For two-photon point scanning microscopy, the excitation laser is typically pulsing at a repetition rate of 80 MHz, that is one pulse each 12.5 ns. To avoid aliasing, it was suggested to synchronize the sampling clock to laser pulses. For this, it is important to know over how much time the signal is smeared, that is, to measure the duration of the transient.
The device that smooths the PMT signal over time is the current-to-voltage amplifier. As far as I know, the two most commonly used ones are the Femto DHPCA-100 (variable gain, although mostly used with the 80 MHz bandwidth setting) and the Thorlabs model (60 MHz fixed bandwidth).
Observing single transients for different preamplifiers
However, 80 MHz bandwidth does not mean that everything below 80 MHz is transmitted and everything beyond suppressed. The companies provied frequency response curves, but in order to get a better feeling, I measured the transients of the above-mentioned preamplifiers when they amplified a single photon detected by a PMT. All transients are rescaled in y-direction (left-hand plot). I also determined a sort of gain for the single events by measuring the amplitude (right-hand plot). I also used two preamplifiers for each model, but could not make out any performance difference between two of the same kind.
For the 80 MHz bandwidth setting (Femto), the transient does not fully decay even after 15 ns or later; the Thorlabs preamp is even slower than this. Both exhibit a smooth multi-step shape during the decay phase.
At first glance, the Thorlabs preamp seems to be the obvious best choice, since the bandwidth is similar to the Femto 80 MHz and the gain is 3x higher. But for functional imaging, electrical noise is not a big problem, since the main source of noise is simply photon shot noise. In my hands, neither of both clearly outperformed the other, although this might be different for a completely different set of PMT/fluorophor/SNR.
The 200 MHz Femto setting would be perfect for lock-in sampling (and I already used it for that purpose), but the gain is, at least for my PMT, at the limit where electrical noise can become dominant (see figure on the right side). The 15 MHz setting, on the other hand, does not give any advantage, except if one samples with much less than 80 MHz.
Counting photons using an oscilloscope vs. using Poisson statistics
Looking at the raw oscilloscope traces when the microscope is scanning a biological sample, one can make another interesting observation. Shown here is a time window spanning 4000 ns, that is, 320 laser pulses. But those laser pulses only manage to elicit 14 photon-detecting events.
Let’s put this into some context. Normally, I bin every 8 laser pulses to one pixel; therefore, counting the detected photons per 8 pulses gives the photons per pixel. The outcome of doing this for each oscilloscope trace gives a couple of numbers (# photons per pixel bin), which are plotted below as single data points and histogram (figure to the right). Here, the number of photons per pixel bin is between 0.1 and 0.9 photons. In other words, only every 10-50th laser pulse creates a fluorescence photon that is detected lateron.
For the same sample, I also recorded an image and determined the number of photons per pixel as described earlier (based on a Labrigger blog post). As far as I can tell from the small statistics for photon counting using oscilloscope traces, there is a surprisingly close agreement between the two independent measurements. It might be off by a factor 1.5 or less.
On a side note, the reason for the low number of photons compared to this earlier example is a different fluorophor. In the previously shown example, it was the artificial calcium sensor rhod-2 which features a high baseline fluorescence; here, the tissue is weakly labeled with GCaMP6f, which has a much lower baseline fluorescence (actually, the tissue shown here is actually probably dead and therefore a little bit brighter than normal GCaMP6f-labeled neurons).
What I learned from this:
- Neither the Femto DHCP-100 at 80 MHz nor the Thorlabs preamplifier are very well-suited for lock-in sampling to 80 MHz laser pulses.
- For normal samples (by which I mean: functional calcium imaging), despite a difference in gain (factor 3), both preamps perform similarly, since for both the signal is stronger than electrical noise.
- Normally, I (and certainly others with dim samples) get < 1 photon per pixel for GCaMP, and << 1 photon per laser pulse. Given by how many infrared photons the tissue is hit (around 10^9 photons per pulse!), this is astounding.
- Calculating the gain of the system and the number of photons per pixel using the method mentioned before yields results that are consistent with (oscilloscope) photon counting. It appears as if one really can rely on this easy method to estimate the number of photons per pixel. This can be an important piece of information when one needs to decide whether to implement a photon counting hardware solution for a given applicatoin or not.
Update [ 2016-09-22 ] :
Upon Dario’s comment, I looked into what happens if you sample at 80 MHz (i.e., synced to the laser pulses), but at different phase lags, as described on the Scanbox blog by Tobias Rose. Using the transients measured above, one can simulate the phase-dependence of the signal intensity. The intensity ratio of best timing vs. worst timing is around 3 for the Femto 80 MHz, and <2 for the Thorlabs 60 MHz. This means that the sampling phase relative to the laser pulses is definitely important, and more for the Femto preamp than for the Thorlabs one. (I do not see this strong dependence with my microscope, but this may be due to a badly behaving laser sync signal.)
