Inside WI > Biocomputing > Graphics > Photoshop > Techniques


Photoshop Techniques 

Scanning and Resolution

Starting with a good scan is important. The more adjustments you have to make in Photoshop, the more pixelated the image becomes. If you have questions about our user room scanners, please see the scanning pages. If you are unclear about resolution and dpi, see resolution on the scanning page.

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Rotating the entire canvas:

Large Adjustment: Go to Image > Rotate Canvas. You can choose to rotate 90 degrees, 180 degrees, clockwise (CW) or counter clockwise (CCW), flip horizontal, or flip vertical.

Subtle Adjustment: If you finish scanning your image and you find that it is tilted somewhat or if you scan a gel and the lanes are a little slanted, you can straighten them in Photoshop using the Image > rotate canvas command. This technique rotates the entire page:

Using the Measure Tool ,

(you will find it underneath the eyedropper) draw a line along an edge that should be horizontal/vertical line but isn't.

-The Info palette will open. You will see the length and the angle of your line.
- Go to the Image > Rotate canvas > Arbitrary window. Based on the angle value you got in the previous step, Photoshop automatically calculates how much you will need to rotate the image to bring your line to an angle of 0 for a horizontal line, or 90 for a vertical line. This value will appear in the angle window. Clockwise or Counterclockwise will also be selected for you. Click OK.


Rotating a selection:
Make a selection and go to Edit > Free Transform. You can then click and drag on the transform handles to skew, rotate, or scale. You can also choose Edit > Transform > Rotate to be able to rotate the image only.


Scaling .............Back to top

(See resolution for more detail on this topic)

Go to Image > Image Size. A dialog box will open where you can choose to alter the dimensions and resolution of your file. The best option for scaling is to increase the dimensions of the image by reducing the resolution. To do this, make sure the "resample image" box is NOT checked, and just change the dimensions as you like.

However, if this procedure gives you a resolution under 200 ppi, your image will look blurry when printed. If this is the case, reset the dialog box by clicking on the option button (the cancel button will change to reset). The values should go back to the way they were when you started. This time check the "resample image" box and put in the dimensions you desire.

If you don't have any extra resolution to take advantage of, you can scale an image by choosing "Edit > Free Transform or Edit > Transform > Scale. By holding shift while placing the cursor over the corner of the image, you can scale proportionally. Photoshop resamples the image when you use this method, which can cause some distortion.

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To make a selection, choose the marquee tool (You can choose the rectangular shape or the oval shaped marquee) or the lasso tool . Click and drag to make a selection.

To move a selection, you must choose the move tool (or hold down the command key to change the selection tool into the move tool), .

To copy a selection, choose the move tool and hold down the option key, then click and drag. (You can also hold down command and option if you have the selection tool.) If you hold the shift key at the same time, the selection will move or copy in a straight line. Always click and drag with the mouse last in this sequence.

To soften the edges of a selection, choose Select > Feather. You can type in the number of pixels you want to feather. This will blur the edges of a selection to make it blend in better.

To change the shape, size, or rotation of a selection outline (not what is inside it) choose Select > Transform. When you have the selection you want, click the selection tool again to apply the change.

To save a selection outline, choose Select > Save Selection. This places the selection in a non-color channel or "alpha channel". You can view it on the channels palette. When you need that selection again, choose Select > Load Selection. You can scroll down the list of selections to find the one you want and click OK.

To get the last selection back again choose Select > Reselect

To reverse your selection, choose Select > Inverse. Instead of selecting what is inside of your selection outline, you will have selected everything outside it.


Cropping .............Back to top

There are a few choices:

A. Use the selection tool to make a selection ,change to the move tool and choose Image > Crop.

B. Hold down the mouse button on the selection tool until the cropping icon appears . Draw a box with this tool, reshape as needed, and then hit return to crop. Cropping will affect all layers of your file.

C. Alternatively, you can make a selection with the marquee tool, choose Select > Inverse and then hit delete. Instead of deleting what is inside the selection, all surrounding pixels will be deleted. The advantage to this method is that, unlike cropping, choosing select inverse only affects the layer you have selected. If you have several images on separate layers that you want to crop individually, this is a good method to use.

