LHA Imaging Protocol

After examining the test images and crunching the numbers, here's the image acquisition protocol for the LHA project:

Image Acquisition:

Acquire images at 10X, 2048x2048, 8 bit, spd 7, Avg 2, 5x4 tiles (15% overlap), with an optical slice thickness of 8.5 µm, which gives 4z in 25 µm sections or 6z in 40 µm sections.

z-Stack:
The slice thickness is based on a pinhole of 40.9 µm, which is approx. 1 A in the Far Red (FR), 1.08 A in the Red, 1.25 A in the Green and 1.66 A in the Blue channels.

Acquisition parameters are optimized across all 4 channels, even though we will rarely use the 4th channel. The alternative is optimizing each combination of tracers for the longest wavelength, which would mean that the x, y and z resolution (lateral resolution and z-step) of a channel would change depending on the combination of channels.

All of these parameters can be found in the 2nd and 3rd sheets of the Image Acquisition Google Document.

Imaging Strategy

The file size, acquisition time, and the number of immuno series to be imaged makes it impractical to image all available LHA sections (except perhaps in the 40 µm 1:6 series). Instead sections to be imaged will be based on Atlas levels prioritized by region of interest. For us (as opposed to UTEP), we will initially focus on the sections corresponding to Atlas level 26, then levels 24 and 25, followed by levels 29 and 30 and finishing with levels 23 and 32. These levels represent the iconic view of the LHA (26), the ACB-recipient LHA (24 and 25), levels central to Arshad's region of interest (29 and 30) and the rostral and caudal  LHA  ends (23 and 32; not quite the pole, but easier to find/identify). This strategy covers 8 of 11 levels of the LHA. Exactly how this translates into images acquired is difficult to estimate because it is likely that more than one image will to deal with plane-of-section issues.

Immuno Strategy

Assuming that the same antibody raised in a different species gives identical results, our goal is to prepare series for 11 different antibodies (plus AVP), replicating one or more labels in each brain to facilitate analysis of the patterns of expression across brain. Ensuring that each brain is in register with the others will require 5 brains of 4 series each, for a total of 20 immuno series, which works out to be 2 series each for 9 of the 11 series, and 1 series for the last 2 (which will likely be AChE and vGluT). Because we want a single-labeled series for each peptide, this also means that 11 of 20 will be single labeling (plus DAPI), and 9 of 20 can be double or triple labeled (plus DAPI).

Files Sizes & Acq Time

 Single Images:
2 Channels: 
10x, 2048, spd 7, Avg 2, 5x4x4x2 (25 µm) = 800 MB and 52 min/image
10x, 2048, spd 7, Avg 2, 5x4x6x2 (40 µm) = 1120 MB and 73 min/image

3 Channels:
10x, 2048, spd 7, Avg 2, 5x4x4x3 (25 µm) = 960 MB and 62 min/image
10x, 2048, spd 7, Avg 2, 5x4x6x3 (40 µm) = 1440 MB and 93 min/image

Level 26:
Let's assume we'll acquire 2 images (not counting the Nissl) to capture level 26 X 20 series to cover all the immuno = a library of 40 images.

If each image is 800 to 1400 MB, and takes somewhere between 52 to 93 minutes to acquire, imaging level 26 take anywhere from 35 to 62 hours and generate between 31 to 56 GB of images. 

All Images/Levels:
Let's assume it will take 12 images to cover the 8 levels of interest. That's 240 images to to cover all series. Using the same numbers for size and duration, it will take roughly 200 to 372 hours (8.5 to 15.5 days) and generate between 187 and 338 GB of images—not including the Nissls. Overall, these numbers aren't as bad as I expected, but clearly the sooner we start, the better off we'll be

 Next Steps

Three things are needed before we start generating data: 

• Work out the DAPI (or NeuroTrace) protocol and get accustomed to using it
• Generate the Nissls and figure out the correspondence between section number and atlas level
• Finalize our list of brains to use, 1º combinations (both which peptides go with each brain and which will be singles & which labels will be combined)

And only two things are needed until acquiring the data is all that remains:

• Optimize the remaining antibodies (5-HT, vGluT2, DYN, and any species variants such as sh ∝ GAD)
• Run the immuno...