Confocal Assistant

ImageJ

MetaMorph

Adobe Photoshop

Canvas 5



Confocal Assistant

Confocal Assistant is an easy to use program for viewing BioRad confocal images on an IBM compatible computer. Instructions for downloading this free program are at the end of this section.


Confocal assistant can be used to:

-merge side-by-side images (Image | Side-by-Side Merge...) - left is green and right is red

-merge images from separate files (File | Merge...)

-add color to single channel or full screen images (File | Merge...)

-load a z-series (File | Open...)

-play a z-series as a movie (Use the arrows directly above the image or Series | Movie...)

-project a z-series (Series | Project)

-separate out images from a z-series (Scroll through you z-series and File | Save As... the image to save)

-make a montage of your z-series (convert montage to Bitmap to save as a tif)

-adjust contrast

-sharpen an image


Images can be saved as a tiff file to subsequently open directly in Adobe Photoshop (Canvas,etc).


Prints made directly from Confocal Assistant will only be 72 dpi. Adobe Photoshop should be used for higher quality prints.



Download Confocal Assistant from ftp.genetics.bio-rad.com and login as anonymous. Go to the directory: Public/confocal/cas and get (as a binary file) SETUP402.exe. Run this file to setup the program in Windows.

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ImageJ

Introduction

ImageJ is a new Java image processing program inspired by NIH Image for the Macintosh. It runs, either as an on-line applet or as a downloadable application, on any computer with a Java 1.1 or later virtual machine.

It can display, edit, analyze, process, save and print 8-bit, 16-bit and 32-bit images. It can read common file formats including TIFF, GIF, JPEG, BMP, DICOM, FITS and "raw". It supports "stacks", a series of images that share a single window. It is multithreaded, so time-consuming operations such as image file reading can be performed in parallel with other operations.

It can calculate area and pixel value statistics of user-defined selections. It can measure distances and angles. It can create density histograms and line profile plots. It supports standard image processing functions such as contrast manipulation, sharpening, smoothing, edge detection and median filtering.

It does geometric transformations such as scaling, rotation and flips. Image can be zoomed up to 32:1 and down to 1:32. All analysis and processing functions are available at any magnification factor. The program supports any number of windows (images) simultaneously, limited only by available memory.

Spatial calibration is available to provide real world dimensional measurements in units such as millimeters. Density or gray scale calibration will also be available in a future version. A scripting language is planned.

ImageJ was designed with an open architecture that provides extensibility via Java plug-ins. Custom acquisition, analysis and processing plug-ins can be developed using any Java development system. User-written plug-ins make it possible to solve almost any image processing or analysis problem.

ImageJ is being developed using Metrowerks CodeWarrior, and the source code is freely available. The author, Wayne Rasband (wayne@codon.nih.gov), is at the Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA.

Download the program from http://rsb.info.nih.gov/nih-image. This program will open Leica scanner files (i.e.multiple image .tiff files). A plug-in is available specifically for Leica files.

MetaMorph

MetaMorph is an image analysis program from Universal Imaging Corporation

This program will directly read images from the BioRad confocal (.pic), Princeton Instruments Winview (.spe) as well as most graphics files (.tif, .bmp, .jpg, etc.)





Adobe Photoshop

The Image Processing Toolkit is a set of image analysis plug-ins for Photoshop developed by Chris and John Russ. Detailed information is available at http://hometown.aol.com/ImagProcTK

Bio-Rad .pic files

Merging BioRad Split Screen Images

Contrast and Brightness

Sizing for Prints and Slides

BioRad Color images - 2 channels, side-by-side

BioRad Color images - single channel

Confocal Assistant is a easy way to merge, project, resave, etc Bio-Rad files.

Open BioRad .pic files by:

File | Open as...
Open as - select Raw and the File Name and Open
Then type in 768 for the width, 512 for the height, channels count=1, the depth is 8 bit and 76 is the header size.

Ignore the message that the specified image is smaller than the file; open anyway - OK.

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Merging a BioRad split screen image:

It is easier to do this in Confocal Assistant.

In Photoshop: split the image into two separate files by:

1. Double click on the rectangle in the upper left of the tool bar

2. Select Style: Fixed Size - width 384 pixels, height 512 pixels and feather=0

3. Carefully click in the upper left of either image (if the box doesn't enclose the image properly then click on Select | None at the top of the menu and retry the selection)

4. Edit | Copy

5. File | New with the following dimensions:

Width: 384 pixels
Height: 512 pixels
Resolution: 72 pixels/inch
Mode: grayscale

6. Edit | Paste into the new file and then Layers | Flatten Image

7. Click on the original image. Select the other half of the image and Image | Crop

8. Select the black background

9. Make a third image of the same dimensions as above:

File | New :

Width: 384 pixels
Height: 512 pixels
Resolution: 72 pixels/inch
Mode: grayscale
Content: Background Color

If necessary, open the channels window: Window | Show Channels

10. Select the arrow in the upper right of the channels window.


11. Select Merge Channels...

12. Change the Mode to RGB Color


13. Click OK

14. Load the images into the appropriate color channel. The black image goes in the blue channel.OK


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Adjust contrast and brightness of color or grayscale images by using:

Image | Adjust | Levels...

Select the color to edit under channel.



Use the upper triangle in input levels for highlights and the lower triangle for shadows. Make small adjustments with the middle triangle.



DO NOT DROP OUT THE MIDDLE VALUES.

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Before Printing:

Adjust the image size to make sure it is within the printable area and to change the resolution:

Image | Image Size...

