Ch 4 Flashcards
Have previous papers used TFs for depth resolved monitoring?
Yes, Pisano 2019 show the first use of the TFs for monitoring fibre photometry signals where they assess dopamine signals in the striatum as mice perform a behaviour task.
To my knowledge, nobody else has publishable results using these.
What are the main findings of Pisanello 2014?
Pisanello 2017?
- gold plated TF with windows to deliver light
- establih what angles of light are required for multomode derlivery of light regions fat away from the tip
- light derluvery length increases with increased NA and decreased TA
- the smaller the angle of entre the light will come out the top of TF, the larger, it will reflect inside and propagate
- illustrated site illumination in FITC and brain slices
- shown site with multi wavelength in dual fluorescent solution
- checked if shaking/twisting patch effected the light
- in vivo in striatum with silicon probe showed site detective excitation
)_____-
to show it could be whole brain or region specific optogenetics
- TF with no fold plating
- did ray simulations to confirm light patterns
- monitored inflammationn
- see increased modulation over intentiy and depth compared to FF
- implemented the galvo mittor for continuous sweeping
site directed opto in striatum
What are the main findings of Pisano 2019?
- did raster scanning along tf in FITC and monitored response with two hotodetectors to determine signal collection field
- found unform and depth specific collection field in fitxc and human tissue
- equalised power density along TF
- monitored in vivo dopamine in striatum woth depth-resolved differences.
How exactly is the TF made?
A heat and pull process where the tip of the fibre is heated and stretched until the ti is ~500 nm,
They set this with a NA of 0.37 and taper of ~2.4
How can the TF record fluorescence?
There is a change in the refractice index (the light refeaction level) from tissue to TF which makes it couple into the fibre.
Then the modes of light pass the same way as they fo on the way down – the site of entry is correlated with the angle of light entering the TF.
How does the taper angle determine the active region?
the tpaer angle decreased will increase the length of th e active region.
Pisanello found that increasing this shortened it most likely because the internal reflectance was too hgih.
How were those images of the fibres acquired?
The fibres were placed into a solution of high concentration fluorescein in a cuvette.
Then light was delivered through the system.
A camera was set up focussed though the cuvette and images were taken.
Why on the images are there some spects of fluorescence?
It is most likley the fact these fibres have been previously used so there may be some residue on them.
What was the function of the galvo mirror?
As we have 1.8mm of TF to work with, we needed a way to modulate where the light was being propagated out each depth of the fibre instead of it being along the whole shank.
This mirror modulates the direction of the laser beam in order to change where the light enters and leaves the TF.
Why for this system did you need to use a laser?
Because we are changing the focus of light on a rapid time scale and lasers are known to have a more focussed beam and a faster operation speed.
Why have you now got an additional photodetector channel?
Because lasers are more unstable than LEDs and can fluctate due to head differences. We also found that perhaps the way the laser was being driven - through the DAQ was causing an unstable power voltage and therefore, we implemented a photodetector to monitor this read out.
Did you use the 405 nm read out for analysis?
No, as we had out control group we did not use this. Also the changes were minor so shouldn’t have effected this profiles.
Why did the original laser deterioate?
This was purchased from a cheaper supplier so it is thought that there was some electrical faults that were perhaps not picked up as they do not have as vigiourous quality control.
Also, this laser was not based on a heat-sink which is important for temperature control in order to prevent damage.
Why was a glass slide ok for focussing onto a detector and not a lens for 405 nm channel?
Because the signals on here were very high as it was direct from the laser so even capturing a small part of this signal would be high enough.
What is a neutral density filter and what type did you use?
Why did you use this?
This is a filter that across all wavelengths, will reduce the light power at equal amounts.
*** need to check system
We used this because this laser was very high power and produced alot of light. To reduce it to working and safety limit we used this.
Why didi you use an aspheric lens that was length 50 mm focal length?
To generate a focal spot of <500 um and the appropriate distance of the system.
Why are some of the focal length of lenses different throughout the system?
As some were being coupled into the fibre they needed to be smaller.
What mirrors were used for first reflection in the system?
Broadband mirrors. These will reflect all wavelengths with equal transmission. These allow direction of the light path.
How is the light directed towards the photodetectors?
The light passes back through the fibre before reaching a 425 short pass mirror where light below 425 will pass and light above will be reflected. This removes the excitation light from our emission channels.