Touch-ups .............Back to top

To clean up dust and scratches, make small feathered selections near the area you want to cover using the lasso tool. Use the move tool to copy the selection (hold the option key) and place it over the blemish. Also try the rubber stamp tool or the smudge tool.


Erasing the Background .............Back to top

Magic Eraser

Double clicking on the magic eraser tool will open the magic eraser options dialog box. There you can set a tolerance (higher for a more varied background, lower for a solid background) and an opacity level.

Before eraser After eraser


Background Eraser .............Back to top

Double click on the Background Eraser icon, shown above. This will open the Background Eraser Options:

You can change the tolerance depending on how much variability there is in your background. If you change the opacity, you can erase to varying degrees of transparency. Anti-aliased will soften the borders of your selection, and contiguous will select only areas that touch each other. If you want to erase a particular color wherever it is in the image, don't check the box.

Click once in the background to select the color that is supposed to be erased. Then begin erasing.

The eraser size is determined by your selection in the Brushes palette. (Window > Show Brushes)

Even if the eraser touches the foreground image, it will not erase it....

unless the color in the foreground is close to the background color.

Near the bottom right of this embryo image, the dark purple color is getting erased.


Preventing foreground erasure:

To prevent the erasure of foreground as shown in the last frame above, you can check the box in the Options dialog box (above) marked "Protect Foreground Color".

Then, by using the eyedropper tool , you can click on the color you want to protect. The color will then appear in the toolbox as the foreground color:

Select foreground color that you want to protect. Now when you erase, the foreground will not be damaged.

Extract: Erasing background without an eraser

Go to Image > Extract

Using the highlighting tool, draw a line around the edge of the foreground image so that the line overlaps with both foreground and background. Once the outline is complete, click with the paint bucket tool in the center of the image to fill it. You can then preview the image and click OK. This is the result, probably the best of the three erasing options.



Color Channels ...... .............Back to top

It is often desirable to assign a separate transparent color to each of two or three images so that when you combine them, you can easily identify the areas where overlap occurs. The way to do this is to put each image on its own color channel. The Channel Mixer automates this process, and makes aligning the images much easier. Here is how to do it:

1. Open your images. They should be saved as TIFF and the mode should be set to grayscale. (Go to Image>Mode and choose grayscale if this is not already the case). If you have only two images to merge, open a third anyway just as a placeholder.

2. Select one of the images and choose "merge channels" from the channel palette menu. (Find channel palette under the Windows menu, and then click on the arrow at the top right of the palette to see options).

3. Choose RGB color mode.


4. Assign a file to each color channel
Click on the scroll bars until the correct file is matched with the desired channel. If you are using a placeholder for your third file, assign it the extra color. When you are finished, click OK.



A new file will be created with your images in separate color channels.

5. If you are using a placeholder for a third file, you can now delete it. To do this, select the channel, click with your move tool in the work area, and choose command-a (or Edit > Select All). Then click the delete button. The next step is to fill this area with black, by selecting the entire work area with that channel selected, going to the Edit menu, and choosing Fill > with black.

6. Adjustment: Once your images are combined, you can select individual channels and use Levels or Curves adjustments on them to vary the relative brightness or contrast of that channel. If there is a lot of background noise on a channel, a Levels adjustment can bring the background back to black. (see Levels)


Tonal and Color Adjustments .............Back to top


There are many reasons to adjust images in Photoshop; the most common two are:

1. Matching the look of the original. Scanning is imperfect, so you may have to adjust the image just to make it look like what you started with.

2. Increasing the clarity of the image. Increasing contrast or adjusting colors are methods for making an more readable.

Currently there are no official guidelines for scientists using Photoshop. As is true in many stages of the research process, you will have to use your own judgment to keep your work ethical. Some generally accepted guidelines to follow are these:

Tonal and color adjustments are fine if they are applied to an entire image and not just selective parts of it. However, the use of the curves adjustment is not recommended for gels, as it increases the nonlinear relationship of the gray tones, which should ideally be linear. Levels and Brightness and Contrast are fine.

Removing distractions such as dust that occurs during scanning or scratches on the film is okay as long as it doesn't alter data. Any blemishes that occur during the experiment and appear on the original gel, blot, etc., should not be altered.

Reordering lanes on a gel image is okay as long as all the lanes remain fixed in reference to markers.

