image of EAT, which obtained from the patient with/without CAD/DM. We used 740nm and 890nm infrared light to excite the auto-fluorescence of metabolic molecules NADH and FAD respectively. We collected the fluorescence signal at wavelength 450nm to 500nm and 500nm to 550nm to obtain the metabolic image. Through the image, we computed the redox ratio (NADH/FAD) by analyzing the intensity. The preliminary result showed that the redox ratio increase in the patients with CAD. It indicates EAT adipocytes of patient with CAD have decreased cellular metabolic activity. But there were no significant variation of redox ratio in the patients with DM.

Photodynamic therapy (PDT) is a light-activated chemotherapeutic treatment that utilizes singlet oxygen and reactive oxygen species induced oxidative reactions to react with surrounding biological substrates, which either kills or irreversibly damages malignant cells. We used multiphoton nonlinear optical microscopy to observe the photo-dynamic effects of TBO-AuNR-in-shell NPs. Excited by femtosecond Cr:forsterite laser operating at 1230nm, singlet oxygen were generated through a plasmon-enhanced two-photon nonlinear optical process. For cells took up NPs, this photodynamic effect can kill the cell. From nonlinear optical microscopy images, we found they shrunk after 3 minutes of illumination.

We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. Grana and starch are the major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two photon fluorescence (2PF) and second harmonic generation (SHG) signals. Consequently, SHG is found on both grana and starch while 2PF from chlorophyll indicates the identity of grana.

Without a labeling, we demonstrated that lipid granules in leukocytes have distinctive third harmonic generation (THG) contrast. Excited by a 1230nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order smaller. These characteristic THG features can also be observed to trace the newly recruited leukocytes following in vivolipopolysaccharide (LPS) challenge. Furtherm ore, using video-rate TH G microscopy, we also captured images of blood cells in human capillaries. Quite different from red-blood-ce lls, every now and then, round and granule rich blood cells with strong THG contrast appeared in circulation. The corresponding volume densities in blood, evaluated from their frequencies of appearance and the velocity of circulation, fall within the physiological range of human white blood cell counts. These results suggested that labeling-free THG imag ing may provide timely tracing of leukocyte movement and hematology inspection without disturbing the normal cellular or physiological status. Keywords: Cell tracking, harmonic generation microscopy, third harmonic generation, leukocyte, lymphocyte, neutrophil, monocyte, red blood cells

Simultaneous imaging of cells expressing multiple fluorescent proteins (FPs) is of particular interest in applications such as mapping neural circuits, tracking multiple immune cell populations, etc. To visualize both in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissues, two-photon fluorescence microscopy (2PM) is a powerful tool that has found wide applications. However, simultaneous imaging of multiple FPs with 2PM is greatly hampered by the lack of proper ultrafast lasers offering multi-color femtosecond pulses, each targeting the two-photon absorption peak of a different FP. Here we demonstrate simultaneous two-photon fluorescence excitation of RFP, YFP, and CFP in human melanoma cells engineered to express a “rainbow” pallet of colors, using a novel fiber-based source with energetic, three-color femtosecond pulses. The three-color pulses, centered at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation of the 1550 nm pump laser and SHG of the solitons at 1728 nm and 1900 nm generated through soliton self-frequency shift (SSFS) of the pump laser in a large-mode-area (LMA) fiber. The resulting wavelengths are well matched to the two-photon absorption peaks of the three FPs for efficient excitation. Our results demonstrate that multi-color femtosecond pulse generation using SSFS and a turn-key, fiber-based femtosecond laser can fulfill the requirements for simultaneous imaging of multiple FPs in 2PM, opening new opportunities for a wide range of biological applications where non-invasive, high-resolution imaging of multiple fluorescent indicators is required.

The understanding of the interaction between tumors and surrounding microenvironment in vivo is an important first step and basis for pathway-targeting cancer therapy. To in vivo observe the dynamic development of tumor cells and validate the efficacy of therapy in microscopic scales, people commonly performed multi-photon fluorescence microscopy through an invasive window chamber setup. However, under such system, the cancer cells can't be identified and long-term tracked without a fluorescence labeling. Exploiting the intrinsic third harmonic generation (THG) and two-photon fluorescence (2PF) contrasts of melanin, we demonstrated in vivo identification of melanoma and tracked its development without labeling. It was achieved with a least invasive femtosecond Cr:forsterite laser and a laser scanning nonlinear microscopy system with 3D sub-micron spatial resolution. Combined with molecular probes or reporters, we anticipate thus developed platform a powerful tool to reveal molecular insights of tumor microenvironments, enhance our understanding of tumor biology, and trigger new therapeutic approaches.

To investigate whether endogenous biliverdins can serve as a fluorescence metabolic marker in cancer diagnosis, we measured their multiphoton fluorescence spectra and lifetimes with femtosecond Cr:forsterite laser. Excited at 1230nm, the two-photon fluorescence of biliverdins peaks around 670nm. The corresponding lifetime (

We demonstrated that the intensity levels and lifetimes of two-photon autofluorescences (2PAF) in human liver tissues can be exploited to diagnose hepatocellular carcinoma (HCC). Excited by an infrared femtosecond laser, we suppressed the two-photon autofluorescences of most endogenous fluorophores and made red autofluorescences more specific to particular molecules in the cryo-sectioned human livers. Using such fluorescence contrast, we found HCC tissues have reduced levels of 2PAF and longer fluorescence lifetime.

