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.