Antigens around Swanson level 15

Below are images surveying the anterior hypothalamus that will help Georgina find her way through the preoptic area. Images stitched at x20.

GAD67 green GAD red

nNOS green AVP red

nNOS green PNMT red

Picks Up!

Acquiring Images with Volocity

Acquiring images is time consuming—acquisition times can be long and there are a lot of images to be acquired—and fluorescence signals are perishable. Therefore the goals is to acquire the best images possible and get everything needed the first time around, reducing the time spent imaging while preserving the signal for anything that does need to be imaged a second time. 

Before Turning on the Microscope

• Sign up to reserve the system on the calendar in the hallway
• Decide beforehand what will be imaged: which series, sections, which labels and how many channels
• For fluorescence, take an empty slide tray and collect the sections to be imaged from the freezer
• Allow time for the slide to warm to room temperature
• After the imaging session, return all slides to their home tray

Start Up the Hardware and Software

• Turn on the hardware and computer.
• Turn on the hardware from left to right, in the order numbered
• Sign in on the sign in sheet
• Boot the computer under Windows (hold down the option key during startup and select the Windows startup HD)
• Make sure all hardware, including the camera, are on before launching Volocity
• Launch Volocity and log in
• Select either 'Volocity' or 'Acquisition Only' from the pull down menu
• Open the Image Acquisition Log and the Landmark DB
• From the Image Acquisition Log spreadsheet, select 'Go to Live Form' under the 'Form' menu
• In the Landmark DB note the date imaged on the sheet for that series, in the cell for that section 

Before Putting a Slide on the Stage

• Check that the camera is set to monochrome (Fluorescence) or RGB (Nissls)
• Configure the Acquisition Setup
• Select Acquisition Setup from the Video menu, or double click the small green 'Acquisition Protocol Feedback Display .

• Under the Channels tab:
• Name the file (see the Working with LightRoom entry for naming conventions). Do this for every image.
• Add all channels to be acquired. Select 'Change Channels with Light Paths', choose channel from the pulldown menu. Click the '+' to add another channel
• Manage shutter speeds. Use 'Balanced Sample Protection' 
• Unless acquiring a z-stack, no other options be selected
• Under the Stitch tab,
• Capture XY using Ludl Stage Controller 
• Set the overlap to 15% 
• Select Create Stitched Image and Auto Align Fields (only
• Select 'Save Raw Tile'
• Click 'Ok' to return to the Video Preview window

• Configure a Light Path for each channel.
• For now, verify that the filters match the channel, that "Fuor' is showing in the Channel Controls pull down, and Monochrome in the camera pulldown menu
• For the channel to be use to scan the overview, set the objective to 5X and the gain to around 5

• 

• Calibrate the Stage
• Switch to XY View
• Select 'Calibrate Stage' from the 'Stage' menu. Note that there is no need to move the stage to the load position if there is no slide

Create an Overview

• Looking through the ocular, find the slide and section to be imaged and preview all (visible) channels
• For each channel make a mental note of the brightest parts, which should be used for setting the correct exposure
• Close the shutter and switch the light path to the camera tube by pulling out the silver post on the side of the microscope
• From the Live Video view, at 5X w/gain of about 5X, AutoExpose and focus
• Return to XY View and drag the marquee tool over the approximate area to be imaged
• From the Stage menu, select 'Scan Selected Area'
• When the overview is generated, double click on the part to be used to set exposure for that channel

Capture the Image

• Return to Live Video. Switch to the desired objective open the shutter and click the AutoExpose button. Leave the the gain high for focusing. Find the best focus. 
• AutoExpose again after reducing the gain and determining the offset. See below for typical settings, and Exposure, Gain and Offset in Volocity for more detailed explanation, but in general, keep exposure times as short as possible)
• Save these settings by clicking the disk icon
• Switch to the next channel verify the focus and objective, set gain, offset and exposure. Save the settings. 
• Repeat for all channels, switching back to XY View as needed to move the stage to the best area for calculating exposure for each channel.
• When each 'Light Path' has been set up, return to the XY View and drag the Marquee tool over the area to be imaged
• Hit the red Record button to acquire the image
• Create snapshots (under the Image menu) of the composite image and each channel individually. 
• Export each snapshot as an OME Tiff.

Tips & Troubleshooting

General:
 Keep the shutter closed as much as possible. Note that Volocity will at times open the shutter  automatically when switching between 'Light Paths'

Stitching

10% is recommended, and 12% usually works well, but 15% should allow the software to stitch lower contrast sections and is more forgiving when the camera is not perfectly aligned

Light Paths

The objective, exposure, gain, offset, fluorescence filters and condenser can be changed for each channel—which means that each of these values must be set for each channel. All changes must be saved before switching to different channel or the changes will be lost.

Quick Scan

Generate a low magnification view to precisely control image content, and to navigate to a specific part of a section or slide.

The 'best' channel is the most abundant (helps recognize location) and most stable (slowest to bleach). If background is very low in all channels, it can be artificially raised by increasing the offset (but remember to change it back)

It is helpful to set the gain high to keep exposure times low. This reduces potential bleaching of the signal, but mainly it reduces the  video lag making it easier to focus

Exposure, Gain and Offset
The rule of thumb is keep the exposure as low as possible but long exposure is better than high gain. For acquisition, an Exposure between 50 and 300 ms is fine, and 300 is preferable to repeatedly tweaking values and re-calculating exposure. For any exposure less than 100 msec, Gain should be 1.0X. The problems with long exposures are 1) the signal may bleach and 2) it adds to the acquisition time. Neither become an issue until well past 500 msec. Gain: The rule: Feel free to use up to 2.5X gain to control exposure time. Use 2.5 to 5X  gain only when exposure are greater than 300 msec at 2.5X, and use  higher than 5X only in extreme cases. Increasing gain always adds noise, it is just that below a certain level it is undetectable. We always want to be well below that level. Offset determines black levels. Blacks should be kept as dark as possible but no darker.

