Traditional drug sensitivity assay confirms the cell death by time-consuming fixation and labeling. This snapshot evaluation overlooks the valuable time-course pharmacodynamics that could offer new insights for accurate drug screening. In this study, we have developed a label-free method to promptly detect cell senescence using endogenous lipofuscin-like autofluorescence. Following drug treatment, we observed that damaged mitochondria release molecules emitting lipofuscin-like red autofluorescence. This corresponding fluorescence intensity significantly increases in apoptotic and necrotic cells. Such innovative approach enables the real-time observation of treatment outcomes in 3D tumor organoids and has the potential to determine drug sensitivity earlier than the Annexin V/PI assay. This metabolic fluorescence signature could substantially enhance the efficiency of drug sensitivity testing.
In intensive care units (ICU), vital signs and biomarkers of critically ill patients provide a set of operational parameters for doctors to assess the severity of organ dysfunction and optimize the supporting treatment. Among those organs monitored, the gut is less accessible, and its latent risk is not manageable. There is an emerging need for sensitive and easily measured biomarkers of early intestinal injury. Here, we found plasma fluorochromics can be used to assess the severity of intestinal injury using label-free methods of quantification. In acute mesenteric ischemia-reperusion animal models, ischemia-reperfusion damage can lead to multiple times increase of NADH, flavins, and porphyrin auto-fluorescence in blood. The intensity ratio between NADH and flavin fluorescence can capture early signatures before the occurrence of shock. Using liquid chromatography and mass spectroscopy, we confirmed that riboflavin is primarily responsible for the increased flavin fluorescence. Since endogenous riboflavin in humans is absorbed from the intestine, its increase in plasma validates its association with intestinal injury. In the future, blood fluorochrome detection could serve as a time-course monitoring modality in the emergency department or ICU to assess intestinal damage in various acute illnesses and critical care conditions.
Label-free visualizing the prediabetic microenvironment of adipose tissues provides a less invasive alternative for the characterization of insulin resistance (IR) and inflammatory pathology. Here, we successfully identified the differentiable features of prediabetic adipose tissues by employing the metabolic imaging of three endogenous fluorophores NAD(P)H, FAD, and lipofuscin-like pigments. We discovered that 1040-nm excited lipofuscin-like autofluorescence could mark the location of macrophages. This unique feature helps separate the metabolic fluorescence signals of macrophages from those of adipocytes. In prediabetes fat tissues with IR, we found only adipocytes exhibited a metabolic fluorescence profile different from that of normal adipocytes.When mice have inflamed fat tissues, both adipocytes and macrophages possess this kind of metabolic change. Based on spatial fluorescence metabolomics, we developed an innovative approach to diagnose prediabetes, providing insights into diabetes prevention strategies.
Traditional drug sensitivity assay confirms the cell death by time-consuming fixation and labeling. This snapshot evaluation neglects valuable time-course details that may provide new insight for the speed-up of drug screening. Here we develop a label-free method to early report cell senescence by the endogenous lipofuscin autofluorescence. After drug treatment, we found the lipofuscin red autofluorescence greatly increased in apoptotic and necrotic cells. This approach allows the time-course observation of pharmacodynamics in 3D tumor organoids and could determine the drug sensitivity earlier than Annexin V/PI assay. This metabolic fluorescence hallmark could improve the throughput of drug sensitivity test.
Emerging advances in iron oxide nanoparticles exploit their high magnetization for various applications, such as catalysis, bioseparation, hyperthermia, and magnetic resonance imaging (MRI). In contrast to the excellent magnetic performance, their upconverted photoluminescence have not been thoroughly explored, thus limiting their development as a tool in photomedicine. In this work, we develop a seed/growth-inspired synthesis combined with primary mineralization and a ligand-assisted secondary growth strategy to prepare mesostructured α-FeOOH nanorods (NRs). Because α-FeOOH rods are all iron-based composites, they exhibit low cytotoxicity towards cells. Surprisingly, these mesoporous α-FeOOH mesostructures display strong third harmonic generation (THG) signals under near-infrared excited wavelength at 1230 nm. They exhibited a much stronger THG intensity compared to naked α-FeOOH NRs. Using these unique nonlinear optical properties, we demonstrate that α-FeOOH rods can serve as contrast agents in THG microscopy for the cell tracking as well as angiography in vivo. Vessel walls can be revealed after the clearance of particles. Our results provide a new strategy of material synthesis for obtaining high THG imaging contrast.
