Fluorescence Microscopy Pictures - How to process in Photoshop? (Dec/05/2008 )

hallo,

I aquired my pictures as black and white ones. what can i do step by step in photoshop to get printable pictures for publication.

contrast/brightness
color curve adjustments (green, red, blue)...

thanks for help

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if it is 8bit (black/white), turn the mode to RGB or CMYK; without, you will not get colored images...

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yes, i know. but after that? how shoud i change contrast, brightness, and colour curves (input/output)...
before i can make an overlay or screen.

thanks

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Try and see !

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you may find it difficult to convert a grayscale to a color image. you will have to perform the colorization by hand (assigning colors to specific parts).

if you have a sophisticated colorization program then you can assign colors to specific shades of gray and let the computer do the rest (this is how they colorize b&w movies).

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you shouldnt

changes of that sort should be done on the microscope - not later in photoshop

as for the overlay - its been a while - i'll have to look it up

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I capture the images in gray scale and immediatly assign colors.
For eg. DAPI stained nuclei when we capture in gray scale only nuclei will be bright. Then we can assign blue color and later merge the pictures.
Not like one shade one color or photoshop.

Hope this helps.

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This is the same way I do my IF photos. As far as I know, there is no way to specifically assign a color in photoshop. You can change contrast/brightness and color scale in photoshop but there is no way (as far as I know) to assign the entire photo one color. I capture the photo in black and white and the software on the computer attached to the camera can assign color. We use SimplePCI software which allows me to assign colors, merge and then adjust brightness/contrast to the image as a whole. I then save each layer (individual color) that is shown in the merge and use these for figures. This way each image is adjusted evenly and the data is not misrepresented. It's important to try to capture the absolute best images possible so you don't have to manipulate the image much, if at all. Editors and scientists in general are becoming more and more skeptical of images that appear to have been altered at all. In fact, some journals are requiring you to submit the original, unaltered images along with the finished figure to judge how much the images have been manipulated. I often shoot photos in three exposures so I can merge the best exposures of each color and not have to adjust the end product.

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You can change colour in Photoshop, but I can only do it for picture with one colour. That is, I can't change merged pictures using this method. You can do as followed:

If you have a coloured pic already, say green, and you want to change to red colour instead:
- Open the pic in Photoshop. Choose Image (on the top bar, next to the Edit button). In the drop menu, choose Mode -> Grayscale. The colour will be discarded. Green now becomes white. If your pic is already in grayscale, ignore this step.
- Image again. Then Mode ->RGB colour. Now it is ready to be assigned colour.
- On the left side of the screen, there is a menu for all kind of altering, like choosing pixels, moving, add text... In that, near the bottom, you see one tap to assign colour for the background and foreground. That tab is the biggest tap in the list, with two big squares overlapping each other. Click onto one square, say the top one (the foreground), and assign the colour you want (red in this case) by choosing red in the "Colour Picker" window. You click the general colour you want (red) in the slim bar at the center of the window, then on the left of that bar, move the little circle in the window there to the shade of red that you want. The chosen colour will appear on the top half of the small window on the right of the bar. Click OK
- Then click Edit ->Fill. In the Fill window that opens, in Contents, choose Use: Foreground colour. On the Blending mode: choose Darken. Then OK. Your picture is now red.

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For overlay of two pictures in Photoshop, to create Merged Pic:
- Open picture 1. Ctrl+A to choose all of that pic. Then Ctrl+C, or Edit -> Copy
-Open picture 2. Ctrl+V or Edit -> Paste.
- Then, on the Layers window (which is usually locate on the right side of the screen. It can also be open by click on Window -> Show Layers), under the Layers tab, there is a box which normally show the word 'Normal'. This box is next to the Opacity box, and it is right under the Layers tab. Click on the box, and choose 'Lighten' in the drop-down menu.
- Then choose from the top bar (the bar with File, Edit... buttons): Layer -> Merge Down. That is the merged picture.

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