Then the light passes a 525 nm short pass dichroic mirror where light above 525 nm will be reflected before passing a 550 nm bandpass filter which is focused onto the photodetector by an aspheric lens. Light below 525nm will pass through the mirror before a 440 nm bandoass filter and being focussed onto a PD.
How is the light seperated for each photodetector? Is this not a mirror instead of a filter?
Yes, the diagram is wrong.
The light is seperated using a 525 nm shortpass mirror.
This seperates the light pass for 440 and 550 nm.
Why did you have a 1.25 mm ferrule setting for this system?
This is the setting that the TF can be made in and it works better as they are lightweight for the chronically implanted headcap.
Why did you have a 470 laer in the first system?
Because at first we discussed the possibility of using this as the control as this system was deisgned before we established the system needed reconfigured.
Why do you need to focus light onto the galvo mirror with a lens?
To make the beam focussed so that the light path is modulated appropriately.
What does the heat sink do?
The heat sink works as a fan to remove heat by dissapating it into air.
Therefore, it prevents the overheating of the laser and damage.
Why did you not need as many mirrrs once the new laser was put in?
Because we did not need to combine the two light paths
Why was the laser grounded?
The laser was grounded because we were seeing low/high frequencu noise. We grounded the laser and the DAQ to remove any outside encirtonmental contributions to this noise.
What do you mean by saying that the galvo mirro is a single axis?
The galvo only moves across a sinve direction. Some have capabilities to move in more than one.
How big is the angle range that the galvo mirror can go?
Do not know the exact angle, but the voltage range is -5 to 5v. I think this was around 12.5 degrees.
Why will the TF form these uniform rings?
Because the light should be moving up the TF shaft in a uniform manner and therefore from the tip, the light cone should form a unifrom circle.
What is the pupose of looking at these illumination rings on white paper?
It illustrates if there is uniform light being propagated at the TF and is representative of how well the light is aligned.
If the circle is dim, damaged it can represent dust on the ferrule or that one of the axis is not aligned appropriately.
What light output from the patch cable were we looking for and how could we tell if the system alignment was good from these values?
~2mW at the highest driving voltage as this was our safety limit.
We would look for consistent values across GVs maintained across days.
How did you monitor the voltage read-out on the 405 nm photodetector?
We had a built in online function which processed the signals as the others are.
How exactly was this system recalibration completed?
The lightoutput at the end of the patch cable with and without the TF attached was measured at increasing gv and laser power. The laser power was kept within the ange used for in vivo experiments as this gave the best estimate.
How was a linear regression modle for each GV formed?
The light output measured for the patch cable was plotted against the laser voltage. Then a linear model was fitted and the slope and y-intercept were used for all recordings.
Why was an initial bleaching of >8 hours required?
Because this would remove the autofluorescent particicles from the systwsm to a baseline level which would only need topped up before each recording.
Why did the bleaching protocol progress over time?
Because we realised that the af varied across galvo voltages which is why we added on the galvo sweep.
Then, we noticed some inconcsistently of baseline fluorescence across day and thought this was perhaps due to the af state of the system being varied so we implemented the bleaching threshold to try make this as consistent as possible.
What was wrong with bleaching at one galvo voltage?
Because there was varied AF at different galvo voaltages and bleachigna t only one votlage meant some af was not being bleached.
Why was bleaching completed at different voltages for each laser?
Because these lasers had a different max votlage.
Why was the glavo mirror drieven at 1Hz for bleaching?
Because this sweeped through all galvoes over 1 second which didnt require a rapid effect and so wouldnt be taxxing on the system.
If this bleaching threshold changed overtime, why and would this effect the results and day-to-day consistency?
Because occasionally the baseline laser intensity would be increased which meant if we continued with the original bleaching threshold, this wouldbe lower than usual.
This wouldnt have affected results as we didnt change this throughout a particular recording apradigm. This was more common if the laser had not been used in a few days.
For bleaching, why was a sampling frequency of 1000Hz used?
Because we do not need rapid signal detection. Sampling every 1 s provides a good enough image of how the af state is.
What is meant by syaing analogue signals were digitised for analysis?
This means we used a data acquisition board such as the NI DAQ to convert the analogue signals that exist in 0-1 from the system over to digital so that data can be processed appropriately.
On the other hand, signals were converted to analogue to drive appropriate systems.
Why was an AF recovery experiment completed?