To make things more complicated, this factor of up to 3 means a threefold increase in signal, but not a threefold increase in SNR. SNR is mainly determined by shot noise, not voltage amplitude; however, I would bet that electrical noise can become quite prominent in the trough of the phase plot, and phase optimization still increases SNR.
“the transient does not fully decay even after 15 ns or later”. But the issue, it seems to me, is how much variability you would get by sampling at a random phase with respect to the laser with a bandwidth of 80MHz. In our hands, and it seems from your own measurements too, that the signal can change by a factor of 2. That would be adding unnecessary noise to the image that can be prevented by synchronizing the sampling to the laser.
Yes, thanks for pointing this out. I agree, the shape of the transient indicates that one should sample at 80 MHz locked to the laser pulses. For real measurements, I see a factor of less than 2, but also clearly more than 1. This difference to your measurement might depend on small technical differences, for example the precision/jitter of the laser sync signal.
However, if one could constrain the transient to e.g. 5 ns, one could sample during this short time-window, thereby excluding electrical noise or electron noise of the PMT which has accumulated during the previous couple of nanoseconds. This is not possible with the Thorlabs preamp or the Femto 80 MHz setting, and this is what I meant with “lock-in sampling”.
I just modified the blog text in order to make it (hopefully) less prone to missunderstandings …
It is a helpful topic for me. I am using the FEMTO DHPCA 100 for preamplifying the signal from the PMT ( R5929 – Hamamatsu) – the scanning speed is 8 kHz , but I don’t know which mode of the FEMTO DHPCA 100 (AC/DC) should I use; if possible, please guide me to a typical setting of the transimpedance amplifier.
I am using the FEMTO amplifier with a resonant scanning two photon microscopy system. But I don’t know how to setup the parameter of the FEMTO amplifier correctly. Would you mind to guide me to a typical setting of the amplifier ?
Hi – of course the setting depends on your application (not only scan speed, but also pixel size or pixel dwell time, or the fluorophore).
Most of my experience with this amplifier comes from two-photon imaging of calcium sensors like GCaMP. I would start with:
– GND or Bias: you can use both; try whatever gives you less noise in your microscope or setup
– My preferred V/A gain setting is high (H)
– For bandwidth (BW), I use 80 MHz, although 14 MHz might be worth a try (80 MHz is used more commonly, see e.g. the comments here: https://scanbox.org/2014/03/18/synchronize-to-the-laser/).
– FBW (full bandwidth)
– I would prefer DC setting; AC would create some unwanted effects like strong flourescence at one spot in your image affecting other locations (some sort of ringing of the AC filter). In the end, the DC setting will give you a more reliable image of the true fluorophore densities compared to the AC setting.
Hope this helps,
Hi Peter, thanks so much for your kindly replying.
I have some more questions:
1. I have a 120 MHz internal clock digitizer and @80MHz femtosecond laser pulse. The pixel dwell times are typically 45 ns (for 1024 pixels resolution) and 90 ns (for 512 pixels resolution). Do you think I should use the laser syn clock for the acquisition or 120MHz internal clock is better ?
2. When I set the DC mode for the FEMTO amplifier (V/A gain is High; bandwidth 80MHz), I got high offset signal and it turns out to be overloading for the FEMTO amplifier. Could you please guide me how to adjust the offset for this problem ? I can see the offset screw on the amplifier, should I tune it ?
1. Using the laser sync clock might be the better option. I have been using both (100/120 MHz internal clock or 80 MHz external laser sync signal), and both worked similarly for me, and I did not see a big difference of signal (but e.g. Dario Ringach observed for his microscope a big signal increase when using the the 80 MHz sync signal, https://scanbox.org/2014/03/18/synchronize-to-the-laser/).
A side-effect of using 80 MHz vs. 120 MHz can occur, depending on how you acquire data points for each line: Either you record every single data point during the line scan period and bin afterwards (then you get more datapoints with 120 MHz and have to bin down to the same pixel number), or you use a fixed number of sampling points per line (for example, I always sample 4096 data points per line, which I lateron bin to 1024 or 512 pixels). For the latter case, using 120 MHz sampling basically reduces the field of view in x-direction (fast scan direction) and therefore the fraction of the scan that is used for acquisition, since the 4096 data points are recorded in a shorter time compared to the 80 MHz scenario. Hope this is more or less clear to you …
2. I do not believe that switching to DC mode really overloads the amplifier. More likely, you have to adjust the lookup table in your acquisition software (white and black values). Alternatively, the input range of the DAQ board is saturated/overloaded by the input. If this is the case, you can simply use – as you mentioned – the offset screw of the amplifier to tune the offset – that’s totally safe and will not affect anything except your offset.
Thank you so much Peter. It would help me a lot.
I’m glad that I could help!