Title: Photoshop for the Microscopist A tutorial for processing images
1Photoshop for the MicroscopistA tutorial for
processing images
- Nancy Kleene
- Center for Biological Microscopy
- University of Cincinnati
- 9/10/04
2- One of the most common needs of the
microscopist is to make a montage of multiple
images. The goal of this tutorial is to teach the
user how to make a montage of three separate
images using Adobe Photoshop (Version 7). This
tutorial uses three images (green.tif, red.tif,
and combo.tif) that can be downloaded, along with
this tutorial, from our website
(http//microscopy.uc.edu/ia/image_analysis.php). - The images were acquired on a confocal
microscope by Bob Hennigan. The samples were
prepared by Robert Brackenbury and Amy Koshoffer.
Please send comments to Nancy.Kleene_at_uc.edu.
3Steps in Making a Montage
- Improve/Add the Scale Bar
- Crop the Images
- Adjust the Resolution and Size
- Make a Montage
- Adjust the Intensities
- Add Labels
- Crop the Montage
- Save Layered Image
- Flatten Before Printing
4Open Photoshop
Open Adobe Photoshop by double clicking on the
icon.
5Setup Photoshop
- Select Window.
- Make sure that the following windows are
checked Tools, Options, Navigator, Layers, and
Status bar.
6Open the Images
- Select FileOpen
- Note that hitting the control key and letter O
at the same time would have also opened the Open
dialog box. Photoshop has many such keyboard
shortcuts. It pays to learn them if you will be
doing a great deal of image processing.
7Open the Images
- Locate the following images combo.tif,
green.tif, and red.tif. - To open all three images at once, select the
first and third image while depressing the Shift
key. - Select Open.
8Open the Images
If the image is obscured by a fine grid, select
View. Click on Extras to remove the grid.
9Improve/Add the Scale Bar
Tool Bar
- The scale bars on images from the LSM browser and
other software often have end bars and text that
are not desired. - You can use the old scale bar to draw a new one.
Then, you can move the new bar to a new position
before cropping the image to remove the old scale
bar. - To zoom in on the old scale bar, click on the
magnifying glass tool
10Improve/Add the Scale Bar
- Draw a box around the scale bar in Combo.tif.
(To draw a box, move the cursor to the upper left
corner of the area that you would like zoom in
on. Click and hold the left mouse button while
moving the cursor to the lower right corner of
the desired region.) - When you release the left mouse button, the
image will zoom as show in the image to the right.
11Improve/Add the Scale Bar
- Look for the button on the tool bar that has one
of the shape tools shown in the image to the left
of this text. - If the Line Tool is not showing, click and hold
on this button to reveal a list of available
shape tools and select the Line Tool.
12Improve/Add the Scale Bar
- If the Indicator of Foreground Color is not
white, click on it to open the color picker
dialog box. - In the color picker dialog box, put 255 in the
boxes next to R, G, and B. Click OK.
Indicator of Foreground Color
13Improve/Add the Scale Bar
- Click on the down arrow that is just under the
main menu to look at the Line options. - Make sure the arrowhead boxes are unchecked.
- Select the weight of the line to be 5 px.
- Draw a line the same length as the scale bar by
clicking and holding at one end of the bar and
dragging the cursor to the other end of the bar
before releasing the left mouse button.
14Improve/Add the Scale Bar
Zoomed In
Zoomed Out
- Click on the Navigator tab.
- In preparation for repositioning the bar, slide
the triangle on the Navigator dialog box to the
left to zoom out until you can view the entire
image.
15Improve/Add the Scale Bar
Layers Dialog Box
- Select the Move tool on the tool bar.
- In the Layers dialog box, select the layer with
the scale bar. - Click and hold on the new scale bar and move it
closer to the center of the image as shown above.