As we were tracking a time-resolved profile, we needed to see what profile AF was recoverign to see if this overlapped withour recoridng. As if so, this would make it difficult to confirm if our signal was truely m04.
This helped establish the time profile of AF across depth for further analysis.
How long was the bleach and AF for this experiment?
How was it completed?
Bleach was 30-mins through sweeping galvo at max voltage.
AF recv was every 30-s for 12-h.
Why for AF recv did you have only one illumination repetition?
Because we were just wanting a snapshot of the fluorescence at this point and wanted to limit the bleaching thorugh this protocol.
Why was a sampling freq of 5000 Hz used?
Because we were modulating the laser with on and of periods in a ms range so in order to capture alll vital data, we sampled at this frequency range.
Why did you need to implement light protocols?
Because the light profile from the tf was not equal alongt the shaft which would make it hard to infer what the fluorescence data means as the brain tissue would not be excited at the same powers.
Why did you need to know the coupling efficiency of the TF?
Because this is a measure of the light transfer of the tf and allows us to better estimate and modulate the light powerr to suit the particular fibre for a better estimate of the light power across mice.
Why do the alignment limits with TFs exist which cause the dips in light power?
As the light will internally reflect more at higher GVs, more of the light energy will be lost in the cladding.
Why did you choose light protocols for
- light power
- power density
Because light power is the most conventional way as this means the same tissue is being illuminated at equal levels so we can get a more reliable comparison.
Power density was done as the light cone of the tf is different across tissue so this equalises across tissue which we thought would be desirable to try.
How will the light protocols compare to when the TF is implanted in brain tissue?
We at first did not consider the changes this would infer. However, using 2p slice images we see that the light from tf scatters differently in the brain tissue which may mean the estimated light protocols may have some error.
Why was 60/80 uW used?
Because this was in the low range which provided a good m04 signal.
Why did you use fluorescein for the image protocol?
Because we needed a larger volume of liquid and this was cheaper. Also, this protocol had already been established for fluorescein.
How did you determine an optimal voltage range for the image light protocol?
We would usually use one of the lower limit of voltages used for in vivo experiments, but we would re run this protcol adjusting the voltage appropriately if it appeared to high or low.
We wanted to see fluorescence along the fibre but not a saturated signal as this would be out with the pizel intensity range of the camera.
Why did you sweep from -4.5-4.5 for image protocol when you never use these values?
Just to get a full emission profile but we do not use that data.
Why do you complete the image protocol at a reoslution of 0.1V galvo?
This matches what we sample at in vivo. It also over samples for the estimation of the req power ratio.
Why were the images for image light protocl taken every 0.5 s?
Because there was a lag between the system and camera so while the galvo changed at 1000 Hz, sampling the camera at 500 Hz ensured we acquried the appropriate image.
What type of camera was used for image protocol
This was a small USB powered optical camera that had a magnification lens.
How was the active reguon of the TF labelled for image light protocol? How was it visually inspected?
A line was manuall drawn along the TF shaft where light was propagated. The goal was to mark along the whole active region of the TF so we can measure the light intensity.
We created a video of all frames with this line drawn on and we accessed if this followed along all regions where light propagated.
What do you mean pixel intenstieis were calculated for image protocol?
Along the line drawn along the TF shaft, the fluorescence intensity was measured by determining the pixel strength at regions along this line.
What do you mean by saying that the light distribution is different along the TF?
At higher galvos, the light dissipates in a downwards manner which means the light can pass over more GVs.
How does the in vitro light protocol calculation work?
The fluroescence has been recorded at each depth.
Due to rules of fluorescence, this is a direct measure of light output.
Now we want to create a ratio scale which will represent the depths with the greatest and lowest power across depth.
This is done by calculating the reciprocal value of fluorescence and normalising this against the minimum.
How does the image light protocol calculation work?
This measures the power density at each depth through complex calculations that determine the area of beam based on tha taper angle - sypplied by our collaborators.
We then need to normalise the power density values to be standardised and comparible which is done by subtracting the minimum PD at each depth.
Then, the recirprocal power density is calculated to illustrate the ratio required to equalise it across depth.
For fibre calibration why did you use those powers?
I used powers that were within the range used for in vivo experiments as this provided the best estimated power.
Why do you think 3V galvo provided the most accurate power estimations?
This was the centre galvo so most likely took a good balance between the higher signals at lower galvos and lower signals at higher galvos.
