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

this is probably more of the same but

to brighten an image
image /mode / rgb colour
image / adjustments / levels - adjust input levels

to turn two pics into one (combine)
open both files
image /mode / rgb colour (both pics)
layer / duplicate layer (box opens)
document (filename 1 [v]) change to document (filename 2 [v] )
[ok]
select other picture
goto "layers,channels,paths" box - change "normal" into "screen"

hey presto

now you see why i took my time

works wonders tho - you can use these to enhance a nuclear stain then combine the pics.

but like i said before - if you want a brighter image - sort it on the microscope - sometimes photoshop can bite you on yer arse if you pretend you got a pic you didnt

have fun

dom

1 million times easier: USE IMAGE J. (free, downloadable and does 99% of everything you need) Only caveat, it gets a bit complicated using 12 Bit images (see below for fix).

Simple:
Import your picture.
Flie>open OR Import an image sequence (if you are using confocal stacks).
I do hope you are taking and keeping Tiff files. These are your backup.

Assign a color (your picture is gray scale, you can HONESTLY assign a green color scale):
Image>Lookup tables> green (or any other color you see).
Your image is now: 8 bit color (see Image>Type)

Convert to RGB or CMYK
Image>Type> move from 8-bit color to RGB (yep. that is all).

Now, do you want to mix two different channels? Did you acquire two pictures from different emissions? (EI:green and red?), that is also simple, but do keep in mind that your images MUST be of the same size.
Open both images using IMAGEJ. Go to Image>RGB merge and assign the correct image to the correct color.

Save your images as Tiff files, since these might be asked for by the reviewers (Yes, we do request them if there is a doubt of how much an image was manipulated)


REMEMBER THAT PHOTOSHOP IS A SOFTWARE DESIGNED FOR IMAGE MANIPULATION, IT DOES NOT CARE ABOUT KEEPING THE VALUE OF YOUR PICTURE LINEAR AND WILL CHANGE THE VALUES IN MANNERS THAT ARE INAPPROPRIATE FOR SCIENTIFIC JOURNALS CHECK OUT
What's in a picture? The temptation of image manipulation. Rossner M, Yamada KM.
J Cell Biol. 2004 Jul 5;166(1):11-5.
Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. North AJ. J Cell Biol. 2006 Jan 2;172(1):9-18. Review.

Brightness and contrast can also be correctly adjusted in ImageJ

Really, restrict your use of photoshop to the final composition of the figure (pictures and tables, graphs, arrows, etc). That is what it is for (and do not use power point except for presentations). Your image took time and effort, respect it. All though, you can change Brightness and Contrast in photoshop, be careful not to use other alteratios (gain) or filters. IF YOU MUST USE THEM, then you must specify such usage in the legend or in the methods section.


Just to stress the point

REMEMBER THAT PHOTOSHOP IS A SOFTWARE DESIGNED FOR IMAGE MANIPULATION, IT DOES NOT CARE ABOUT KEEPING THE VALUE OF YOUR PICTURE LINEAR AND WILL CHANGE THE VALUES IN MANNERS THAT ARE INAPPROPRIATE FOR SCIENTIFIC JOURNALS CHECK OUT What's in a picture? The temptation of image manipulation. Rossner M, Yamada KM.
J Cell Biol. 2004 Jul 5;166(1):11-5. Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. North AJ. J Cell Biol. 2006 Jan 2;172(1):9-18. Review.