Nonlinear Optical Imaging by TpVChV

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									Nonlinear Optical Imaging
       Microscopy

       LM/EM Imaging Core Facility
    RM362, Biomedical Research Building
         West Virginia University
NLOM Bench
               NLO Facility
• Two Photon Microscopy

• Second Harmonic Generation Microscopy (SHG)

• Third Harmonic Generation Microscopy (THG)

• Coherent anti-Stokes Raman Scattering (CARS)
  Microscopy
Advantage of 2-P vs OPF (Confocal)

  No pinhole necessary meaning more fluorescence
    light collected.
  Nondescan setup meaning scanning mirror is out of
    fluorescence light path, detector closer to specimen.
  Laser tunable; one wavelength can simultaneously
    excite many probes
  Less out-of-focus photobleaching/photodamage good
    for live cell/tissue imaging
  Less scattering for deeper tissue penetration
                   TPF Probes

1. Organic dye – immunostaining, colors ranging
  from Blue to NIR

2. Fluorescent protein (GFP, CFP YFP, RFP,
  mCherry, tdTomato, etc.)

3. Nanoparticle (Quantum dots, Carbon nanotube)
4. Autofluorescence (NAD(P)H, flavoprotein,
  elastin, etc.)
            Two Platforms


• Inverted Microscope Configuration


• Upright Microscope Configuration
IX71 Inverted Microscope
                Features:
                Inverted; slow and fast
                    scanner; Equipped with
                    TC incubator/CO2
                    System

                Epi-detection:
                1. Two-channel 2P/3P
                   fluorescence
                2. CARS
                Forward detection:
                1. Second harmonic
                   generation (SHG)
                2. Third harmonic
                   generation (THG)
                3. CARS
MOM Upright Microscope

              Unique Features of MOM:
              Upright; objective is xyz
              motorized; electrophysiology and
              imaging combined

              Epi-detection:
              4 channel capability, 3 channel
              simultaneous 2P or 3P imaging,
              SHG or CARS
              Forward detection:
              1 channel, SHG, THG, or CARS

              Equiped with perfusion
              system:
              equipped for in vitro and in vivo
              imaging
TPF: In Vitro and In Vivo Study




    Barry Masters and Peter So, Handbook of Biomedical Nonlinear Optical Microscopy
                          TPF Applications
 Mammalian neurobiology
  Intracellular imaging of calcium dynamics
 in dendritic spine
  Long-term imaging of neurodegeneration
    measurement of vascular hemodynamics
  Targeted intracellular recording In Vivo



Tumor biology
Chronic in vivo models for Dynamic
imaging of
 Tumor-associated blood and
lymphatic vessels
 Tumor Vessel permeability
 Tumor/host cell interaction in vessels
and stroma
TPF Applications cont’d


Immunology
 Visualization of
lymphocyte mobility,
chemotaxis

 Visualization of
antigen recognition in
isolated lymphoid
organs

 Visualization of In
Vivo lymph nodes in
anesthetized mice
                     SHG Applications

SHG from Endogenous
Tissue (non-
centrosymmetric)

Collagen, acto-myosin
complexes, mitotic
spindles, microtubules,
and cellulose.

Such as in skin, bone,
tendon, cartilage, teeth,
brain tissue, cornea, etc.
            SHG Applications                                cont’d


SHG from
Exogenous
Chromophores

 Molecular “Flip-Flop”
Dynamics in Membranes

 Intermembrane Separation
Measurement                  Figure. High resolution SHG recording of action
                             potentials deep in hippocampal brain slices. (A) SHG
                             image of a neuron patch clamped and filled with FM4-
 Membrane Potential         64 in slice. (B) SHG line-scan recording of elicited
Imaging (using voltage       action potentials (55 line scans were averaged). (C)
                             Electrical patch pipette recording of action potentials
sensitive dyes)              seen in B. Calibrations: (A) 20 µm; (B) 2.5%
                             ΔSHG/SHG, 50 ms; (C) 20 mV, 50 ms. (W. Webb lab)
 SHG Applications




Figure. Rat hippocampal neurons are imaged by SHG
(green pseudocolor) after 5 days (left) and 7 days (right)
in culture. At the later development stage,
protoprocesses have presumably matured into dendrites
whose microtubules are of mixed polarity and cannot
produce SHG. Only the axon microtubules, which
maintain a uniform polarity, are revealed by SHG (red
pseudocolor represents autofluorescence). (W. Web lab)
                THG Applications

Changing the Look
of Malaria
The optical effect
called third harmonic
generation causes
malaria secretions to
glow blue in infected
blood cells (left),
promising a faster,
more efficient
diagnosis than
traditional
microscopy imagery
(right)
Coherent anti-Stokes Raman
    Scattering (CARS)
         Microscopy
        CARS Applications
Tissue imaging (chemical selectivity & noninvasive)

                                 CARS: very sensitive to
                                      lipids
                                     To study dynamic
                                      processes in living
                                      cells;
                                     Lipid rafts on
                                      membrane
                                     Trafficking and
                                      growth of lipid
                                      droplets
                                     Intracellular water
                                      diffusion
                         CARS Images

                                  CARS                                              TPF




Lipid droplet of A Nile red-stained daf-4 mutant (C. elegans) arrested in its dauer stage for
3 weeks. Hellerer T et al. PNAS 2007; 104:14658-14663
Optical Parametric Oscillator
     Technology (OPO)
 Deep Tissue Imaging cont’d




Kobat et al. 2009 / Vol. 17, No. 16 / OPTICS EXPRESS 13354
Deep Tissue Imaging




Helmchen et al. Nature Methods 2005, 2(12), 932
Data Visualization & Image Analysis
 2D image processing
 Photoshop, MATLAB, ImageJ

 3D/4D image visualization & measurement
 (3D – stack images; 4D – stack images + time lapse)
 Commercial (Amira, Imaris, MATLAB), Free (ImageJ, Voxx, VisBio)
        Technology Development

Administrative supplement to Neuro CoBRE grant supports:
     Optical Parametric Amplification
     Long wavelength imaging
     Develop new applications for SHG, CARS

Neuro CoBRE grant also supports:
     Develop in vivo imaging capability
     Set up chamber for time-lapse in vitro imaging
     Imaging Discovery Grants to
      facilitate purchase of reagents and use of facility
Imaging Discover Grants

								
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