TITLE: Mesuring JF dye kinetics in the brain of C57/Bl mice
AUTHORS: Boaz Mohar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to compare the intake and clerance kinatics of JF dye systemic delivery using retro orbital injections.</div><div class = "text-block">We image a mouse with a crenial window before and after systemic dye injection.</div><div class = "text-block">We want to fit the clearance of the dye with a double exponanatial decay function:</div><div style = "text-align :; float : ;"><span style = "">y = \textbf a*e^{-\frac{1}{\textbf b*x}}+\textbf c*e^{-\frac{1}{\textbf d*x}} + \textbf e</span></div></div>

[STEPS]
?. [Baseline]
Use a mouse with a window (dosen't have to be in ALM)
?. [Baseline]
Move the mouse to an induction chamber with 3% ISO and flow of ~2L.min. Wait until the mouse takes ~ 1 breaths per second
?. [Baseline]
Move the mose to the microscope and clean the window with Q-tips and 70% ethnol.
?. [Baseline]
Select if you want to use a 4x or 20x objective.If using a 20x add water above the window.Lower the objecive using room light or white light illumenation and focus on the top of the cortex.
?. [Baseline]
Pick the approprite filter cube and illumnation source for the dye
?. [Baseline]
Setup the imaging conditions in a way that baseline image takes ~ 10-20% of the dynamic range but is above read noise level. We use 50ms exposure with 40 frames per timepoint totaling ~2s duraion per timepoint. Or 200ms with 10 frames if signal is lower.
?. [Baseline]
Recorde baseine image at a few illumenation settings to make sure that the first timepoints after injection are not saturated.For exmaple with a LED use 100mA, 500mA 1A and 2A driving current for the same aquisition settings.When imaging the first image after injection try using 500mA but change if it is too dark / saturated.
?. [Dye injection and clearance ]
Take the animal out of the imaging rig and back to the induction box if it is breathing faster then 1/s
?. [Baseline]
Record a white light referance image so you don't have to bleach your signal to return to the same field of view. If possible record the objective's location.
?. [Dye injection and clearance ]
Prepare a fresh dye aliqoute
?. [Dye injection and clearance ]
Inject into the retro-orbital sinus
?. [Dye injection and clearance ]
Return the animal to the rig and return to the same field of view using the white light image as referance
?. [Dye injection and clearance ]
Start imaging using the fluorescence light source at fast interval at first (~1-3min) to catch the rise and fast decay phases and depending on the dynamics start reducing the intervals (5-20min) after ~ 1h you should get a sense of the amount of change you see and start seperating time points by more then 30min.
?. [Dye injection and clearance ]
After ~4h you can usually take the animal out of the rig and preform recurdings at >2h intervals.
?. [Dye injection and clearance ]
After 24h usually no more dynamics is obsureved.
?. [Analysis]
Load images and mean each timepoint, save the mean image
?. [Analysis]
Load the mean images in Fiji and using the stack sorter move the first (baseline) image to be the last.
?. [Analysis]
Use the Linear Stack Alignment with SIFT plugin to align the stack of images.
?. [Analysis]
Save the aligned stack and load in Matlab
?. [Analysis]
Define a mask (elipse or otherwise) of the imaged region of interest (imellipse).Median the pixels there, substract the baseline timepoint
?. [Analysis]
Fit a double exponant model to the rest if the median pixels (excluding baseline) using the file creation date as x axis.y = \textbf a*e^{-\frac{1}{\textbf b*x}}+\textbf c*e^{-\frac{1}{\textbf d*x}}Add a lower bound  of 0 to all parameters. If cell are expressing a HaloTag, use:y = \textbf a*e^{-\frac{1}{\textbf b*x}}+\textbf c*e^{-\frac{1}{\textbf d*x}} + \textbf eFirst point used will be the peak (Most times the first as the rise is very fast)