Imaging nerves in vivo with harmonic generation microscopes

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Jon Moulton
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Imaging nerves in vivo with harmonic generation microscopes

Hsieh CS, Ko CY, Chen SY, Liu TM, Wu JS, Hu CH, Sun CK. In vivo long-term continuous observation of gene expression in zebrafish embryo nerve systems by using harmonic generation microscopy and morphant technology. J Biomed Opt. 2008 Nov-Dec;13(6):064041.

http://www.ncbi.nlm.nih.gov/pubmed/19123687

"Optical higher-HGM can continuously observe zebrafish embryos, including cell morphology of the nervous system, for over 20 h with no photodamage effects, making it ideal for morphant technology research"

Fraser Moss
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Jon

Jon

Can you give us an explanation of harmonic generation microscopy?

Jon Moulton
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Hi Frasermoss,

Hi Frasermoss,

I'm intrigued but out of my field. Here's what I have picked up so far. Take all of this with a big crystal of salt -- I just learned about this technique this morning.

The microscope resembles a scanning confocal setup, but has emission filters tuned to an integer multiple of the photon excitation energy. For second harmonic generation microscopy, the emission is filtered for photons of twice the excitation photon energy (half-wavelength), while for third harmonic generation microscopy the emission is filtered for photons three times the excitation photon energy (1/3 wavelength). For the paper cited in the previous post, they excite with a Cr:forsterite laser with a center wavelength of 1230 nm.

When the excitation energy is focused into a volume with the appropriate optical properties for second harmonic generation, two long-wave photons are converted to a single half-wavelength photon (I suspect there are physicists who would howl at that explanation -- I invite them to attempt to set me straight!). Similarly, there are focal environments that can convert three photons into a single 1/3-wavelength photon (the third harmonic).

It is the properties of the materials in the focal volume that determines whether the second harmonic or third harmonic photons are emitted. Some biological structures that can emit second harmonic photons are spindles and nerve fibers. Collagen can also emit second harmonic light. Third harmonic signals are emitted where two media of different optical indices have an interface, such as a membrane surface.

It appears that this system can be used to get rid of a lot of noise. If few structures in your sample of interest will produce a signal detectable by the harmonic generation microscope, you get a simpler image than the usual confocal image. Another advantage is that the excitation photons are fairly low-energy, allowing longer exposure to the excitation beams before the sample is significantly damaged. This property is especially useful for in vivo imaging, for example of embryos.

Materials such as fluorescein or hemoglobin can produce harmonic signals. I am interested in delving farther into the harmonic microscopy literature to see what sort of compounds can be used as harmonic dyes. I have glanced at papers reporting measurements of cell membrane potential using this technique. There is much more to learn.

Try Googling "harmonic generation microscopy" and "harmonic microscopy".
Also:
http://en.wikipedia.org/wiki/Second_harmonic_generation
http://en.wikipedia.org/wiki/Harmonic_generation

Fraser Moss
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I did find this video that

I did find this video that may (or may not) explain some of the physics. I have not had time to watch the whole thing through yet though.

If anyone who knows there stuff on this can chime in it would be great

Jon Moulton
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OK, I watched it -- that's an

OK, I watched it -- that's an interesting lecture. The technology described produces many higher harmonics; using higher harmonic generation a laser can produce a comb of peaks at higher energies, through the high energy UV and into the low energy X-ray band. This can potentially generate high-energy photons for very small scale lithography, applicable to semiconductor manufacturing. The higher order harmonic generation occurs where the laser beam is briefly pulsed and focused into a gas, usually an inert gas. Atoms of the gas are ionized by the intense laser irradiation, losing inner shell electrons as well as valence electrons, then as the electrical field of the photon reverses it accelerates electrons back at the ionized nucleus and as an electron is drawn electrostatically to the nucleus it is sharply accelerated, emitting a very high frequency photon. Requiring high intensities around 10^15 watts/cm^2, this specific application is too energetic for imaging biological samples without significant damage.

As I understand it, the usefulness of the SHG and THG microscopy applications in biology derives from two factors: the low excitation energy (allowing observations in vivo over time) and the structure/material-dependent generation of the harmonics, allowing mapping of SHG generating materials or THG-dependent structures. The high-energy environment in the laser focal region of the higher-harmonic generator would rip electrons from most any atom, so the structure/material discrimination of SHG and THG microscopy would not be available using the high-energy harmonic generation scheme described in the YouTube lecture.

Online video lecture by: Prof. Attwood
Prof. Attwood presented additional material by:
Prof. Henry C. Kapteyn (U Colorado Boulder)
Prof. Chang Hee Nam (KAIST Univ. Korea)

http://www.youtube.com/watch?v=hclQ2mb1HG0

Fraser Moss
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Thanks for that excellent

Thanks for that excellent reply Jon.