16Improve/Add the Scale Bar
- If there is no scale bar but you know how many
microns per pixel there were, you can draw a
scale bar. - For example, if you want a 20 ?m bar and the
image is 0.23 ?m/pixel, you will need to draw a
line that is 87.0 pixels long. - You need to draw this line before you change the
resolution of the image.
17Improve/Add the Scale Bar
- Click on the Info tab and place cursor on image.
See if the X and Y coordinates in the Info
dialog box are in pixels (whole numbers). - If your program is not set to show pixels, you
will need to select Edit PreferencesUnits
Rulers
18Improve/Add the Scale Bar
- In the Preferences dialog box, click on the down
arrow to reveal the selections for Rulers
units. - Select pixels.
- Click OK.
19Improve/Add the Scale Bar
- Select the line tool and draw a line of the
desired length while watching the D (Distance)
value in the Info dialog box. - Make the line straight and as close to 87 pixels
as possible. - The interior of the line will become solid white
when you release the left mouse button.
20Improve/Add the Scale Bar
- Because you do not need two scale bars and to
practice recovering from mistakes, select
EditUndo Line Tool to remove the line that you
just drew. - Note also that Photoshop will allow you to Step
Backwards multiple times to reverse a series
mistakes. - You are not adding a scale bar to the red and
green images because you only need one bar for
the montage.
21Crop the Images
- Select Edit PreferencesUnits Rulers
- In the Preferences dialog box, select inches
for the ruler units. - Click OK.
22Crop the Images
- Find the tool bar button with a dashed shape
(Marquee). - If that shape is not a rectangle, click and hold
until the rectangle option is visible. Select it. - Examine the marquees Style by looking just
under the main menu. If the style is not
Normal, click on the down arrow and select
Normal.
23Crop the Images
- Click and hold on the combo image in the
position that you wish to become the upper left
corner of the cropped image. - While keeping the left mouse button depressed,
drag the cursor to the desired lower right corner
and release the left mouse button. - You can use the arrow keys to nudge the marquee
in various directions. You can click and hold
inside the marquee and drag it to a new position.
24Crop the Images
- Because you will want to crop the green and red
images to the same size, click on the Info tab
and note the width (W) and height (H) of the
marquee. - Because you will want to position the marquee
similarly in the other images, place the cursor
in the upper left corner of the marquee and
record the X and Y coordinates that are shown
in the Info dialog box.
25Crop the Images
- Select Image Crop to crop the image.
26Crop the Images
Marquee icon
- With the following steps, you will transfer the
marquee from Combo.tif to Green.tif - Bring the Green.tif image to a position near the
Combo.tif image. - Confirm that the marquee icon is still selected.
- Click the left mouse key while the cursor is
inside the marquee in Combo.tif. - Keep the key depressed as you drag the marquee
to the green.tif image. Then, release the left
mouse key.
27Crop the Images
To get guides to help you accurately position the
marquee, select View New Guide.
28Crop the Images
- In the New Guide dialog box, select Vertical
and enter the X coordinate for the upper right
corner of the marquee that was used to crop
Combo.tif. - Click OK
- A vertical, blue line will appear on the image.
- Use the arrow key to position the marquee so
that the left edge is on the blue guide line.
29Crop the Images
- To position the marquee vertically, select
View New guide - In the New Guide dialog box, select
Horizontal and enter the Y coordinate for the
upper right corner of the marquee that was used
to crop Combo.tif. - Click OK
- A horizontal, blue line will appear on the
image. - Use the arrow key to position the marquee so
that the upper edge is on the blue guide line.
30Crop the Images
- Select Image Crop to crop green.tif.
- Crop red.tif using the same procedure that you
used with green.tif.
31Crop the Images
- Save the three cropped images by selecting
File Save As - Add the wordcrop to the end of the file name.
- In the Save As dialog box, select the
Photoshop (PSD extension) format so that the
layer that was created by the new scale bar is
maintained.
32Adjust the Resolution Size
- Select the Combocrop.psd image.
