Stefan Wittea,b,1, Adrian Negreana,b,c, Johannes C. Lodderb,c, Christiaan P. J. de Kockb,c, Guilherme Testa Silvab,c, Huibert D. Mansvelderb,c,2, and Marie Louise Groota,b,2

aBiophysics Group, Institute for Lasers, Life, and Biophotonics Amsterdam, VU University, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands; c Integrative Neurophysiology, Centre for Neurogenomics and Cognitive Research, VU University, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands; and bNeuroscience Campus Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands

Procedures of the National Academy of SciencesPNAS April 12, 2011 vol. 108 no. 15 5970-5975

Raul SotoBME502

Label-free live brain imaging and targeted patching with third-harmonic generation (THG) microscopy

THG : Principle of Operation[A] Fluorescence

microscopy:• Single photon excitation of

outer electron in fluorescent dye caused by continuous photon flux

[B] Multi-photon microscopy:

• excitation by high energy pulses of multiple photons w/ longer λ

• 1st photon induces virtual state on outer electron (no dye needed)

• 2nd, 3rd photons of pulsed high energy light absorbed by electron before virtual state is de-excited (~ 10 -15 sec)

• 3 photons are converted to 1 photon with 3x energy and 1/3 λ

(Friedl, et al 2007)

Improvements• THG images can be improved by using

aberration correction

Drosophila embryo(Olivier, et al 2009)

Pollen Grain(Olivier, et al 2009)

Mouse embryo(Debarre, et al 2009)

Set-up Schematic

“The Story”THG Imaging of Live Brain Tissue Figure 1B, C,

D

Label-Free Imaging of Various Brain Structures

Figure 2, S3Movies S1, S2

Combined THG and Fluorescence Imaging Figure 3

Deep Tissue Imaging and Automated Cell Detection

Figure 4, Movies S3, S4

Label-Free Targeted Patching of Live Neurons

Figure 5,Movie S5

THG Imaging of Live Brain Tissue

[B] Axons and dentrites have very high lipid concentrations, small diameters (0.3 – 2 µm)

• Laser focal volume >> diameter : very good THG signal

[C] Neuron cell bodies (somata) contain organelles, much smaller diameters (20 – 100 nm)

• THG signal not good

[D] Result: “shadow contrast” : neural tissue THG image shows dark cell bodies and illuminated axons / dentrites

Label-Free Imaging of Various Brain

Structures

THG Signal is significant in lipid-rich structures

[A] THG Image of mouse neocortex

[B] 2-photon fluorescence image of same sample• sample stained with Nile Red dye, binds to ER (lipid

rich)

Label-Free Imaging of Various Brain

StructuresTHG provides high resolution images of white matter structures (lipid-rich myelin sheaths)

[C] dentate gyrus brain region

[D] corpus callosum, axon bundles visible

[E] striatum in mouse brain (coronal section)

THG can also visualize vasculature[F] blood vessels in cerebral

cortex• Hemoglobin in RBCs

provides good THC signal!

Label-Free Imaging of Various Brain

Structures

Single RBCs in blood vessel in the top layer of the cerebral cortex of a live, anesthesized mouse

Label-Free Imaging of Various Brain

Structures

THG can produce 3D projections of live neuronal tissue(6 sec)

Combined THG and Fluorescence Imaging

THG and 2PLSM can be combined to obtain more information about brain structures [i.e. ID specific cell types]

[A] THG image: pre-frontal cortex [PFC]

[B] 2PLSM image of same tissue, using SR-101 dye. • Astrocyte cell bodies = green

[C] Overlay A&B: shows distribution of astrocytes in neuronal network

Combined THG and Fluorescence Imaging

THG and 2PLSM can be combined to obtain more information about brain structures [i.e. ID specific cell types]

[D] THG image : PFC

[E] 2PLSM image of same tissue; transgenic mouse expresses GFP in somatostatin-positive neurons

[F] Overlay D&E: shows distribution of somatostatin-positive neurons in neocortex, hippocampus

Deep Tissue Imaging and Automated Cell

DetectionTest of achievable depth range

• used mouse PFC• A, B, C show images

of tissue sample at various depthso A : 100 µmo B : 200 µmo C : 300 µm

• Neurons still visible at depth > 300 µm

• Limit reached around 350 µm

Deep Tissue Imaging and Automated Cell

Detection

THG Depth scanfrom surface to 350 µm, in 2 µm steps (23 sec)

Deep Tissue Imaging and Automated Cell

DetectionQuantify Cell Visibility• THC can be used for high-

speed automated cell detection

• Red pixels = cell detection algorithm identifies as cell bodies

THG signal vs depth• Red: cell visibility contrast vs

depth• Black: THG signal intensity vs

depth• Once depth reaches 350 µm

both cell visibility and THG signal intensity drop

• Too deep for back-scattered photons to reach detector

Deep Tissue Imaging and Automated Cell

DetectionCritique:• Cell counting algorithm

should be tested at various depths and beam intensities (mW) to validate its reliability

