Imaging deeper with 3p
One of the factors that limits the depth at which one can image brain tissue in vivo with two-photon imaging is out-of-plane fluorescence. Although the excitation falls off as the square of the distance from the focal plane, some highly fluorescent samples can add to background even if they’re out-of-plane. For example, the dura mater fluoresces quite well with 810 nm excitation, and as power is increased to see deeper into the brain, the dura can generate green photons even if it is not in the plane of focus.
Experimenters using brightly labeled layer 5 neurons have noted that they can easily image deep into the brain, partially due to the decreased background fluorescence at 910 nm.
So why not use three photon excitation? The excitation photons have even lower energy (1500+ nm), and the probability of excitation falls off as the power of 4. Horton, Wang, Kobat et al. did just that.
http://www.ncbi.nlm.nih.gov/pubmed/22029361
this group was imaging vasculature down to 1.6mm
So the obvious follow-up question is: what prevents n-photon excitation to image at arbitrary depth?
(I’m sure there’s a physics answer I don’t know; my optics knowledge is terrible)
Alfred- Thanks for the reference.
Neuroecology- The short, 2-part answer is that it gets harder and harder to deliver enough excitation photons, and the gain in depth is not linear. In this case, a lot of the gains are though reduced out-of-plane fluorescence, i.e., noise.
Also, one catch is that the pixel clock is 15 us because the laser pulses at a rate 1/80th of a standard Ti:Sapph laser. So the frame rate isn’t great. At least not with their setup (they used about 20 seconds/frame).
along the lines of neuroecology’s question, does there exist a situation where one would be able to image arbitrarily deep?
Ed Callaway showed images down to 1.5 mm in Neuron, see 10.1016/j.neuron.2011.07.005.
i could only find up to 750um in that paper?
jason kerr’s lab has hit 1000um using regenerative amplification doi:10.1038/nn.2879
but many people are routinely reaching layer5/6 without particularly special methods. a lot of structures lay just beyond the 1000um mark.
Look at Fig 2.
huh. turns out to be an erratum
http://www.cell.com/neuron/fulltext/S0896-6273(12)00283-8
The reason the article is posted here is because it’s using 3p imaging to image deep inside the brain and they got it working fairly well. It’s not the first deep imaging paper, nor is it the first 3p imaging paper.
Imaging depth cannot be directly compared across preparations. For example, imaging dye in capillaries is a much lower bar than imaging GFP in somata, which is itself a lower bar than imaging dendritic spines.
Several variables that differ among preparations can significantly influence imaging depth, including the brightness of the object being imaged and out-of-field (or background) fluorescence.
The references given above by the commenters are excellent. They’re all very interesting papers with a lot of value. However, their value cannot be summarized as “imaged to X depth”.
Isn’t it not only the delivery of light to the fluorescent target, but also in the collection of the emitted light? The deeper you go, the more material the emitted light has to traverse to get back out. So there is scattering and now the wavelength of light (emitted) is again visible so scattering events are significant. I would think this plays a major role in the difficulty of imaging deep in tissue.
True. Which is why the fluorophore being imaged matters and makes comparisons difficult.
Water absorption!!:
http://en.wikipedia.org/wiki/Electromagnetic_absorption_by_water