- Select Image Image Size
- Make sure that the Resample image box is NOT
checked. - This image is 5.2 inches wide at 72 pixels per
inch (ppi). Printing at 72 ppi will look very
bad. - A good resolution for printing is 320 ppi.
33Adjust the Resolution Size
- Change the resolution to 320 ppi.
- Note that the size of the image is smaller now.
- If you had decreased the size of the image, the
resolution would have increased. - Click OK.
- Change the resolution of redcrop.psd and
greencrop.psd to 320 ppi.
34Lost Resolution
- If you decrease the image size while the
Resample image box is checked, you will lose
pixels (the original was 380 by 292 pixels) and
your image will probably look horrible. - If you increase the resolution while the
Resample image box is checked, you will
artificially increase the number of pixels by
subdividing the existing pixels.
Decreased image size
Result of decreasing image size with Resample
image box checked
35Make a Montage
- Select File New.
- Enter 5 and 2 inches as the Width and
Height, respectively. We are choosing a size
that will hold all three images with enough room
to add labels if needed. Well crop the excess
away at the end.
- The Resolution needs to be the same as the
resolution of the images that you will place in
the montage. For this tutorial, that is 320 ppi. - The Mode should be RGB Color.
- Click OK.
36Make a Montage
- Select the Combocrop.psd image by clicking
anywhere within the image. - Select Select All to have a marquee surround
the entire image. - Select Edit Copy Merged to copy both the
image and the layer with the new scale bar.
37Make a Montage
- Select the new image by clicking anywhere within
the new image. - Select Edit Paste to place the combo image in
the new image.
38Make a Montage
- Select the move tool in the Tool bar.
- Each pasted image creates a new layer. In the
Layers dialog box, select the layer with the
combo image. - Click on the combo image and slide it to the
desired position as third in what will be a row
of images.
The final arrangement of images
39Make a Montage
- Select and copy the green and red images and
paste them into the montage. - Because they do not have layers, the red and
green images will use the command Copy instead
of Copy Merged. - Use the Move tool to arrange the images in a row
as shown above.
40Make a Montage
- Close the combocrop, redcrop, and greencrop
images by clicking on the X in the upper right
corner of each image. - Select Yes to save the resolution changes.
41Make a Montage
- Save the montage by selecting File Save As
- Call the file montage.
- Use the Photoshop (psd) Format.
- Select Save.
42Adjust the Intensities
- There are many ways to adjust the appearance of
images. The next few slides will show one of the
most common methods. If this method does not
work for your images, consult the Photoshop help
guide or contact us for more ideas. - In the Layers dialog box, select the layer
with the red image. Now, the adjustments that
you make will only affect the red image.
43Adjust the Intensities
- Select Image Adjustments Levels
- Click on the down arrow and select Red for
Channel. - The Y axis of the graph shows the number of
pixels in the image with a particular intensity.
Intensity is graphed on the X axis.
of Pixels
Intensity
44Adjust the Intensities
- Initially, the X axis range is 0 (black) to 255
(bright red) for most images. - The black triangle on the left of the X axis
marks the black level (BL), also called
shadows. The BL value is shown as the left most
Input Level.
BL
HL
BL
HL
- The white triangle on the right of the X axis
marks the highlights (HL). The HL value is shown
as the rightmost Input Level. - Generally, one should use as much of the
intensity range as possible. This image is not
using the entire range.
45Adjust the Intensities
- The gray triangle in the middle of the X axis
marks the gamma. The value of gamma is shown as
the middle Input Level. - For a fluorescent image, increasing the gamma
above one will increase the midtones to a greater
degree than it will increase the very bright and
very dim intensities. - Adjusting gamma can be very helpful for images
of fluorescent specimens.
