V. Advanced topics
1. Merging Pairs Of Double Labeled Sections
One of the most valuable and commonly used features of confocal microscopy is the ease of obtaining images of double and triple labeled histological sections. There are two major obstacles that may cause difficulties when attempting to merge two images:
A) The contrast and brightness values of one section may be markedly different from the other(s). This is best dealt with by careful evaluation of the images at the time of original data collection, and modifying your means of collection.
B) The two series of images of such a pair may not be in perfect register with each other. This may be due to various factors, including misalignment of the PMTs, the mirrors, and filter blocks. Most of the errors appear to occur in translation (X- and Y-axes) and not in rotation. A shift of a few pixels may not be noticed, but occasionally the error results in a marked shift from one color plane to the second. Simple translation errors can be corrected by shifting the images one or more pixels at a time. Rotational errors are more difficult to correct, take longer, and frequently result in image warping. If you find that you have marked rotational errors, the service personnel from the manufacturer of your confocal microscope should deal with this.
NIH Image provides an alignment operation, "Register". This can be used on sections in a Stack (see below).
A) NIH Image And Adobe Photoshop
Two or three gray scale images of different fluorophores can be combined into a single colored image, with each fluorophore represented by a different color. The color chosen to represent each fluorophore is arbitrary, and can differ from the original one. There are two different programs that can be used successfully for this purpose, NIH Image and Adobe Photoshop. There are benefits and disadvantages in the use of each program. Adobe Photoshop is relatively expensive, but a superb commercial program. It supports 24-bit images and allows almost instantaneous adjustment of the individual red, green and blue planes of a merged image. NIH Image produces an 8-bit custom palette of the merged image. It takes 5-10 seconds to produce this image on a Macintosh IIfx, and is correspondingly faster on a Quadra 950 or a PowerPC. Although this is an 8-bit color image, the result is often satisfactory, and occasionally even comparable to that obtainable with Adobe Photoshop. However, NIH Image may produce excessive dithering of the resultant image, and you cannot make small adjustments in brightness or contrast of the final color image obtained with NIH Image . Instead, you have to go back to the original gray scale images, modify them, and then once again Merge the images.
Since each "Indexed Color" image produced with NIH Image has its own unique LUT, you cannot directly do a side-by-side comparison of two different color images if you merged using "Custom Colors", as the color values shift markedly as you change windows. If you selected "System Colors", the quality of the color is more limited, but you will be able to compare results with other windows merged using the same system LUT. This is a major disadvantage in relying exclusively on NIH Image . However, for routine operations, NIH Image is satisfactory.
In comparison, Adobe Photoshop, using a full 24-bit window, allows you to compare multiple colored images simultaneously on a single screen. Adobe Photoshop also provides an excellent range of filters and convolutions. The more immediate advantages of NIH Image are manifest in measurement capability, generating Stacks, Z-series projections, and 3D-projections and rotations. Adobe Photoshop does not provide such facilities.
Until recently (prior to version 1.56), NIH Image could only run under an 8-bit monitor setting. If you wanted to shift back and forth from NIH Image to Adobe Photoshop, you had to reset the monitor to 24-bit. Beginning with version 1.56, NIH Image operates satisfactorily with the monitor set to 24-bits.
NIH Image version 1.59 now directly allows the user to save an RGB stack of three sections in a format that can directly be read by Adobe Photoshop 3.0. If the file is modified in Adobe Photoshop, then saved as an Adobe Photoshop TIFF file, it can be re-opened by NIH Image as a three slice stack. However, if you add additional Layers or Channels in Adobe Photoshop, it forces you to Save the file in Adobe Photoshop format. This cannot be read by NIH Image . An NIH Image Z-series containing Merged color slices cannot be read by Adobe Photoshop.
B) Double Labeled Sections: Building A Stack (Best Done Using A Macro)
- Color Merge In NIHImage
Two color plate: If you wish to combine only two plates, open the two files.
Use the macro "Color Merge Two Images" contained in the sample "Confocal Macros" that is provided with this manual. If you examine the sequence of commands in the macro, you will find the following operations:
A) Open a fresh stack
B) Paste the "red" image into the first slice
C) AddSlice to add an additional slice
D) Paste the "green" image into the second slice
E) AddSlice to add a black (empty) third slice
F) Merge from RGB to 8-bit color using a custom LUT
G) Open a new window containing the merged color image
Alternately, you can do this operation manually to familiarize yourself with the procedure. The first file opened will become the red plane, the second file the green plane. Under menu item Stack choose Windows to Stack . This will put the two plates into a stack labeled "RGB". Any further operations will not alter your original data files, so you can always go back and start again.
Save a copy of the NIH Image Stack. The following section describes how to use this Stack with Adobe Photoshop 3.0.
If you want to alter the contrast or brightness of either of the color planes, you have to modify either the original images or those in the RGB stack. I suggest that you limit your initial attempts to the slices in the RGB stack. When you change the Brightness or Contrast of any single image, you must then Apply LUT (Enhance menu) to that single image. Do not use the Apply LUT to "Stack" macro as this will modify all the images in the Stack.