Critique:• # times this experiment was

repeated (n = ?) not disclosed

• Beam intensity (mW) should be varied as well

• Use various types of neural tissue

Deep Tissue Imaging and Automated Cell

Detection

THG Inverted Shadow ImageShows 3D image of neurons in PFC (6 sec)

Label-Free Targeted Patching of Live

Neurons• THG can visualize and guide

small electrodes, glass pipettes, microsurgery tools

• THG can be used to patch a specific target neuron w/out using fluorescent dyes (potentially toxic)

• We can pick target neuron before entering tissue

[A] THG image: before live patching

[B] Neuron patched (25µm)[C] Larger field view of the same

image (100µm)[D] action potential response of

patched neuron, as response to stimulation with 100 pA current pulse

Label-Free Targeted Patching of Live

Neurons

SM05.mov

• THG can be used to guide microscopic instruments

• Can guide, target and patch specific neurons accurately

• Transverse image dimensions are 150 x 150 μm

• (52 sec video)

Label-Free Targeted Patching of Live

NeuronsDetermine if high-intensity laser radiation has effect on neurons• Monitored neuron membrane resting

potential during THG image acquisition

• Effect of laser in cell is observed as deviation from equilibrium value

• Damage is observed as irreversible change in membrane potential

• [E] Small and reversible change observed (4% change in voltage) at higher power (200 mW); [ n > 30 cells; 7 at depth > 250µm ]

• NO photo damage or photo bleaching

Critique: • Experiment should consider

multiple brain cell types.• Exposure time of cells to THG only

described as “over 5 minutes”.

Label-Free Targeted Patching of Live

Neurons• Maximum observed change in

membrane resting potential (mV) vs incident laser power (mW)

• Linear response

• They claim to have tested patching neurons for over 5 minutes without adverse light-related effects

Critique: • Experiment should consider other

variables : depth, cell type.• # times this experiment was

repeated (n = ?) not disclosed

• Data supporting 5+ minutes claim is not presented in the paper

Possible Biomedical Uses

o Real-time diagnostic tissue screening during brain surgery

o Guide microscopic surgical instruments with sub-cellular precision

o Visualize & study structures with high lipid content such as brain tumors, cholesterol plaques in Alzheimer’s patients

o Preclinical research (animal models) : significantly reduce number of animals sacrificed to take brain samples.

http://www.bel.cs.ucla.edu/images/hplab1.GIF

Suggested ExperimentAlzheimer’s pre-clinical research in animal models

• Use THG to monitor , in vivo, changes in prefrontal cortex :o neuron proliferation / apoptosis (imaging, cell

counting)o Aβ plaque formation (imaging)o as a function of dose and time

• beta-Amyloid peptides destabilize calcium homeostasis, render human cortical neurons vulnerable to excitotoxicity

http://dementiasos.files.wordpress.com/2012/01/dem-amyloidvshealthycells.jpg

Suggested Experiment• G-CSF (granolocyte colony-

stimulating factor) when administered to mouse models of Alzheimer’s provides:o Cognitive improvementso Decrease in Aβ plaque (beta

amyloid) => can be viewed with THG

o Increase in neurogenesis

• Studies so far mostly in vitro, compare tissue samples from WT vs Alzheimer’s model mice

http://www.omicsonline.org/ArchiveJPB/2009/December/03/images/JPB2.505Figure1.gif

Suggested ExperimentWT AD

Low G-CSF dose

Med G-CSF dose

High G-CSF dose

NO G-CSF dose

THG baseline images & cell count

per unit volume before 1st dose

THG images & cell count per unit

volumeAt 0 / 4 / 12 / 24 / 48

hrsTap neurons and measure changes in membrane resting potential as a function of dose, time

Negative controlsAnalysis: graph and

model

Suggested Experiment

Hrs: 0 4 8 12 24

NO

Low

Med

High

Neuron proliferation / apoptosis (cell count per unit volume) as a function of dose and time

Suggested ExperimentResponse Surface model of neuron resting potential, as a function of dose and time

Resting potential (m

V)

Time (hours) Dose (mg/

g body

weight)

Conclusions• THG is a powerful alternative to fluorescent

dye-based imaging• Can visualize structures with high lipid content• Non – invasive, high resolution• Can be used for in vivo brain imaging, where

dyes may be toxic• Can produce 3D images up to 350µ depth• Can be combined with other imaging techniques• High speed, real time cell counting• Causes no irreversible damage to neurons (i.e.

no photobleach)

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targeted patching with third-harmonic generation microscopy." PNAS. 108.15 (2011): 5970-5975. Web. 30 Jul. 2012. <http://www.pnas.org/content/108/15/5970.short>