Setting Exposure:
After the Quick scan, set the Gain to 1.0x,  the Offset to around 0 and AutoExpose. If the Exposure is greater than 100 ms, feel free to increase the Gain

Working with LightRoom

LightRoom (LR) is the program that keeps track of the image files for us—how they are stored, which files belong together (e.g., individual channels and the merge, tiles in a mosaic, slices in a stack, etc.) and all edits of the original. LightRoom is also going to play a big part in the analysis because it has powerful tools for selecting, grouping and comparing images. 

To get the most out of LR, here's what we need to do:

Exporting Images: Import only whole images (stitched mosaics or projections). If Volocity cannot stitch the image, assemble it using Adobe Bridge and Photoshop (PS) before importing it. Export the composite image with all channels and each channel individually. Export only one copy of each. Use an R-G-B color scheme when you can, but as long as we have the individual channels we can assign or change colors in PS as needed.

File Naming: Files should be named according to their content, or more to the point, the filename should not refer to anything that is not in the file. To denote the relationship between the composite image (an image of all components) and its component channels (multiple images of individual component), use the following scheme:


Composite:
2013_05_31_001a_DAPI, Cy2-MCH, Cy3-NOS_G04f, lvl 24,25

Channel 1:
2013_05_31_001b_DAPI_G04f, lvl 24,25

Channel 2:
2013_05_31_001c_Cy2-MCH_G04f, lvl 24,25

Channel 3:
2013_05_31_001d_Cy3-NOS_G04f, lvl 24,25


Keywords:
Keywords must come from a defined vocabulary to be useful. Most but not all keywords have already been created. Existing keywords can be found in the "Keyword List" in panel on the right. Note that that most have been organized into folders. Unfortunately the Keyword List can't be used for Keywording, but there is an autocomplete function when typing in keywords. If the keyword you are adding does not show up in the autocomplete list, check the Keyword List for the right keyword or correct spelling.

For each image, add these keywords:

• Peptide/Antibody
• Label (Cy 2, etc.)
• Counterstain (e.g., DAPI, Nissl)
• Tissue & Series (e.g., G1204a)
• Labeling method (IHC, etc.)
• Imaging method (epifluorescence, confocal)
• Image Type (Mosaic, Projection, etc.)
• Test or Data series
• Image content: Brain structures (just the obvious ones: LHA, AHN, VMH, PVH...)
• Atlas Level(s). Should know this before the image is taken (from the landmark database)

Notes:

Vocabulary: "Mosaic" and "Projection" are more accurate and we should use them instead of "Stitch" and "Stack". 

Tags:
In addition to keywords images can also be tagged with Stars, Flags, and Colors. Tags can be assigned from the menu or by right-clicking the image. 

Star Ratings:

1. Original Tile or Component
2. A complete raw image (Stitched, Projected and/or single frames). 
3. Manipulated w/out altering content (Stitched, color corrected, basic levels or curves, etc.) incl. Cropped and/rotated (part of image discarded but not altered)
4. Content Edited (for brightness/contrast, size/resolution, etc.)
5. Output version/figure component

Almost images should get a 2 star rating when first imported, or rather we should avoid adding more 1 star images. Most data series images will make a trip through PS and return with a 3 star rating. We will try to keep PS edits to a minimum but multi-channel composite images will usually get a 4 star rating.

Flags ("Rejected", "Flagged", "Unflagged)

Most images will be unflagged, which is the default. A few images will be flagged under special circumstance:

• Use the "Rejected" flag  to  hide unusable images without deleting them
• Use the "Flag" to mark images that need attention, like missing or non-standard keywords, the file format is not usable or if content is not clear, etc.

Color Labels:An image can be tagged with one of 5 different colors (R, G, B, Y, P) that we can explicitly defined. The obvious use for Color labels is to keep track of channel information (use Purple for Cy 5, Alexa 647, etc., and Yellow for Fluorogold, which isn't relevant to the LHA project).

Metadata:
Metadata opens up another realm of options. For now let's leave it at that: optional. Note though that it migh be convenient to copy the file name into the 'Title' field. Also that the 'Caption' field ccontains all of the microscope info exported with an OME Tiff, which may be useful at some point.

Anti-FITC Test Results

2013_05_26_001
NEI (rb; 1:10k) A488 rb anti FITC 1:750
16 hour incubation in A488

2013_05_26_003
NEI (rb; 1:10k) A488 rb anti FITC 1:750
39 hour incubation in A488

2013_05_26_004
NEI (rb; 1:10k) Cy2ms anti FITC 1:1k
16 hour incubation in Cy2

2013_05_26_005
NEI (rb; 1:10k) Cy2ms anti FITC 1:1k
39 hour incubation in Cy2

2013_05_26_006
NEI (rb; 1:10k) Cy2ms anti FITC 1:2k
16 hour incubation in Cy2

2013_05_26_007
NEI (rb; 1:10k) Cy2ms anti FITC 1:2k
39 hour incubation in Cy2