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NLO Systems

Nonlinear optical (NLO) microscopy uses near infrared (NIR) light to generate two-photon excited fluorescence (TPEF) from UV/VIS absorbing molecules and proteins, second harmonic signals (SHG) from noncentrosymmetric structures, and coherent Raman scattering (CRS) from lipids, proteins and water. The reduced scattering at NIR wavelength allows submicron visualization of cells and tissues at depths of several hundred microns with minimal damage to living systems. NLO microscopy can produce highly detailed maps providing information on important parameters such as general tissue morphology, redox state, fibril orientation and lipid composition. Currently, LAMMP features three NLO platforms: two custom-made systems

Nonlinear Optical Platform 1 (NLO-PC1)

This system consists of two synchronized ultrafast lasers that are interfaced with an inverted microscope. The laser scanning microscope was acquired in 2011 through LAMMP supplement funds, and has replaced our older scanning optics and microscope optics. The system is configured for detecting TPEF, SHG and coherent anti-Stokes Raman scattering (CARS) signals.

System Specifications:
  • 5 W Verdi Laser (Coherent)
  • Mode-locked Ti: Sapphire laser (170 fs pulse width; Mira 900F, Coherent)
  • Picosecond Nd:vanadate laser (10 ps pulse width; PicoTrain, High-Q)
  • Laser synchronization box (High-Q)
  • Laser scanning microscope (Fluoview 300, Olympus)
  • Inverted microscope frame (IX71, Olympus)
  • 3 NLO NDD detection channels (photomultiplier tube, Hamamatsu)
  • SpectraPro-150 spectrograph (300 grooves/mm grating blazed at 500 nm, Acton Research Corp.)
  • MacroMax: 512BFT CCD camera with 512x512 pixel imaging array controlled by ST-133 controller (Princeton Instruments)

Nonlinear Optical Platform 2 (NLO-PC2)

This system is based on a picosecond laser system that consists of a Nd:vanadate laser (1064 nm) and a synchronously pumped optical parametric oscillator (OPO), in addition to a femtosecond Ti:sapphire laser which is synchronized to the Nd:vanadate source. The light sources are coupled into a laser scanning microscope. The platform is configured for recording images with TPEF, SHG, CARS and stimulated Raman scattering (SRS) contrast. In addition, an automated wavelength sweep of the OPO enables the recording of hyperspectral CRS image stacks. The system is also equipped with a 532 nm cw laser for generating spontaneous Raman signals, which are detected by a CCD-based spectrometer.

System Specifications:
  • Picosecond Nd:vanadate laser (10 ps pulse width; PicoTrain, High-Q)
  • Optical parametric oscillator (OPO, Levante Emerald, APE)
  • 5 W Verdi Laser (Coherent)
  • Mode-locked Ti: Sapphire laser (170 fs pulse width; Mira 900F, Coherent)
  • Laser synchronization box (Synchrolock, Coherent)
  • Laser scanning microscope (Fluoview 300, Olympus)
  • Inverted microscope frame (IX71, Olympus)
  • 3 NLO NDD photomultiplier tube detection channels (Hamamatsu) 267
  • 1 photodiode based SRS channel
  • homebuilt demodulator (10 MHz demodulation, 1 µs response time)
  • 100 mW, 532 nm cw laser (CrystaLase)
  • Shamrock spectrometer, fiber coupled (Andor)
  • CCD camera (iDus, Andor)

Zeiss LSM 510 Meta microscopy system

This NLO microscopy system is based on a platform of an Axiovert 200M inverted microscope equipped with standard illumination systems for transmitted light and epi-fluorescence detection and a standard set of visible light lasers (an Argon laser 458/477/488/514 nm/ 30 mW, a Helium: Neon laser 543 nm/ 1 mW and a Helium: Neon laser 633 nm, 5 mW) for confocal microscopy. It is equipped with an NLO interface for a femtosecond Titanium: Sapphire laser excitation source (Chameleon-Ultra, Coherent) for multi-photon excitation with tunability from 690 to 1040 nm. The instrument is equipped with two single channel photomultiplier tube detectors, the META polychromatic detector, the NDD detector for SHG detection in reflected mode, and a transmission light channel detector capable of detecting SHG signal in transmitted mode. The microscope platform is equipped with a motorized X-Y scanning stage and long-working distance and high numerical aperture objectives (10, 20, 40, and 100X).

System Specifications:
  • Mode-locked Ti: Sapphire laser (170 fs pulse width, 690-1040 nm tuning range, Chameleon Ultra, Coherent, Inc.)
  • Zeiss Axiovert 200M inverted microscope
  • 6 lines for confocal scanning 458/477/488/514/532/633 nm
  • Up to 4 channels parallel detection
  • Scanning speed of up to 5 frames/second for acquisition of 512x512-pixel images
  • Motorized X-Y stage with mark and find and tile scan functions and fast piezo objective focus for Z drive with 25 nm smallest increment.
  • User-defined ROI.
  • Multiple acquisition modes such as spot, line, frame, Z-stack, lambda stack, or time series.
  • The polychromatic 32-channel detector (META detector) provides spectral separation of fluorophores within the same sample by spectral fingerprinting and linear unmixing algorithms.

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  • Hanna Kim
    Resource Coordinator

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