Why were sync channels of the laser and galvo needed?
Because this provided us information for post-processing for when the laser was on and at what galvo voltage each measure was at.
Additionally, it can be used to ensure the correct voltages were applied.
Did you monitor the laser voltages you applied?
Yes, we also acquired a mat file that has the laser voltages at each galvo for each light protocol.
More recently they have been used to help towards acquiring correlation.
How did you choose your desired powers?
As we couldn’t choose irradiance due to the inability to determine the area of light of each taper, we were unable to directly compare with FF experiments.
Therefore, for first recordings I started with a wide range of powers and found that below 60 UW, the profile was very low and the signal was noisy.
How does the formulatas for calcularing the desired powers work?
In step 1 for all light protocols we are calculating the required power needed for each galvo. For original this involves using the coupling efficiency so account for differences with each TF. Here, we devide the coupling/100 by the desired power. For models, this involves using the power ratio divited by the desired power to
Next step involves transferring this required power over to voltage usits using the prevosusly established intercept and slope. For models the coupling effciciency is also used by dividing this by req power.
How is slope and y-intercept used?
These are used to transfer the req power over to req voltage so we know at what rate to drive the laser.
What are the two steps in the power calculations for?
step 1 is for establishing the requried power
step 2 is for establishng the required laser
What was the additional project for collaborators?
They took brain samples that were sectioned into 300 um thickness and they put them into a two photon set up where they implanted a fibre and imaged the profile. This was to see the scattering profile and what the fluorescence looked like in live tissue.
What was the implanting issue in FAD44?
The fibre broke upon implantation.
Why would you not know the bleaching protocol for APK?
i have not noted what specific protocol was done, rather just that bleaching was completed.
Why were the powers sand sampling intervals changed for APK?
Because i was struggling to get a reliable signal so various parameters were changed in order to boost this. For wxampke, powers were decreased to reduse bleaching or increased to get a better profile. Sampling intervals were increased to reduce bleaching.
Why is there such a big age range in mice used for head-cap surgery?
Because we wanted mice in the young age range for genus as this is where it seems to be more efficous.
Whereas we were using mice of various ages fro PK in order to acquire a strong signal
How does isoflurane work?
Isoflurane is a general inhalation anesthetic used for induction and maintenance of general anesthesia. It induces muscle relaxation and reduces pains sensitivity by altering tissue excitability. It does so by decreasing the extent of gap junction mediated cell-cell coupling and altering the activity of the channels that underlie the action potential.
MOA of naropin
Why is it used?
Block the generation and conduction of nerve impulses. Specifically, they block the sodium channel and decrease chances of depolarization and consequent action potentials.
Local anaesthetic used to knumb the area of surgery to ease the pain and promote recovery of the aniaml.
MOA of vetrogesic
Why is it useD?
In summary, buprenorphine is a potent, long-acting analgesic acting at opiatereceptors in the central nervous system.
It is used to reduce pain in mice.
MOA of rimadyl
Why is it used?
The mechanism of action of carprofen, like that of other NSAIDs, is believed to be associated with the inhibition of cyclooxygenase activity. Two unique cyclooxygenases have been described in mammals. The constitutive cyclooxygenase, COX-1, synthesizes prostaglandins necessary for normal gastrointestinal and renal function. The inducible cyclooxygenase, COX-2, generates prostaglandins involved in inflammation.
Another pain killer.
Why did you inject saline before chronic surgery?
To hydrate the mouse with fluids since it is losing some throughout the surgery. Helps with recovery.
Why did you choose the ear bar approach?
Because this is a really good way to use the stereotaxic to stabilise the mouses skull.
What is the purpose of betadine?
It is an anti-septic solution which will clear the skin of infectious particles to reduce the risk of infection.
Why did the peristeal membrane need to be removed?
As this is a sticky membrane acting as a protectve barrier to the skull. However, if this is not removed it hinders access for burrholes. Also, the dental cement will not stick to it as well and increases the risk of the headcap falling off.
What were the differences in your implant sites and why?
We moved more lateral as my implants appeared to be sitated more medial than desired so I was trying to offset my error.
I then moved it more lateral and posterior to increase chances of hitting my target.
What were the skull screws needed for?
To stabilise the headcap.
Why was a straw attached to the back of the headcap?
For headfixation. The mouse would be attached to a metal rod by this.
