Ch 3 Flashcards
Why was the system originally set-up in that way?
The original system set-up was used for gcamp monitoring by exciting at 470 nm and 405 nm was the isosbestic signal which is a channel that records the fluorescence devoid of the sensor.
What do the bandpass filters do?
These will allow only the desired wavelength to pass and remove all unwanted wavelengths from the illumination path.
What are LED drivers?
These are the voltage supply of the LEDs by controlling the current that passes the the LED.
When triggered this will allow current flow to a certain rate in order to protect the LED.
What does the f and NA mean for the aspheric lens? What is an aspheric lens?
Aspheric lens are lens that are not a sphere in shape. They are used as they can improve abberations. They allow light to be focussed using the aspheric side with minimal aberration and can be used for collimation by applying light to the other side.
F is the focal length and this is the distance from the lens to where the light converges.
NA is the angle that the light is spread from the lens. The higher NA allows high ligh-gathering.
What does a 425 nm long pass dichroic mirror do?
This will reflect light shorter than 425 nm, such as 405 nm.
Whereas, 470 nm will pass through the mirror.
Therefore, the two beams will occur next to each other.
What is a dichroic mirror?
Glass that can split a beam of light into two beams with differing wavelengths. It does this by reflecing some wavelengths, while letting the selevtive wavelength pass.
Basically its a filter but on a mirror.
Can you use a camera instead of a photodetector for fibre photometry?
For this particular system no.
But with the correct optical devices this has been completed. It allows imaging of a specific field of view.
Why was a 525 nm emission filter used?
This allows 525 nm wavelength of light to pass with a band width of 39 nm.
This was used for the previous set-up where gcamp that has a peak emission of 510 nm. However, this is off-peak for Methoxy-x04.
In any case, this allows only the emitted light to pass and excludes excitation light from the signal.
Why did you choose a 2.5 mm setting for ferrules, and the appropriate parameters for the patch cable and fibre?
We choose 200 um core as this was less invasive and showed good fluorescence in vitro.
We choose 50o as this increased the light cone and was compatible with the low AF cable.
We choose the low AF cable as this would help improve our SNR.
2.5 was compatible with low AF setting at this time.
Where was this Methoxy-x04 spectra acquired?
An online source that allows their data to be shared.
Why were the mirrors swapped for reconfigured system?
We only want 405 nm excitation and we want to collect our signal now at 440 nm. In the first set-up the 498 nm dichroic mirror will reflect light below this, therefore removing our desired signal.
Therefore, this is moved to reflect the LED light to the path as this is desired. Then the 425 nm DC will allow light above this to pass which is desired.
Why were LEDs used?
As they are lower in cost and have a wider availability fo wavelengths.
Why was a 498 nm dichroic mirror used?
This allowed reflection of the excitation light to the launch system and transmission of emitted light through the mirror to the detection pathway.
What are broadband mirrors and why were they used?
These are mirrors that should have near total reflectance of all wavelengths of light and allow modification of the direction of light through the system.
What is a drop-in filter holder?
This was a holder that could attach to the mount rods with screws which can easily be removed and reattached without having to disassemble the whole system.
Why were lens tubes used?
To sheild background light from the emission pathway. Also, we covered this in cardboard to reduce background light.
How was the system realigned?
First, the light output at the end of the patch cable was measured using a light sensor while the launch system was focuseed into the patch cable and the mirror was focused onto the photodetector.
Once this was at its maximum, the fibre was put into high concentration DAPI and these mirrors was modified until the maximum read out was measured on the PD.
After, system calibrations were completed to see if read outs were comparable.
How was the system cleaned?
Weekly the system was sprayed with dust remover and patch cable ferrules were cleaned with lint free tissue and propanol.
Annually the system was cleaned if necessary with propanol, but as this was covered this was not often.
What does photobleaching the cable do?
By leaving light on this causes internal reflectance within the patch cable. As the patch cable contains cladding that can generate autofluorescence, this works to quench these signals before recording.
Does the low AF cable help?
We found that the low AF cable reduced the overall noise/AF of the system and when it was bleached, the AF remained low for the duration of the recording and was relatively stable. This helped retrieve stronger signals.
Why was enzyme remover used?
Some fibres had been used for in vivo measures and therefore, may have some tissue residue. Therefore, enzyme remover helped remove this and clear the fibre.
Why did all recordings have to be completed in the dark?
We did some test recordings with lights on and we found that the photodetector was very sensitive and this background light was picked up by the sensor and therefore reduced our SNR.
