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DIGITALLY IMAGING PARASITIC HYMENOPTERA (or other small, less interesting, insects)

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Capturing digital images (in particular for use in AutoMontage): Using the ProgRes3008

This is primarily a tutorial for the setup at the Chalcidoidea lab at TAMU, it may not be relavent to other setups.

Start the ProgRes plugin

Open Photoshop.
File->Import->ProgRes_v5.0

If you haven't done so lately (once a week) calibrate the camera.

Adjust the light and input/output settings
Turn up the light until the image is visible. If the overexposure and underexposure boxes are checked then areas on the image over/under-exposed will be highlighted (default colors blue=under; green=over). Two very important things: The Progress preview will (almost) always be darker than your final product! The main concern is to not over or underexpose. Levels will be post processed in Photoshop. DO NOT increase the exposure or gain to increase light unless absolutely necessary. I have never found it necessary, even at highest powers to increase the exposure past 2, I never increase the gain. Increasing of both decreases image quality. Tweak the color settings (see below) so that the background is a shade of grey, it doesn't matter what shade, just make it grey. Make the background grey. This should help to ensure that the color balance on/of your wasp is correct. It also makes it much easier to remove the background in post-processing. There are two ways to do this: Click Prescan. You can automatiaclly set the white level with "Auto white". This sometimes works, but should always be used in conjunction with manual adjustment of levels (see below). You can manually set the white level by clicking on Set White. This gives you the eyedropper. Position the dropper over the background and click once. If either of the methods produces ar grey background you've succeeded. If your close but not right on finally tweaks should be made in the "Balance" dialog box (see below). Set black can be done in the same manner, except the darkest point (which should be on the insect) should be selected. Generally this is not as important at setting the background to a grey. DO NOT repeatedly use the set/auto buttons without "Resetting" in-between each undesireable result.

Click "Balance". It might not be necessary to use these if the set-white was completely successful. There are four sections in the balance- in red: 1,2,3,4. "Reset" on the main ProgRes dialong resets 1 and 2 to "5"; 3 and 4 to "95". FIG Setting the background to grey is done primarily with 3. Brief experimentation with these settings, while watching the image in the background, should be enough to find the grey background. 2 should be slightly higher than 1 (around 9). Try taking a sample shot to see if the background is indeed grey when the image is actually captured. If not grey, correct the balance.
The camera should now be configured.

Capturing the images

If only indiviudal images are needed simply focus and capture.
If a Montage is being done first focus to the "highest" point on the insect. Check that the point is in fact in focus by taking a sample shot. Once this focal point is set:

Capture an image
Importat another image (return to Progres)
Increment focus down (counterclockwise turn). Try to make the increments exactly equal. This can be achieved very acurately manually if you attach a ruler printed on paper to your fine focus, with a fixed line beside it on the scope. If the scope has fine focus increments these can obvoiusly be used as well. However, for habitus shots careful manual increments generally work well enough.
repeat...

Do NOT completely focus through to make all the picture in focus, this is unecessary and distracts from the specimen. By watching resulting images the final point in the series can be determined. For habitus shots that will be Automontaged 15-20 works well, though the more the better.
Save the images

DO NOT use a format that compresses the image (.jpg/.gif and others) for your "master" copy.
Use .bmp for images to be processed in AutoMontage.
Remember to add terminal numbers to the filename if AutoMontage will be used (e.g. filename_01.bmp; filename_02.bmp...filename_nn.bmp)
See below for automating the save process. If you don't have Photoshop 6 to speed up the process you can do this (eliminates the use of the mouse) once all images are captured (they will be closed with ctrl-w in order that they were taken):

ctrl-w; enter;
enter the name of your file (filename_01)
copy the string with ctrl-c
set the Format to ".bmp"
enter; enter
ctrl-w
ctrl-v [paste the name]
manually increment the number
enter; enter;
etc...

Tricks for speeding up the image capture process

Make a macro (action) in photoshop that starts the ProgRes plugin automatically. Attach it too a key. [Chalcidoid lab - key is F2].

This minimizes the number of steps to capture an image to 3:

1) Keypress ([F2] - opens ProgRes plugin);
2) Increment focus down;
3) Click to capture image.

Repeat

Automate the saving of your series of images (Photoshop 6.0). This greatly increases the speed in which you can save your images.

Make a macro (action) that does NOTHING. (e.g. select all) [Chalcidoid lab - action is in automotage functions ->"nothing"] .
File->Automate->Batch [better yet...make another action to jump to this screen automatically]
[Fig]
Choose the "nothing" Action that you built.
Set the Source to "Opened Files"
Set the Destination to "Folder"
Click "Choose" to find the folder you want to save the images too.
Set you naming convention.

Give the images a "base" name (in general its a bad idea to use spaces in your names, use an underscore instead).
Choose a 2 or or 3 Digit Serial Number, Automontage needs this to process the input images.
Include the extension

Click Ok
MORE HERE (choose .bmp)

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