46Adjust the Intensities
- Pull the white triangle in to the right edge of
the graphed curve. This will set all the pixels
198 and greater to the maximum value of 255. And,
pixels less than 198 will be mapped to
corresponding higher values. This will brighten
the image. - A longer exposure or higher gain would have
prevented the need for this manipulation and
would have resulted in a better looking image
than what we will get by changing the highlights
now. - Drag the gray triangle to the left to increase
the gamma (brighten the midtones).
47Adjust the Intensities
- Pull the black triangle in to the left most edge
of the graphed curve. This will set all the
pixels from 0 to 14 equal to the minimum value of
0. And, pixels greater than 14 will be mapped to
corresponding lower values. This will make the
background darker. - Note the three Input Levels so that you can use
them with the red channel of the combo image. - Click OK.
48Adjust the Intensities
- To adjust the green image, select the layer that
contains the green image. - Select Image Adjustments Levels
- Click on the down arrow and select Green for
Channel. - This image is in pretty good shape. Adjust the
black level to 4. - Record the three Input Levels and click OK.
49Adjust the Intensities
- To adjust the combo image, select the layer that
contains the combo image. - Select Image Adjustments Levels
- Click on the down arrow and select Red for
Channel. - Enter the black level, gamma, and white level
values that you used in the red image. Do not
click OK yet.
50Adjust the Intensities
- Click on the down arrow and select Green for
Channel. - Enter the black level, gamma, and white level
values that you used in the green image. - Click OK.
51Add Labels
- Every label will be placed in a new layer
automatically. - To make sure that the labels appear on top of
the images, make sure that the top layer is
selected in the Layers dialog box.
52Add Labels
- Find the tool bar button with the letter T.
- Click and hold until the type tools are visible
and select the Horizontal Type Tool. - In the Options bar under the main menu, click
the down arrow next to the font size and select
9 pt. - Click in the upper left corner of the red image.
The cursor will become a blinking vertical line
whose length is similar to the font size.
53Add Labels
- Type the letter A.
- If you move the cursor away from the text, it
will change into a move tool. - Now, if you click the left mouse button and
hold, you can move the text to a new position. - Select the rectangular marquee (or any other
tool) to leave the typing mode.
54Add Labels
- Reselect the type tool
- Place a letter B in the upper left corner of
the green image. - Leave the typing mode by clicking any other
tool. - Reselect the type tool and put a letter C in
the upper left corner of the combo image. Click
any other tool when done typing.
55Add Labels
- To modify text, click on the layer that contains
the letter A. - Then, click on the Type tool.
- Click on the previous text (A). If needed, use
the arrow keys to get to the right edge of the
text. - Type . Propidium Iodide
56Add Labels
- After you have added text, you can modify it.
- To highlight the text, depress the left mouse
button while positioned JUST to the right of the
text and hold the mouse button down while
dragging the cursor across the text.
- Click the down arrow on the font size.
- Select the 6pt font size.
- Click any other tool to leave the text mode.
57Add Labels
Add the labels to the green and combo images as
they are shown here.
58Crop the Montage
- Select the rectangle marquee.
- If the style for the marquee is not Normal,
click on the down arrow and select Normal. - Draw a marquee around the montage of the three
images. - Select Image Crop.
59Save the Multilayered Image
- Saving the image in a Photoshop (PSD) format
will preserve the layers and allow you to make
future changes more easily. - Select File Save to save the changes to
montage.psd.
60Flatten Image Before Printing or Saving as TIFF
- You will save quite a bit of memory if you
flatten the image (i.e., collapse the layers). - Click the right pointing arrow on the Layers
dialog box. - Select Flatten Image
61Save as TIFF
- If you would like to put the image in Powerpoint
or a Word document, you probably want to save the
image as an uncompressed TIFF. - The JPEG format involves compression. If you
save the image as a JPEG, you will have a smaller
file but the image is altered during the
compression.
- To save the image as a TIFF, select File Save
As. - Select TIFF as the format and click Save.
- Select IBM PC in the TIFF Options dialog box
and click OK.