For long-term acute and chronic experiments we used a red light bulb as this interfered with the signal less.
What powers were used for system calibration measurements?
This varied across experiments. First we used 0-5 V. Then, we used 0.1 to 1mW/mm2 to reflect in vivo measures.
Why did you reduce the number of repetitions for the system sensitivity measure?
Methoxy-x04 appears to be quite photo unstable. So in attempt to minimise the photobleaching of the dye throughout the experiment, we reduced the time that the fluorophore was being excited.
Why did you use 6-9-month old mice?
For this part of the project, we really wanted to confirm that this approach was a feasible way to monitor plaque pathology. To do so, we wanted the best chance of monitoring a FP plaque signal. Therefore, using mice that had a strong plaque pathology was desirable.
Why were these experiments performed under terminal anaesthetic?
We wanted to acquire signals at increasing depths. As flat fibres have only one illumination and collection depth, it would not be possible in a waking condition, without using a microdrive. Therefore, terminal anaesthetic was required.
How did you align between bregma and lambda?
By using a glass pipette and stereotax, I measured the depth distance from bregma and lambda. I wanted this <50 um as this meant it was less likely to miss my target.
Why were some recordings performed within the surgery room?
At this point thsi was due to availability of space within the lab. We also wanted depth profile experiments to be only under urethane as this had been shown to be less effective on neuronal activity. Therefore, we moved rigs so the surgery room would be available for others.
What does fluovac do?
Absorbs the anaesthetic.
How was the heatmat temperature controlled?
there was a probe underneath the plastic cover that was weight activated and controlled the termperature.
If you were concerned about the bleaching of M04, why did you maintain the 10 repetitions for DP recordings?
As this was a one-off measurement and not occuring every 5-s, it wasnt as big of a concern.
How did you create a linear regression model?
We measured the light output at the end of the fibre using the light sensor at 1-5 V. By fitting a linear line, we could use the coefficients to determine the voltage of LED required for a desired output.
Why did you record at 5000Hz?
Because we are delivering light pulses at a millisecond range and we want to sample around this.
5000 Hz allows sampling of everything.
Why did you choose 440 and 550 nm?
440 nm is the peak of M04 emission spectra so we expect this to boost the fluorescence recorded.
550 nm should be mostly devoid of a M04 signal and should be high in AF as most AF can occur in this wavelength. So this was a trial control channel.
Why did you choose these implant sites?
These three implant sites pass brain areas that are highly dense in plaque pathology and sparse in plaque pathology. This should provide a signal that has a good comparison between high and low fluorescence to allow confirmation that a signal is being detected.
Why did you inject 24-h before?
As in many previous papers and the landmark study, this was shown to provide the best signals for 2PM as it had the least background fluorescence. Therefore, this suggests this is most reflective of plaque staining.
What did you expect/see with the DP experiments before the APK recording?
Despite Methoxy-x04 not being injected yet, plaques can provide strong autofluorescence and therefore we expected once we began to hit our target, we would see a rise in fluorescence in the 440 and more specifically the 550 nm channel. We done this to be able to have a better idea that we are in the correct area as this is a one-chance experimental protocol.
Did you see this with the DP?
Yes, I tended to see a small increase in fluorescence as I reached my target and if the coordinates and fluorescence matched, this is when I would start the recording.
How do you think the autofluorescence of plaques will interfere with the recording?
As the recording channel is so specific to 440 nm with a small bandwidth of 10 nm I believe this signal should be a strong M04 channel.
Why did you perform a 30-minute baseline recording before injection?
So that we had a measure of the fluorescence within the brain without M04 present as a baseline. This would allow us to normalise against this baseline to see small increases in fluorescence.
Will the mouse being under anaesthetic affect the pharmacokinetic properties of M04?
It remaisn unclear as we couldnt acquire a positive signal in acute to compare.
However, the neurinal activity is meant to be maintained, but I would assume it would be slowed due to a enhanced parasynmpathetic nervous system.
Why did you sample at 30-s?
As the change in fluorescence would be fairly slow, a rapid sampling interval that is required for calcium imaging is not needed here. This was used to identify if a slower sampling interval could be used.
Why are some recordings completed with voltage inputs and other irradiances?
At first, I used voltages. But as this could vary from fibre to fibre depending on their functionality, the light output would not be consistent across mice.
Therefore, I implemented an approach to calculate the voltage requried for a chosen range of irradiances so that we could be consistent across recordings.
