Novel fluorescence detection technique for non-contact temperature sensing in microchip PCR

DNA amplification using Polymerase Chain Reaction (PCR) in a small volume is used in Lab-on-a-chip systems involving DNA manipulation. For few microliters of volume of liquid, it becomes difficult to measure and monitor the thermal profile accurately and reproducibly, which is an essential requirement for successful amplification. Conventional temperature sensors are either not biocompatible or too large and hence positioned away from the liquid leading to calibration errors. In this work we present a fluorescence based detection technique that is completely biocompatible and measures directly the liquid temperature. PCR is demonstrated in a 3 ILL silicon-glass microfabricated device using non-contact induction heating whose temperature is controlled using fluorescence feedback from SYBR green I dye molecules intercalated within sensor DNA. The performance is compared with temperature feedback using a thermocouple sensor. Melting curve followed by gel electrophoresis is used to confirm product specificity after the PCR cycles. (c) 2007 Elsevier B.V. All rights reserved.

Dual Confocal Laser-Induced Fluorescence/Moveable Contactless Conductivity Detector For Capillary Electrophoresis Microchip

A new dual simultaneous detector was developed for capillary electrophoresis microchip. Confocal laser-induced fluorescence (LIF) and moveable contactless conductivity detection (MCCD) were combined together for the first time. The two detection systems shared a common detection cell and could respond simultaneously. They were mutually independent and advantageous in analyses of mixtures containing organic and inorganic ions. The confocal LIF had high sensitivity and the MCCD could move along the separation channel and detect in different positions of the channel. The detection conditions of the dual detector were optimized. Rhodamine B was used to evaluate the performance of the dual detector. The limit of detection of the confocal LIF was < 5 nM, and that of the MCCD was 0.1 mu M. The dual detector had highly sensitivity and could offer response easily, rapidly and simultaneously. 

Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Radially polarized and pulsed output from passively Q-switched Nd:YAG ceramic microchip laser

For the first time, to the best of our knowledge, a radially polarized laser pulse was produced from a passively Q-switched Nd:YAG ceramic microchip laser with a piece of Cr4+:YAG crystal as the saturable absorber and multilayer concentric subwavelength grating as the polarization-selective output coupler. The averaged laser power reached 450 mW with a slope efficiency of 30.2%. The laser pulse had a maximum peak power of 759 W, a minimum pulse duration of 86 ns, and a 6.7 kHz repetition rate at 3.7 W absorbed pump power. The polarization degree of the radially polarized pulse was measured to be as high as 97.4%. Such a radially polarized laser pulse with a high peak power and a short width is important to numerous applications such as metal cutting. (C) 2008 Optical Society of America

Efficient excitations of radially and azimuthally polarized Nd 3+:YAG ceramic microchip laser by use of subwavelength multilayer concentric gratings composed of Nb2O5/SiO2

Cylindrical vector beams were produced from laser diode end-pumped Nd:YAG ceramic microchip laser by use of two types of subwavelength multilayer gratings as the axisymmetric-polarization output couplers respectively. The grating mirrors are composed of high- and low-refractive-index (Nb2O5/SiO2) layers alternately while each layer is shaped into triangle and concentric corrugations. For radially polarized laser output, the beam power reached 610mW with a polarization extinction ratio ( PER) of 61: 1 and a slope efficiency of 68.2%; for azimuthally polarized laser output, the beam power reached 626mW with a PER of 58: 1 and a slope efficiency of 47.6%. In both cases, the laser beams had near-diffraction limited quality. Small differences of beam power, PER and slope efficiency between radially and azimuthally polarized laser outputs were not critical, and could be minimized by further optimized adjustment to laser cavity and the reflectances of respective grating mirrors. The results manifested, by use of the photonic crystal gratings mirrors and end-pumped microchip laser configuration, CVBs can be generated efficiently with high modal symmetry and polarization purity. (C) 2008 Optical Society of America.

Rare-earth-doped silica microchip laser fabricated by sintering nanoporous glass

We report a new method for fabricating rare-earth-doped silica glasses for laser materials obtained by sintering nanoporous silica glasses impregnated with rare-earth-doped ions. The fabricated materials have no residual pores and show good optical and mechanical properties. Good performance from a Nd3+-doped silica microchip laser operating at 1.064 mum is successfully demonstrated, suggesting that the fabricated silica glasses have potential for use as active materials for high-power solid-state lasers. (C) 2005 Optical Society of America.

