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Photoshop for the Microscopist A tutorial for processing images

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Title: Photoshop for the Microscopist A tutorial for processing images


1
Photoshop 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.

3
Steps 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

4
Open Photoshop
Open Adobe Photoshop by double clicking on the
icon.
5
Setup Photoshop
  • Select Window.
  • Make sure that the following windows are
    checked Tools, Options, Navigator, Layers, and
    Status bar.

6
Open 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.

7
Open 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.

8
Open the Images
If the image is obscured by a fine grid, select
View. Click on Extras to remove the grid.
9
Improve/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

10
Improve/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.

11
Improve/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.

12
Improve/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
13
Improve/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.

14
Improve/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.

15
Improve/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.

16
Improve/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.

17
Improve/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

18
Improve/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.

19
Improve/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.

20
Improve/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.

21
Crop the Images
  • Select Edit PreferencesUnits Rulers
  • In the Preferences dialog box, select inches
    for the ruler units.
  • Click OK.

22
Crop 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.

23
Crop 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.

24
Crop 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.

25
Crop the Images
  • Select Image Crop to crop the image.

26
Crop 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.

27
Crop the Images
To get guides to help you accurately position the
marquee, select View New Guide.
28
Crop 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.

29
Crop 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.

30
Crop the Images
  • Select Image Crop to crop green.tif.
  • Crop red.tif using the same procedure that you
    used with green.tif.

31
Crop 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.

32
Adjust 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.

33
Adjust 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.

34
Lost 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
35
Make 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.

36
Make 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.

37
Make 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.

38
Make 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
39
Make 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.

40
Make 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.

41
Make a Montage
  • Save the montage by selecting File Save As
  • Call the file montage.
  • Use the Photoshop (psd) Format.
  • Select Save.

42
Adjust 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.

43
Adjust 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
44
Adjust 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.

45
Adjust 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.

46
Adjust 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).

47
Adjust 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.

48
Adjust 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.

49
Adjust 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.

50
Adjust 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.

51
Add 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.

52
Add 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.

53
Add 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.

54
Add 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.

55
Add 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

56
Add 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.

57
Add Labels
Add the labels to the green and combo images as
they are shown here.
58
Crop 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.

59
Save 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.

60
Flatten 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

61
Save 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.
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