By using a frequency-controlled narrow band THz source, a Fourier Transform Infrared (FTIR) spectroscopy system, and a frequency-controlled terahertz (THz) emitter, for the first time, we studied the THz photon absorption related to the THz confined acoustic vibrations in semiconductor nanocrystals. Through a specific charge separation in the CdSe/CdTe type-II nanocrystals and a piezoelectric coupling in the wurtzite CdSe nanocrystals, the THz photons can be resonantly coupled with (l=1) dipolar modes and the (l=0) breathing modes, respectively. Our results provide new mechanisms for low dimensional systems to convert a THz photon into a phonon of the same frequency.

In this talk, we will review our recent works about the GHz-repetition-rate (GRR) femtosecond lasers. These works make GRR femtosecond lasers more flexible in the manipulation of pulse repetition-rate and the operating wavelength. We first demonstrate a phase insensitive way to multiply the repetition-rate of a passive mode-locked laser in femtosecond regime. By inserting an intracavity flat surface with low reflectivity, we multiplied the repetition-rate of a femtosecond Cr:forsterite laser by ten times. It provides a simple and stable way to modify MHz-repetition-rate femtosecond lasers into GRR lasers. To achieve desired wavelength, which can't be directly generated by a gain medium, nonlinear conversion is required. But for GRR femtosecond lasers, the efficiency of single-pass conversion is low due to its low pulse energy. In order to increase the yield, we adopt the method of resonant-enhanced external cavity. With a resonant cavity matched to a 2-GHz repetition-rate Ti:sapphire laser, we demonstrated a high power femtosecond blue source at 2-GHz repetition-rate.

For the purpose of functional third harmonic optical microscopy, it is necessary to find a method to locally enhance third harmonic generation at specific cellular site. We have demonstrated that by matching the third harmonic generation frequency of a Cr:forsterite laser and the surface plasmon resonance frequency of

Multiple quantum well (MQW) structure piezoelectric semiconductor can be treated as a piezoelectric transducer to generate nanometer wavelength and THz frequency acoustic waves. The generation mechanism of nano acoustic wave (NAW) in quantum wells induced by femtosecond optical pulses can be modeled by a macroscopic elastic continuum theory. The absorption of the MQW's modulated by NAW's through quantum confined Franz-Keldysh (QCFK) effect allows another femtosecond optical probe pulses to monitor the propagating NAW. Many applications in typical ultrasonics can be achieved by NAWs. The simultaneous waveform synthesis is demonstrated by an optical coherent control technique. The phase of the totally reflected NAW is studied. Acoustic coherent control can be achieved by designing the thickness of the cap layer on the MQW. We also demonstrated the feasibility to apply THz NAWs to acoustically control an electronic device with higher operation speed and spatial accuracy. Seismology, which is the first step toward ultrasonic imaging, was also demonstrated. The arrival time of the echo is obtained by processing the transient transmission changes of the probe. Ultrafast technique and nano technology are ready for nano ultrasonics.

The problem of weak signal intensity due to the low incident average intensity limited by photodamage probability in common nonlinear light microscopy and spectroscopy can be fundamentally solved by increasing the repetition rate of the excitation light source. Since the possibility of nonlinear photodamage is determined by the incident peak intensity (or pulse energy), increasing the repetition rate of the excitation light source while keeping its peak intensity (or pulse energy) well below than damage threshold will not provoke any optical damage but will augment the average nonlinear signals. We used a femtosecond Ti:sapphire laser with a 2-GHz repetition rate as the light source of a second-harmonic-generation (SHG) microscope and strongly enhanced SHG signal was observed while no photodamage could be identified. Compared with the common 80-MHz Ti:sapphire laser, the microscopic images taken with the 2-GHz laser require shorter acquisition time and exhibit higher contrast, resulting in real-time SHG imaging capability.

Multi-photon fluorescence spectra of a number of commonly used biological probes were measured in this study. Significant spectral variation has been detected between single and multi- photon excitation. The result is important for the proper selection of spectral setting/dichroic beam splitter in the set- up of a multi-photon fluorescence microscope. The information can also be useful in the detection of multi-photon fluorescence in bio-chip technology. In addition, we have investigated a few highly fluorescent bio-molecules commonly found in plant cells.

The non-linear nature of multi-photon fluorescence excitation restricts the fluorescing volume to the vicinity of the focal point. As a result, the technology has the capacity for micro- spectroscopy of biological specimen at high spatial resolution. Chloroplasts in mesophyll protoplast of Arabidopsis thaliana and maize stem sections were used to demonstrate the feasibility of multi-photon fluorescence micro-spectroscopy at subcellular compartments. Time-lapse spectral recording provides a means for studying the response of cell organelles to high intensity illumination.