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 (noncentrosymmetric)
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
 Membrane Potential
Imaging (using voltage
sensitive dyes)
Figure. High resolution SHG recording of action
potentials deep in hippocampal brain slices. (A) SHG
image of a neuron patch clamped and filled with FM464 in slice. (B) SHG line-scan recording of elicited
action potentials (55 line scans were averaged). (C)
Electrical patch pipette recording of action potentials
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