From the Stacks menu select RGB to 8-bit Color. In the resulting dialog box, select "Custom Colors". This will generate an "Indexed Color" window from the Stack, but will not alter the stack itself.
Since each original file may have a different distribution of values (see histogram), the saturation of each color plane may differ markedly. Be creative, try different ways of doing it. (e.g., use Enhance Contrast operation on one slice at a time). Try non-linear LUTs, various filters, etc.
The resulting "Indexed Color" image can be saved with a unique name.
Once you have mastered this sequence, you will have a clearer understanding of the operation of the macro "Color Merge Two Images" contained in the sample "Confocal Macros" that is provided with this manual.
If you now Save the RGB stack (RGB TIFF) in NIH Image , the resulting file can be viewed with Adobe Photoshop as a 24-bit file.
- Merging Files With Adobe Photoshop
Version 1.58/1.59 of NIH Image now permits Stacks to be directly saved in Adobe Photoshop 3.0 TIFF format.
A) The stack must consist of 3 slices.
B) Before saving the stack, open Slice Info in the Stacks menu. Confirm that RGB is selected.
C) Save the file. It will write the file as an RGB TIFF file.
You can then directly open this NIH Image Stack in Adobe Photoshop 3.0.
An alternate means of converting the stack to an RGB TIFF format is to call the RGBtoColor function. This will automatically change the "Slice Info" window to RGB. The resulting "Indexed Color" image will give an approximate (8-bit) preview of the results to be obtained with Adobe Photoshop (24-bit). Save this file.
You will now be able to use the various Adobe Photoshop tools to modify the resulting 24-bit image. You should explore the use of the Levels command (Command+L ), as well as the Brightness/Contrast command (Command+B ).
If you have a quadruple (or more) labeled section (e.g., 512x512, four fluorophores, or three fluorophores and a Nomarski/DIC image), and have stored them in a four slice stack in NIH Image , you can open them in Adobe Photoshop using the method described below.
A) From the Adobe Photoshop File menu, select Open.
B) Using the Adobe Photoshop Open dialog, go to the desired directory containing the NIH Image Stack file of interest.
C) Using the same dialog box, pull down list at the bottom of dialog box, choose Raw file format.
D) This will show all files in the chosen folder.
E) Select the NIH Image Stack containing the four slices. You have to know the dimensions of the slices.
F) The resulting dialog box requests that you fill in:
- Width (e.g., 512 pixels)
- Height (e.g., 512 pixels)
- Number of channels (e.g., 4, for the number of slices in the stack)
- Select "not interleaved"
- Offset =768 (3 X 256 = 768)
H) Save the new Photoshop image using the Save As... dialog box. Do not save as a Raw image. Select TIFF format, and choose a new name for the file, otherwise the simple Save command will overwrite the original NIH Image file and store the data in the Raw format.
The quality of the resulting color image will generally be much better than that provided by NIH Image , as it can utilize a full 24-bit look-up table, and does not require dithering of the image.
The Adobe Photoshop manual and tutorial will provide further guidance in modifying the images.
- Preferred Method
C) Compare Results Of NIH Image 8-Bit Merge With Photoshop 24-Bit Merge
The quality of the color image (assuming your monitor is set for 24-bit color) is generally much better in Adobe Photoshop than the optimized 8-bit image obtained in NIH Image .
D) Adjusting Color Contrast On Sections In A Stack (See Macros)
Do the Enhance Contrast operation separately on each section in the stack. Failure to do so will result in the merging of both saturated and unsaturated images.
If you are dealing with a single RGB section, you will find that Adobe Photoshop provides much better tools for this.
E) Merging A Double Labeled Pair Of Z-Series Using A Macro
- Open the two Stacks. The first stack will be assumed to be the "red" stack. The second stack will be the "green" stack.
- Close all other windows.
- Select the macro "Color Merge Two Stacks".
- Lean back and watch the fun.
The drawback of this procedure is that the resulting images are 8-bits, not 24-bits. I cannot find a way to do this procedure with Adobe Photoshop.
2. Projection Of A Z-Series And 3D Rotations
Many of the most valuable qualities of confocal Z-series are that the data can be manipulated to obtain "through views" of the stack, the stack can be resliced at various angles converting a transverse view into a sagittal or horizontal plane, it can be used to generate 3D images, stereo pairs, and other operations. Indeed, NIH Image is able to perform operations as well as much of the software provided by the manufacturers of confocal microscopes, and equivalent to many of the basic operations provided by very expensive image processing programs such as VoxelView, VolVis and VoxBlast running on a Silicon Graphics Workstation. For more elaborate operations using true Voxels, complex shading and ray-tracing in 24-bits, these latter programs are excellent choices. For the most common operations, however, NIH Image is quite adequate. Optimal performance on these tasks will be achieved using a PowerPC with sufficient memory to handle your typical data sets.
A) Stepping Through A Z-Series Using Stacks
- Open a Z-series in a Stack.