Why did you choose that brain region for APK?
Subiculcum is the most dense region of plaques in the 5xFAD brain so should allow ius to detect the strongest signal available.
Why did you fix the tissue with paraformaldehyde?
Because this maintains the tissue and cell architecture in a life-like state which allows us to stain the tissue. Additionally, they allow good preservation of the tissue and make it sturdy enough for sectioning and processing.
How long do you fix the tissue and why?
We fix it for 24-h after to allow the process to occur. But no longer as this can make the tissue fragile.
Why do you use sucrose in preparing brain tissue?
For cyroprotection where we want to protect the tissue from freezing processes throughout histology to maintain the tissue structure.
Why did you use 100 um sections?
As my fibre was 200 um in width, we wanted to choose a section thickness that would allow identification of the fibre track.
Why did you use sodium azide?
This works to prevent bacterial infection of the sections and allows them to be preserved for longer.
Why did you stain with TS if you had already injected with M04?
At first, we wanted to confirm that the M04 stain had a similar plaque marking as TS.
Additionally, it could be used as a backup channel if something went wrong with the injection as we were still confirming if the system could detect M04.
What does TS stain?
Like M04, they bind to any type of amyloid as their target is beta-pleated sheets.
What does neurotrace red stain and why did you use it?
It is a nissl stain that is used to mark nissl in the cell soma of neurons within the brain. This is important substance for protein synthesis and channels in neurons.
We first used this to help identify the structure of the brain when imaging. However, we later found that imaging just the blue channel was sufficient for identification of brain regions and further analysis.
What are free-floating sections and why were these used?
This is sections that are floating within a well that contains the specific staining agent.
The reason we used this approach rather than fixing them onto the slides is because this is better at maintaining the brain structure and preventing tissue loss as in fixed conditions its common for some of the section to be washed away. Also, it allows better antibody penetration on both sides and reduces background fluorescence due to better washings.
Why did you stain adjacent sections?
We done this because we wanted to identify the full fibre track. Occasionally this could be at a slight angle and as we are quantifying plaques on the contralateral hemisphere, this sometimes was at an angle. So this approach ensured we would have enough brain tissue either side of the implant so no plaques that should be quantified were missed.
Why do you use PBS in triton X?
We used this because it increases the permability of the section and therefore allows better staining.
Why is gelatin used?
To penetrace the section to hydrate it and maintain its structure as well as allowing it to stick to the section appropriately.
Why did you use epifluorescent microscopy rather than others?
As this allows quick imaging of the whole brain section.
Our goal was to identify the fibre track, get images of the whole section and plaque pathology.
Therefore, this is a more suitable approach as we do not require depth analysis of the full section. While this would be nice, like light sheet microscopy this has high acquisition times, and can be expensive for numerous samples.
What is epifluorescence?
An imaging approach where both the excitation and emission light flow through the same objective.
Why x4? Did you try higher objectives?
Becuase this was suitable to get the plaque pathology across the whole brain section.
Occasionally, I would look at x10 to confirm presence of the fibre track damage.
Why is there differences in the exposure times?
At this point, a lab technician took over my histology and he believed imaging at this exposure provided better images. Perhaps as we were only imaging the blue channel, a lower brightness was fine.
Why did data need to be offsetted and how exactly was this completed?
Within the system there was a slight grounding offset where the baseline signals were ~-0.05V rather than at 0.
To abolish this, the system and DAQ channels were grounded and then we would take an average value of the baseline signal of each channel and add this on to each channels to boost signals to have a baseline at 0.
This would allow us to see a true and more understandable value of the fluorescence measured.
Why did you detect signals that were greather than 0.05V?
This was because throughout all recordings all irradiances that we supplied would have a voltage that was greater than this value.
This meant that by detecting the values in the data matrix that had this voltage or greater, we could identify the times in the mastrix when the LED was on.
How was event times and data extracted?
Using ther detected events from the sync channels, we would take the first value as the event onset.
Then, at each of these data points, the photodetector channel was extracted from data points 3 to 9 to accounr for some offsets in LED power on either side of the pulse.
Then, this data was averaged.
How did you acquire a fluorescence signal for a single concentration?
As the concentration was increased every 2-mins, the median fluorescence was calculated in 2-min time bins.
Why did you calculate median and not mean?
We found that the fluorescence signal can provide some outliers. Specifically in this experiment when completed in a plastic well. Therefore, we found calculating the median would be a better reflection of the fluorescence that is not skewed by these measurements.