Substituting or enhancing colors is permitted as long as it doesn't change the data. For example, if a blue dye is very faint, it may be brightened in the image ( and in the control ) in Photoshop as long as it isn't intended to be quantitative.

It is a good practice to retain a copy of your original non-adjusted image for reference.

Adjusting Grayscale and Line Art: Image > Adjust

You will get the best results with these adjustments if you keep them subtle. Repeated dramatic adjustments will eventually make your image look pixelated, because so much original information is discarded in the process. The new adjustment layer feature allows you to make minor adjustments of different kinds and layer them for total effect, without too much pixelation.

Layer > New Adjustment Layer : Instead of making adjustments to the pixels of your image, consider making the adjustments on a layer above your image. This way, if you don't like the way the adjustments look, you can simply make that layer invisible, or delete it. The adjustments work the same way as before. You can view your adjustment layers in the layer palette.


Levels .............Back to top

Go to Image> Adjust > Levels.
This gives you a histogram showing you the amount of each shade of gray ranging from black to white your image contains. You can change the range of grays in your image by determining the beginning and end of this curve.

Black is on the far left, and white on the far right. You can see that the image above has mostly middle gray tones.


Making the lights lighter: By moving the lightness slider left to the edge of the histogram, you can reset the lightest of the middle gray tones white, which also resets all the values in between.


This is what the histogram looks like for the adjusted image. The histogram has basically been stretched to span the range from white to black.

The empty spaces between tones (white lines) indicate that this image might have been adjusted too radically, and the image might begin to look pixelated at this point. This is why beginning with a good image and a good scan is important.

Making the darks darker: The dark tones in the example image are dark enough, but if you wanted to make them darker, you would move the darkness slider on the far left to the right, to towards the left edge of the curve.

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Image > Adjust > Curves

Note: There is some question as to whether this is a legitimate method for adjusting gels, as it increases the non-linearity of the image.

Curves also allows you to change the highlights and shadows, but it also lets you selectively adjust individual midtones in between. Using the pointer, pull the curve gently concave or convex.

The grayscale along the bottom of the graph shows which tones you are affecting. You can change multiple points on the curve. If the preview box is checked, you can view your changes before they are applied. Nothing is applied until you click "OK".


Brightness and Contrast

Instead of changing selective tones in an image, this setting affects all pixels selected. Brightness lightens all pixels, while Contrast pushes the darker pixels toward black, the lighter pixels toward white, reducing middle grays. This is not a very sophisticated tool, but it is simple to use.


Adjustment layers .............Back to top

If you would like to make an adjustment to an image but keep the original intact, try using an adjustment layer. This is a special type of layer that acts a filter over your image and appears in the Layers Palette. If you don't like it, you can make it invisible or delete it without touching your original. If you open the file years later and wonder what the image originally looked like, you can just turn off the adjustment layer to see the original. Most of the same adjustment tools are available to use on an adjustment layer. However only 8 bit channels are supported in this mode.

Layer > New Adjustment Layer

When you open and adjustment layer, you can give it a name and choose to group it in the Layers Palette with the image it acts on.

Make your adjustment as usual.

The example below is what you see in the Layers Palette after you make the adjustment. As you move layer 2, the layer that has been adjusted, the levels adjustment moves with it so you don't accidentally lose it. Click directly on the histogram icon to modify the layers adjustment.


Adjusting Color: .............Back to top

Color photos benefit from the adjustment tools mentioned above, as well as a few others relating specifically to color. However, you may notice color shifts when using the adjustment tools.

Avoiding color changes:

You can avoid the extreme color shifts that sometimes occur when adjusting the contrast of color images by using adjustment layers to make color adjustments. After choosing the desired adjustment, Choose Mode > Luminance in the "New Adjustment Layer" dialog box.


Color Balance: .............Back to top

Image > Adjust > Color Balance presents you with sliders for each additive color, Red, Blue and Green.

You can selectively alter the color balance of shadows, midtones or highlights of an image by moving the sliders to the right to intensify the color, or to the left to decrease that color in the image. Decreasing one color increases its opposite. You can play with different combinations until you are satisfied, and then click OK.