BioRad MRC 600 confocal images are collected at 72 dpi. Changing the resolution to 144 dpi (in Photoshop, not in Canvas) will make the print look less "pixelated".

Even though our printers will print at 300-720 dpi, you won't notice the difference between 144 and 300 dpi with MRC 600 images. The 300 dpi files will be larger and take more time to print.

To change the image size, keep the proportions checked and change the longest dimension.

Printable areas:

Tektronix Phaser II SDX prints:

maximum letter size is 8.6" x 8.1"

maximum legal size is 10.8" x 8.1"

Tektronix Phaser 480 prints:

maximum letter size is 8.2" x 10.1"

Check the Page Setup and select the printer (Keck Center Phaser=Phaser II SDX), page orientation (portrait or landscape) and the paper size.

Save as Tiff or Photoshop(psd)

Slides from HSERC:

Maximum image size is 10.14 x 7.33 for slides

File | Save as...

Save File as format type:

Pict (Macintosh) (Note: CISB may charge an extra $1.00 to convert a file to a pict. Call CISB at 5-1180 to find out what format they prefer from the program you are using on the PC)
Byte order: Macintosh

Instructions for slides (put on HSERC form):

1. extra fine color
2. soft contrast
3. high resolution
4. run on Mark II
5. black background

Notes:

Use Adobe Photoshop for all image adjustments. Use Canvas to make montages and add text or arrows.

If the image has more than one layer then you can only save it as a .psd format. Flatten the layers (Layer | Flatten Image) to save in any other format.

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Adding Color to BioRad Dual Channel Images (side-by-side)

Starting with a split screen image

Starting with a merged image

Starting with a split screen image:

1. Open the image

2. Double click on the rectangle in the upper left of the tool bar

3. Select Style: Fixed Size - Width 384 pixels, Height 512 pixels and Feather = 0

4. A black background should be selected for the rest of the steps. Carefully click in the upper left of either image (if the box doesn't enclose the image properly then click on Select | None at the top of the menu and retry the selection)

5. Edit | Cut

6. File | New with the following dimensions:

Width: 768 pixels
Height: 512 pixels
Resolution: 72 pixels/inch
Mode: grayscale
Contents:background color

7. Edit | Paste into the new file

8. Use the Move tool to move the image to the same position it was in the original image and then Layer | Flatten Image.

9. File | New with the following dimensions:

Width: 768 pixels
Height: 512 pixels
Resolution: 72 pixels/inch
Mode: grayscale
Contents: background color

10. There should be three images on the desktop:


11.If necessary, open the channels window: Window | Show Channels

12.Select the arrow in the upper right of the channels window.

13. Select Merge Channels...

14. Change Mode to RGB Color


15. Click OK

16. Load the images into the appropriate color channel. The black image goes in the blue channel.OK

Starting with merged images:

1a. Follow steps # 11 and #12 above but split the channels

2a. Select a black background. Click on the green image. Under Image| Canvas Size change the width to 768 pixels and move the box in the window to the left. OK.

3a. Select the red image. Under Image| Canvas Size change the width to 768 pixels and move the box in the window to the right. OK.

4a. Select the blue image and change the width to 768 pixels under Image| Canvas Size.

5a. Follow steps #12 - 16 above.


Adding Color to BioRad Single Channel Images

1. open the file
2. Select | All
3. Edit | Copy
4. The background needs to be black
5. File | New - Change Mode=RGB Color, Contents=Background Color. Leave the other sizes the same.
6. Select the color for the image in the Channels box
7. Edit| Paste

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Canvas 5.0.3

Use Canvas to make montages and to add text or arrows to images.

Adjust the image resolution (dpi) in Photoshop before placing images into Canvas.

Start Canvas 5.0.3 and create a blank page if necessary (File| New). Select Layout | Document setup... to set the Illustration size and Orientation of the page. Create a page size for a specific printer by selecting the printer first (File | Printer Setup...) and then select Illustration size: From Printer in Layout | Document setup...

Montages: use File | Place to create a page with several images.

If the image size is incorrect then check the Image | Mode to make sure the image is read correctly (Grayscale or RGB Color). Then check the Image | Area | Resolution... If the resolution is inaccurate, then change the image width and height units to inches (or cm) and type in the correct resolution (res). Do not select a new resolution in Canvas (i.e.other than the original or what was saved in PhotoShop ) Canvas does not interpolate as well as PhotoShop.

Image Adjustments: Canvas 5.0.3 has a number of useful editing features for images.

The selection box around the image determines what features are available: Select ë an image - the surrounding box may have solid squares in the corners : n or it may have "edit" marks : _| in each corner

To change modes, select ë and double click on the image. To exit a mode, double click outside of the image.

Change the brightness and/or contrast of an image by selecting Image | Adjust | Levels... with the image in "edit" mode.

To Crop... (Image|Area) an image you must be in "select" (solid boxes in the corners of the image).

Resize images by selecting Object | Scale... proportional must be checked.

Text: Select the text tool T to type. Modify text (font, size, bold,etc.) by selecting the text ë and double clicking the text tool. Click on Apply to implement changes. Modify the color by selecting the text ë and changing both the foreground (pen) and background (paint bucket) to the desired color.

Use a standard font size for text that will be rotated. You can change to a nonstandard font size after rotating.

Arrows:

To add arrows, first draw a line with the line tool

Double click the strokes tool to open the strokes palette. Change the width of the line and add an arrowhead in this menu.

The pen will change the color of the arrow and line.

Save: Save your plate in the .cvs format if you have mixed text and images. Text will be bitmapped if saved as tiff, bmp, jpg or pict

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