Depths of Detection

A fortuitous chat

The other week I had a chance conversation with a colleague about one of her experiments she was struggling with. It involved recording AgRP neurone activity with in vivo fibre photometry. She was particularly having problems with her fibre placements. Her AAV injections were fine, as she was getting great GCaMP expression in the Arcuate nucleus. But, she was struggling to get good fibre photometry signal. It seemed that she was either overshooting with her fibre and causing damage to the base of the mouse’s brain, or she was not going deep enough to get close enough to the AgRP neurones to pick up the signal.

This led me to wonder about photometry fibre placement. How close do you actually need to get to the fluorescent cells to pick up a good fibre photometry signal? However, it’s difficult to find information about this related to in vivo fibre photometry. The couple of studies I found both used 2-photon excitation for the photometry, but that has a very different excitation profile than “normal” epifluorescent photometry1,2.

Photometry signal detection

After some sleuthing, I found a paper by Simone et al. developing an open-source photometry system. As part of the validation process, they tested the detection power of their system using an artificial setup (small pieces of fluorescent tape submerged in 2% intralipid; Figure 1). They found that detection tailed off dramatically even before 100 µm displacement.

However, the Simone data uses a system that is very different from our in vivo setup. In particular, they used low diameter fibres with intralipid as the confounding medium.

After some further scouring of the internet, I found a thesis from the University of Florida, where the author had set our specifically to investigate and optimise fibre photometry recording4. A quick caveat: as a thesis this work has not been published through peer review. But, the work does look very thorough and will have passed a viva board so I think can be trusted.

Anyway, as part of the thesis, Mansy set up an in vitro system using fluorescent beads obscured by acute brain slices to investigate detection profiles with different fibre optics (Figure 2). Using 400 µm fibres, they found that fluorescent detection dropped off rapidly upon distance from the fibre tip. Interestingly, this was far more pronounced in the .50 NA fibre than the .22 NA fibre (Figure 2A). This surprised me, as we are always told to use the highest NA fibre possible for photometry. The reasoning being to increase the amount of light collection.

However, upon reflection, it makes sense to use lower NA fibres if you think of the detection based not just on the fluorescent collection distance, but also the depth of excitation light penetration (for more info, check out my Depth calculator and blog posts). In that case, it would absolutely make sense for the high NA fibre to have a much decreased detection profile. The difference was even more pronounced when looking at the 3D detection volume (Figure 2B).

How to relate this to our work? I know that my colleague who was having photometry troubles was using a 400 µm .48 NA fibre. These should give an almost identical detection profile to the .50 NA fibre investigated by Mansy (Figure 1A, left). I have since suggested to her that she use lower NA fibres. Switching to the .22 NA fibre should extend her 50% detection depth from about 150 µm to about 300 µm, based on this work (Figure 1A, right).

A note on tapered fibres

Finally, I found a paper which improves the depth of fibre photometry signal detection even further, by moving away from flat-ended fibres2. The problem with imaging from a flat-ended fibre is that the light emission tails of exponentially, and the detection along with that. Furthermore, the detection will also be heavily biased towards the neurones nearer to the fibre. This is dramatically improved by using a tapered-ended fibre to provide more uniform light emission and signal detection (Figure 3).

I had a quick search online, and found that Doric sell tapered photometry fibres (we have a Doric photometry system, and we purchase our photometry fibres from them). My recommendation to my colleague, and anyone else doing photometry, is to try out the tapered fibres provided they will work in your experimental system, and failing that to use lower NA flat-ended fibres.

1. Pisanello et al., Front Neurosci 13(82) 1-16 (2019) The three-dimensional signal collection field for fiber photometry in brain tissue

2. Pisano et al., Nature Methods 16, 1185-1192 (2019) Depth-resolved fiber photometry with a single tapered optical fiber implant

3. Simone et al., Neurophotonics 5(2), 1-10 (2018) Open-source, cost-effective system for low-light in vivo fibre photometry

4. Mansy, PhD Thesis for the University of Florida (2019) A systematic characterization of fiber photometry for optical interrogation of neural circuit dynamics