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.
Nanomedicine can kill tumor cells or block the growth and spread of cancers by maximizing drug accumulation in tumor tissues. How much drug will selectively accumulate in tumor tissues depends on physical parameters such as vessel permeability in tumor microenvironments. On the other hand, by properly choosing the size of nanomedicine, the accumulation of drugs in normal tissues will be reduced. In this study, we measured the permeability rate of vessels in different strain of mice in vivo with two-photon fluorescent angiography. We used FITC / TRITC / TEXAS RED labeled dextran to investigate size-dependent permeation from vessels in normal tissues. We choose nanoparticles of 40kDa, 70 kDa, 150 kDa and 2000 kDa, and selected three strains of wild-type Balb/C, ICR, C2J mice. We found , even at the same size, the vascular permeability rate of dextran still vary with the dye-conjugates and the strain of mice. One of our results shows that ICR mice have the biggest vessel hole in normal tissues. Choosing wrong nanoparticle size will cause their leaking in normal tissues. This study will provide a new threshold and guideline to reduce the accumulation of nanomedicine in normal tissues.
Harmonic generation microscopy (HGM) has become one unique tool of optical virtual biopsy for the diagnosis of cancer and the in vivo cytometry of leukocytes. Without labeling, HGM can reveal the submicron features of tissues and cells in vivo. For deep imaging depth and minimal invasiveness, people commonly adopt 1100- to 1300-nm femtosecond laser sources. However, those lasers are typically based on bulky oscillators whose performances are sensitive to environmental conditions. 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 obtained by a bismuth borate or magnesium-doped periodically poled lithium niobate (MgO:PPLN) mediated frequency doubling of the 2300-nm solitons, generated from an excitation of 1550-nm femtosecond pulses on a large mode area photonic crystal fiber. Combined with a home-built laser scanned microscope and a tailor-made frame grabber, we achieve a pulse-per-pixel HGM imaging in vivo at a 30-Hz frame rate. This integrated solution has the potential to be developed as a stable HGM system for routine clinical use.
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.
Gold-nanorods incorporated with microbubbles (AuMBs) were introduced as a photoacoustic/ultrasound dual-
modality contrast agent in our previous study. The application can be extended to theragnosis purpose. With the unique physical characteristics of AuMBs, we propose an enhanced delivery method for the encapsulated particles. For example, laser thermotherapy mediated by plasmonic nanoparticles can be made more effective by using microbubbles as a
targeted carrier and acoustic cavitation for enhanced sonoporation. The hypothesis was experimentally tested. Firts, these
AuMBs first act as molecular probes with binding to specific ligands. The improved targeting efficacy was
macroscopically observed by an ultrasound system. The extended retention of targeted AuMB was observed and
recorded for 30 minutes in a CT-26 tumor bearing mouse. Secondly, cavitation induced by time-varying acoustic field
was also applied to disrupt the microbubbles and cause increased transient cellular permeability (a.k.a., sonoporation).
Multimodal optical microscope based on a Cr:forsterite laser was used to directly observe these effects. The microscope can acquired third-harmonic generation (THG) and two-photon fluorescent (2PF) signals produced by the AuMBs. In vitro examination shows approximately a 60% improvement in terms of fluorescence signals from the cellular uptake of gold nanoparticles after sonoporation treatment. Therefore, we conclude that the controlled release is feasible and can
further improve the therapeutic effects of the nanoparticles.
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.