High repetition rate Q-switched microchip Nd:YVO4 laser with pulse duration as short as 1.1 ns

We have demonstrated a compact and an efficient passively Q-switched microchip Nd:YVO4 laser by using a composite semiconductor absorber as well as an output coupler. The composite semiconductor absorber was composed of an LT (low-temperature grown) In0.25Ga0.75As absorber and a pure GaAs absorber. To our knowledge, it was the first demonstration of the special absorber for Q-switching operation of microchip lasers. Laser pulses with durations of 1.1 ns were generated with a 350 mu m thick laser crystal and the repetition rate of the pulses was as high as 4.6 MHz. The average output power was 120 mW at the pump power of 700 mW. Pulse duration can be varied from 1.1 to 15.7 ns by changing the cavity length from 0.45 to 5 mm. Pulses with duration of 1.67 and 2.41 ns were also obtained with a 0.7 mm, thick laser crystal and a 1 mm thick laser crystal, respectively. (C) 2007 Elsevier GmbH. All rights reserved.

Antiphase state in passively Q-switched Yb : YAG microchip multimode lasers with a saturable absorber GaAs

We report on recent experimental results of the spontaneous antiphase dynamics that occurs in a laser-diode-pumped multimode passively Q-switched microchip Yb:YAG (where YAG is yttrium aluminum garnet) lasers with a saturable absorber GaAs. We observe that the pulse sequence of the first mode characterized by one, two, and three pulses as a group and all the modes display an antiphase state as the pumping ratio rises. We modify the multimode rate equations to account for nonlinear absorption due to GaAs in the presence of spatial hole burning. We perform numerical simulations based on the proposed rate equations and reproduce the observed antiphase state of two and three active modes.

Observation of antiphase states in a passively Q-switched multimode microchip Yb : YAG laser with a GaAs saturable absorber

Antiphase dynamics has been observed experimentally for the laser modes operation in a laser-diode-pumped Q-switched microchip Yb:YAG laser with GaAs as a saturable absorber in the presence of spatial hole-burning. The Q-switched pulses sequences of two modes at different pump power have been obtained. The experimental results have shown that the pulses sequences displayed classic antiphase dynamics. (C) 2003 Elsevier B.V. All rights reserved.

Simultaneous Q switching and mode locking with narrow envelope width in a microchip Nd : YVO4 laser with a composite semiconductor absorber

By using a composite semiconductor absorber and an output coupler, we demonstrated a Q-switched and mode-locked diode-pumped microchip Nd:YVO4 laser. With a 350-mu m-thick crystal, the width of the Q-switched envelope was as short as 12 ns; the repetition rate of the mode-locked pulses inside the Q-switched pulse was more than 10 GHz. The average output power was 335 mW at a maximum pump power of 1.6 W. Q-switched envelope widths of 21 and 31 ns were also achieved with crystals 0.7 and 1.0 mm thick, respectively.

Diode-pumped passively Q-switched Yb : YAG microchip laser with a GaAs as saturable absorber

A passively Q-switched Yb: YAG microchip laser has been constructed by using a doped GaAs as the saturable absorber as well as the output coupler. At 13.5 W of pump power the device produces high-quality 3.4 muJ 52 ns pulses at 1030nm with a pulse repetition rate of 7.8kHz in a TEM00-mode.

Passive Q-switching of diode-pumped Yb:YAG microchip laser with ion-implanted GaAs

We reported a passive Q-switched diode laser pumped Yb:YAG microchip laser with an ion-implanted semi-insulating GaAs wafer. The wafer was implanted with 400-keV As^(+) in the concentration of 10^(16) ions/cm^(2). To decrease the non-saturable loss, we annealed the ion-implanted GaAs at 500 oC for 5 minutes and coated both sides of the ion-implanted GaAs with antireflection (AR) and highreflection (HR) films, respectively. Using GaAs wafer as an absorber and an output coupler, we obtained 52-ns pulse duration of single pulse.