B) Animating A Z-Series
- From the Special menu, select Video for oscillating movies. This will produce smooth back-and-forth motion of the Z-series animation.
- Command to Animate a series. Number keys 1-9 control the speed of the animation.
This is an extremely useful benefit of the confocal microscope. A stack of Z-series sections are all in optimal focus. If projected onto a single plane, all objects throughout the thickness of the imaged section will now be in sharp focus and spatial relationships may be more evident.
NIHImage version 1.58 and later provides macros to perform the Z-projection. A sample macro to accomplish this operation is included in the accompanying macro files.
- Selection Of Area For Z-Projection
A) Choose an extended Z-series (e.g., more than 10-20 images) with well defined profiles of objects.
B) Run the macro "Project Z Series".
C) Use Project command
D) Set :
Slice Spacing (Pixels): Set with your slice spacing
Initial Angle (0-359): 0
Total Rotation (0-360): 0
Rotation Angle Increment: 0
Lower Transparency Bound: 0
Upper Transparency Bound: 100
Surface Opacity (0-100): 0
Surface Depth-Cueing (0-100): 100
Interior Depth-Cueing (0-100): 0
E) Select "Minimize Window Size"
F) Select "X-Axis"
G) Select "Brightest Point"
After you develop a sense of familiarity with the program, play with different values. Start with the default values. Once you are more familiar with the Thresholding tool (Magic Wand), you will be able to use that to set the range of values desired for your Z-series.
This will generate a single Z-projection of all the plates in the Stack, viewed from directly in front of the stack.
Experiment with different view angles by using different values for the "Initial Angle", while leaving "Total Rotation" at 0.
If you want to make a quick and dirty stereo pair of images from a Z-series, then the values should be something such as:
Initial Angle (0-359): 356
Total Rotation (0-360): 8
Rotation Angle Increment: 8
Select the "Y-Axis" as your "Axis of Rotation". This will cause the resulting image to rotate left to right (i.e., around the Y-axis). Choose "Brightest Point" as the "Projection Method".
This will produce a new Stack containing two images at an 8 increment. This is a common angle for stereo pairs. In order to visualize this, you can do two different procedures.
- From the Stacks menu, Animate the stack, and the image will rock back and forth around the Y-axis.
- Make a Montage using this function from the Stacks menu. You can experiment with different values for the scaling factor. The smaller the scaling factor, the easier you will find it to fuse the images. Ideally, for people with limited experience in "Free Fusing" stereo pairs of images on the screen, matching points on the screen should be less than 50 mm apart. With further experience, you will be able to fuse larger images with matching points further apart.
- Optimizing Settings For Project Function Of Z-Series
A) Make sure that "Invert Pixel Values" are not selected in the Edit/Preferences menu item. If selected, de-select this item. Then Select File/Record Preferences.QuitNIH Image in order to implement the changes in preferences. If this item is "selected", you will be easily confused by the various values displayed in the info, mapping and histogram windows.
B) There are a number of parameters in the Project item under the Stacks menu. Understanding the proper use of this item is critical to obtaining pleasing results in Z-projections and rotations.
C) The first of several items include obvious settings regarding "Slice Spacing", "Initial Angle", "Total Rotation" and "Rotation Angle Increment". These values are all obvious and pertain to the sampling interval of the resulting projection.
The remaining items on the list, "Lower" and "Upper Transparency Bounds", "Surface Opacity", "Surface-Depth Cueing", and "Interior-Depth Cueing", however, are very confusing, and cannot be easily understood without some explanation.
One of the greatest difficulties in mastering these functions is because once you have selected specific settings, obtained an image, and then try other settings, your screen will be cluttered with images, and the particular parameters used to obtain them has been forgotten.
Start with the "Lower" and "Upper Transparency Bounds" in relationship to the stereo pairs you generated a few minutes ago.
When making a simple single view Z-projection, the slice spacing is irrelevant. However, depending upon the setting of the "Lower" and "Upper Transparency Bounds", and "Interior Depth-Cueing", or the resulting image is likely to appear somewhat lacking in brightness.
"Lower Transparency Bound" must be set to 0. (Note that this value is expressed from 0-254). This value determines the cut-off point of displayed pixels. All pixel values below the setting selected will be discarded and modified to a darker value. Remember the whitest/brightest pixel on the Macintosh has a value of 0, the black pixel a value of 255. "Lower Transparency Bound" greater than 0 (e.g., n ), will modify all pixels from zero to n in your image and replace them with darker pixels. This results in dark gray to black holes in the midst of a bright object. The setting of this value is quite simple. The most effective way to appreciate the consequences of this choice is to look at histograms of the images produced with "Lower Transparency Bound" set to zero, and compare that with a histogram generated with a "Lower Transparency Bound" of perhaps 50. Make sure that your starting image has prominent objects with histogram brightness index values of 1-10 (out of a range of 0-255).
For the "Upper Transparency Bound", I suggest that you start with a value of 254. After using this for a while, play with other values.
The three following functions in the Project dialog box are expressed as values of 0-100%.
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