Optical detection via second harmonic generation
There’s been some work recently on looking at second harmonic generation for optical readout of action potentials… any opinions on this work?
First a brief primer on SHG (from Yuste’s recent Nature Methods paper on fluorescence microscopy):
In SHG, high-infrared light intensity drives the lowest-order nonlinear polarizability of molecules (or groups of molecules) in the specimen so that coherent light of exactly double frequency (or half the wavelength) is emitted. Because the process can occur away from resonance frequencies, there is no absorption of light, thus avoiding complications of photochemistry. This phenomenon is rare and requires, like two-photon excitation, a high concentration of photons at the focal point, something that also gives it optical sectioning. SHG is particularly interesting because it only occurs where chromophores are oriented in noncentrosymmetric arrays, such as chromophores adsorbed to biological membranes or other chemical interfaces. Thus, SHG is perhaps the only optical technique that is truly sensitive to biological membranes, something which makes it ideal for detecting changes in membrane potential. As many important biological processes, such as electrophysiological communication, detection and transduction of external molecules and cell-cell interactions occur at plasma membranes, SHG is likely to become a very useful tool for biologists.
Seed papers:
- Overcoming photodamage in second-harmonic generation microscopy: Real-time optical recording of neuronal action potentials [PNAS Open Access, Feb 2006] , L. Sacconi, D. A. Dombeck, and W. W. Webb
- Imaging membrane potential in dendritic spines [PNAS, Jan 2006] , Mutsuo Nuriya, Jiang Jiang, Boaz Nemet, Kenneth B. Eisenthal, and Rafael Yuste
April 4th, 2006 at 7:31 pm
I don’t know anyone who’s gotten it to work with a mammalian neuron, with external dye loading.
April 4th, 2006 at 9:24 pm
guest, thanks for the info. in fact i’m working with external dye loading into mammalian neurons right now (for voltage-sensitive imaging, or maybe just calcium), and i was wondering if i should check out this second harmonic stuff. i guess i’ll put it pretty low on the ol’ to-do list.
April 5th, 2006 at 12:19 pm
As for the issue with loading in mammalian neurons en masse, I wonder if there’s a way to genetically express a dye with SHG properties. Something similar to the FM dyes?
April 5th, 2006 at 4:18 pm
Some people have found luck with SHG and V-dep dyes by adding antioxidants, e.g. in Watt Webb’s group. But I believe they’re mostly focused on Aplysia neurons. Mammalian neurons may need extra help.
July 6th, 2006 at 8:02 pm
S. Baconnier and S.B. Lang, “Calcite microcrystals in the pineal gland of the human brain: second harmonic generators and possible piezoelectric transducers, “ IEEE Transactions on Dielectrics and Electrical Insulation 11, 203-209 (2004).