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 in vivo to trace the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. Furthermore, using video-rate THG microscopy, we also captured images of blood cells in human capillaries. Quite different from red-blood-cells, 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 imaging may provide timely tracing of leukocyte movement and hematology inspection without disturbing the normal cellular or physiological status.
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 (<100ps) was much shorter
than those of porphyrins (~10ns), which is another commonly present metabolites in living cells. Further mixing
biliverdins with proteins like fetal bovine serum (FBS), biliverdins reductase A (BVRA), or heme oxygenase-1 (HO-1),
the yields of red autofluorescences didn't change a lot, but the corresponding lifetimes with HO-1 and BSA were
lengthened to 200~300ps. This indicates that biliverdin can have an association with these proteins and change its
lifetime. These spectral and temporal characteristics of fluorescence make biliverdin a potential marker fluorophore for
hyperspectral diagnosis on the heme catabolism in human cells or tissues.
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.
As a novel modality of optical microscopy, second-harmonic generation (SHG) provides attractive features including intrinsic optical sectioning, noninvasiveness, high specificity, and high penetrability. For a biomedical application, the epicollection of backward propagating SHG is necessary. But due to phase-matching constraint, SHG from thick tissues is preferentially forward propagation. Myosin and collagen are two of the most abundant fibrous proteins in vertebrates, and both exhibit a strong second-harmonic response. We find that the radiation patterns of myosin-based muscle fibers and collagen fibrils are distinct due to coherence effects. Based on these asymmetric radiation patterns, we demonstrate selective imaging between intertwining muscle fibers and type I collagen fibrils with forward and backward SHG modalities, respectively. Thick muscle fibers dominate the forward signal, while collagen fibril distribution is preferentially resolved in the backward channel without strong interference from muscle. Moreover, we find that well-formed collagen fibrils are highlighted by forward SHG, while loosely arranged collagen matrix is outlined by backward signal.
Gene expression plays an important role in embryo development and organ function. Previous studies have shown that harmonic generation microscopy (HGM) can be used as a fluorescence signal-independent, minimally invasive method with a subcellular 3-D resolution and a penetration depth in the order of millimeters for long-term continuous imaging of vertebrate embryos. We show that it is ideal to combine in vivo HGM with the morphant technology for minimally invasive, long-term continuous observation of gene expression in the nervous system of vertebrate embryos. Since second- and third-harmonic generations (SHG, THG) are virtual-state-transition-based systems that depend only on the structure of the organisms, they are not temporally limited by the expression of the fluorescence proteins. We successfully identified the expression of the zarnt2a and the hif-1, 2, and 3 genes in the nervous system of zebrafish embryos with specific knockdown genes by microscopically observing the embryos from the early stages of embryogenesis. The results from a combination of the two different modalities, i.e., SHG microscopy and THG microscopy, successfully revealed the weak cell adhesion, cell apoptosis, nerve formation reduction, and neural tube distortion in the morphant zebrafish embryos.
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.
We demonstrate a compact and self-starting fiber-delivered femtosecond Cr:forsterite laser for nonlinear light microscopy. A semiconductor saturable absorber mirror provides the self-starting mechanism and maintains long-term stability in the laser cavity. Four double-chirped mirrors are employed to reduce the size of the cavity and to compensate for group velocity dispersion. Delivered by a large-mode-area photonic crystal fiber, the generated laser pulses can be compressed down to be with a nearly transform-limited pulse width with 2.2-nJ fiber-output pulse energy. Based on this fiber-delivered Cr:forsterite laser source, a compact and reliable two-photon fluorescence microscopy system can thus be realized.
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 <50-nm silver nanoparticles, localized enhancement of third harmonic intensity of more than 100-folds can be achieved both in phantom and in real biological tissues. This strongly enhanced third harmonic signal can then be applied to specific molecule imaging by attaching the nanoparticles to the target molecule with the advantages of noninvasiveness and deep penetration capability.
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.
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.
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.
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