What is kwikcast?
silicon gel that allows the brain tissue to be protected from dental cement.
What is meant by 4-day summary analysis?
The full 4 day recording data.
How will having different implant sites differ the results?
Potentially one of our strong signals are offsetting by a weaker signal which reduces the intensity of our collected signals.
Why are some bleaching in PK exps at 3.5V?
Perhaps when laser was dying or when I was trying to establish appropriate bleaching protocol.
Why did you increase the number of reps?
Because the laser stabi;lity varied so to get a better median signal.
Why did you increase the sampling interval?
Becaue the dynamics of the plaques was very slow so we did not need rapid sampling. And to reduce bleaching.
What is meant by:
- investigative time samples
- inaccurate power estimation
- laser dying
- needed further habituation
1) We just started the recordings so were unsure how long we would want to record to see the fluorescent profile. So we did various types like 8 hours seperated bu a cap and so on.
2) due to the inaccurate y-intercpet/slope, the powere applied was not estimated correctly.
3) the first laser had an electrical fault and began to deteriorate in power throughout recordings.
4) mouse was not comfortable being head fixed or being in the open chamber so needed to be habituated to this process more.
Why was a red bulbed light used?
Because its wavelength is out of the mouses spectra so they cant see it and because it means I can still see them and the system without the light itnerfering with the recorderd signal.
How was the head-fixation process?
Using the straw to attach the mouses head to a metal rod.
However, later just scruffed the mouse.
How did mice behave in this open recording chamber?
The bejaved reasonably normaly and slept, groomed, ate.
Why did you want to investigate the pharmacokinetic profile?
Because we wanted to see the time profile of the increase in fluorescence and how long it took to return to baseline when recording. Additionally we wanted to see if the system was able to detect changes in the concentration of m04 as a sign if it would be able to detect changes due to plaque modifying effects.
For chronic recordings why did you have a day 0 baseline?
Because this was a fluorescence signal of the brain tissue without m04 so could be used as a baseline signal for a control and normalisation.
For pharmacokinetic, why the 30-min baseline?
This provides us with a baseline signal on the day for normalisation.
How did you decide the duration of recordings?
After several recordings of monitoring for 10+ hours, we seen that the maximum signal appeared around 2/3 hours and therefore minimised the recording protocol for ease and to increase reps possible.
What is meant by an incomplete recording?
That recordings were not completed across every day - sometimes happen due to unpredictable events like i got an appointment, i was sick, there waa a fire drill etc.
Why did you investigate the re-dose protocol?
Because our goal was to do longitudinal recordings so we needed to see if we could maintain a strong fluorescent signal.
Did all recordings follow the same recording schedule?
The majority did bay the first few animals as this was recordings where I was trying to see what the best lengths were.
What do you think the fluorecence would look like a week after this protocol?
After PK - I think the signal would be returned to baseline with potentially some bleaching of the AF of plaques.
After RD - I think the fluorescence would be maintained, but after longer peiods like a month this may be limited as binding sites would be limited.
Do you think less redosing could be completed?
If the length of recording was reduced yes as the binding seems to be stable over lengths of time as long as the signal is not bleached.
What do you think happens after these 4.5-h after injection?
It seems like the max signal has been reached as the fluorescence doesnt increase on the next day so it seems to stay stabley bound.
When this signal decreases, do you think its bleaching or clearance? How would it clear?
There is not much information on the clearance of m04, in fact other studies suggest that once it is bound it remains bound for several days +90 days.
So this to me suggests that this is bleaching of the signsal.
By redosing like this, how would this affect teh binding site? Could the binding become saturated?
Yes, this will eventually bind and prevent alongation of the fibril which will prevent plaque growth.
How long were mouse recovering?
We allowed 5 days for recovery but usually I wouldnt record for over a week after surgery.
How were mice habituated to this?
Allowed to explore the chamber for several days.
Habituated to handling and scruffing and fake injections. and touching of the head cap
Habituated to being tethered.
Why were the photodetectors recorded from continuously?
So we can see the change when the LED is on and off.
Why did you only go up to 4.5V galvo?
As we wanted to include some negative values to account for some variations in initialisation but wanted to maintain the same pulse duration to prevent bleaching.
Also, as this is only a small distance this was suitable and still was able to mornitro forom our desired brain region.
Why was the perfusion now completed with naropin?
Because our onsite vet would rather we use this as it is stronger and more effective than lidocaine.