Hue and Saturation: .............Back to top

Image > Adjust > Hue and Saturation. This very useful tool allows you to change the Hue (color), Saturation (purity or intensity of the color) and Lightness ( amount of white or black ) of your image.

You can either select "master" which adjusts all the color channels in an image, or you can choose to change individual color channels.


Replace Color: .............Back to top

Image > Adjust > Replace Color gives you an eyedropper (different from the tool palette eyedropper) to select a color from your image, and replace that color everywhere it is found in the image with another color of your choosing.

In the above image, the highlighted areas represent my selection, a certain shade of green in my image.

The fuzziness slider was used to make my selection less tightly defined, increasing the range of green that would fall into my selection.

The Hue, Saturation, and Lightness sliders were then used to change that color into a new one (red) to replace the green with.

When Converting to CMYK makes the image gray:

Select the grayish background with the eyedropper. The fuzziness slider should be at or close to zero to prevent making changes within the images. Look at the selection to make sure it is only affecting the background. Then move the lightness slider all the way to the right.

Creating a figure .............Back to top

Once you have finished adjusting your images, you may want to combine, arrange crop, add text or move them into other applications.

Combining images from different files:

1. First make sure the resolution and mode are the same in both files. (To check the mode, go to Image > Mode)

2. Then select the image from one file with the move tool, and click and drag the image to the new file. A copy will automatically be made as you drag.

Aligning your images:

If your images and text are all on separate layers, you can align them by using Layer > Align Linked function. To do this, link all the layers you want to align by clicking the box to the left of each layer in the layers palette. You will see the linking symbol. Then go to Layer > Align Linked.


While the layers are linked, you can also move them as a unit. To unlink them, click the linking icon to the left of the layer in the layers palette.

Adding text to images:
You can add text by choosing the Type Tool from the tool box. Photoshop places each new text block on a separate layer. When that layer is selected, you can move it using the move tool just as you would any other object. You can also use the arrow keys to make subtle adjustments. This makes it easier to arrange your figure. Edit the text by clicking on it with the Type Tool.

Many text options are available on the options bar at the top of the screen when the Type tool is selected, but you can access more options by clicking on the "palettes" button on the right. This will open the Character Palette:

You can also select text add effects such as glowing letters and drop shadows (Layers > Effects).


Importing Photoshop files into other applications ............Back to top

If you plan to arrange your figure in another program, you will need to save it in a more universal format. Be aware that Photoshop format is the only one that supports layers--in all the formats below, layers will automatically be flattened. This means you should save your original in case you need to edit it later.

Illustrator and InDesign: Because these are also Adobe programs, you can import native Photoshop files (.psd) into them. No need to save as another format.

FreeHand, Canvas, or QuarkXPress or other vector drawing program: save as TIFF. Once you have a TIFF image, you can open it in your graphics application using a command such as "place" or "import".

PowerPoint, Dreamweaver, and web applications: save as JPEG. Keep in mind that this format is not ideal for print.

For more information about formats and when to use them, see formats


Creating Contact Sheets ............Back to top

If you find yourself with a folder full of images, and you can't remember which ones you wanted to use, this feature might be helpful. It automatically creates thumbnails of your images using the file names as labels.

1. Go to File > Automate > Contact Sheet II

2. Click Choose to specify the folder containing your images.

3. Under Document, specify the dimensions, resolution, and color mode.

4. Under Thumbnails, specify layout options for the previews.

5. Click OK


Web Image Galleries .............Back to top

To take the contact sheet one step further, this option creates a web page with thumbnail images that you can click on to go to the larger image.

For more information regarding web page creation and organization, see the Web Design page.


Web Optimization .............Back to top

File > Save for Web

Original shows you the image without optimization

Optimized gives you a preview of your optimized image

2-up lets you compare side by side

4-up lets you compare the tradeoffs between file size and detail.


Printing your images from Photoshop .............Back to top

The biggest issue that comes up with printing is color accuracy. This topic is covered in detail in Color Management. Please read this page--you shouldn't use Photoshop without having the correct color settings or you could unknowingly do permanent damage to your files.

For information regarding how to print to our color printers, please visit the Printing page.

ImageReady .............Back to top

Photoshop now comes bundled with ImageReady, a software application that can help you create web pages and optimize images for the web. For tutorials on how to use ImageReady, check out this tutorial site: