I"m using a Nikon A1R multiphoton system to image GFP-labeled cells below the brain surface, such as in cortical layer II/III (100+ µm deep). The system indicates the power output at a given wavelength (i.e. ~1700 mW for 920 nm, or something similar), which I presume is the amount of power delivered to the surface at 100% intensity. Most of the literature I've found suggests the laser power at the sample should be kept below ~50-75 mW to prevent bleaching or phototoxicity, which is a simple intensity adjustment at the surface since scattering is presumably minimal. By ~100 µm deep however, I find I require substantial increases in intensity to see my cells, usually 4-6 times higher than at the surface.
Is there any way to figure out how much power is actually reaching this deeper tissue?
Hey Thomas,
Firstly, I wouldn't trust the power output indicator of your scope, as it will be based on some measurement taken well before the objective, hence, any misalignment will render the output inaccurate. You need to put a power meter after the objective to measure the real power at the sample.
While there may, theoretically, be ways to measure the power at the focal point, deep in the tissue, I have never heard of anyone using them. You just kinda need to eye ball it, that is to say, you know how bright your tissue is at the surface with a known power, so the power should *roughly* be similar if you get the same brightness deep in the tissue.
Thanks William. From asking several other researchers I've definitely gotten the impression that it's mostly qualitative, but then I'll see publications where an actual measurement appears to have been made, and I don't know what to think. For example, in Kleinfeld et al 1998, PNAS 95(26) they write "The total average power delivered to the cortex ranged from 30 to 300 mW; higher values were required to image at greater depths in the brain to compensate for the scattering of incident. The total average power delivered to the focus was
Hi again Thomas,
I'm really pretty confident that when Kleinfield says "Average power delivered to the cortex ranged from 30 to 300 mW" they are talking about the power after the objective, measured with a power meter, and not what the literal power is deep in the tissue.
You can measure the power output from the objective (using a handheld meter like a thermometer) and then calculate for the tissue's transmittivity for the particular wavelength. Alternatively, deliver a known concentration of fluorophore to the depth you want to measure from and calculate in the reverse based on the signal output you get. Because the signals are usually variable between experiments, it is better to use a control flourophore in each case, that way you can normalize to it.
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