6.1 Image Handling - Tiff Files Created by LeicaTCS-NT
Tiff File Images
The LeicaTCS-NT confocal microscope system creates image files in the tiff format. Tiff stands "tagged image file format" and has its origins on Apple computers! Normally tiff file names end with the file extension .tiff but on PCs, which used to have a 3 character limit on the file name extension, tiff format file names still end with ".tif". Some of the idiosyncrasies of the tiff format are discussed below and will help in determining how best to save image data on the LeicaTCS-NT.
Standard versus Multipaged Tiff Files:
The internal structure of a tiff image file can be varied and versatile, which also leads to difficulties in handling them. One tiff capability, which the LeicaTCS-NT software makes use of, is having multiple images in a single tiff file. Such files are referred to as multipaged tiff files. Unfortunately this format is not commonly used. Most image processing software can not handle multipaged tiff files, and just display and operate on the first image and gives no indication that the subsequent images exist. This creates a complication when dealing with multiple channel scans or z-series or time-series tiff files saved on the Leica confocal, which are saved as multipaged tiff image files. This problem does not apply to single images of single channel data and files saved using "file -> save_selected".
The LeicaTCS-NT system can create either standard tiff files or multipaged tiff files depending on how many fluorescence channels are gathered and on how the image is saved:
- Standard Tiff Files:
- Single channel fluorescence - use "file -> save as" to save a single page tiff. Readable by most image software.
- Multichannel fluorescence - use "file ->save selected" to create on RGB tiff, which has overlapping red/green/blue channels and is not a multipaged tiff. Readable by most image software.
- Multipaged Tiff Files:
- multichannel fluorescence, Z-series or Time-series data saved using "file ->save as" creates a multipaged tiff file. Using "file->save selected" with z-series or time-series files always creates a multipage tiff file. Only the first channel and first slice or time image can be displayed or operated upon by most image software. Fortunately LeicaTCS-NT, WangImage and ThumbsPlus do handle multipaged tiff files.
Choosing the Tiff Format - Recommendations on how to save images:
"Save as": Using "file -> save as" saves the essential images and acquisition parameters and creates files, which can be reopened by the LeicaTCS-NT software and processed to add calibration bars, and in the case of z-series data analyzed in 3-D. Information about the pinhole size, objective used, filters used, laser, PMT voltage and offset, zoom, date, time and more are embedded as text in the tiff file. This information may be displayed with Adobe PhotoShop using "file->info" and can also be extracted as a text format .inf file using tiffsplit.exe or tiffsplit4.exe, which are PC command line prompt programs. Text information, once extracted, can be viewed with any text editor such as Notepad and Word.
"Save Selected": Using "file -> save selected" to save an image in RGB tiff format does not save any of the acquisition parameters, including lens and size calibration information. Such files can not be reopened in LeicaTCS-NT. However they do contain red/green/blue information and can be opened in PhotoShop and most image processing software easily. With files saved this way using LeicaTCS-NT the first channel is displayed in green, the second in red and third in blue. This assignment of colors can not be changed in LeicaTCS-NT but can, later, be in PhotoShop. For example, if an image of a single Texas Red labeled sample is saved using "file -> save selected" the image will be saved in the green channel, with the red and blue channels being black (blank). Another limitation with using "save->selected" is that four channel or more data can not be saved.
Choosing one format over the other involves compromising ease of opening the image and seeing all channels over loosing the essential image data, acquisition parameters and ability to reopen the image data in LeicaTCS-NT. Some users save files initially using "files -> save as" and then save the data, possibly at a later time, using "file -> save selected". If in doubt, saving the data using "file -> save as" saves the essential information and an RGB image can be generated later using tiffsplit3.exe with the -e command line option.
Working With Multipaged Tiff Files:
Converting a multipage tiff file into individual images allows all the images to be viewed and operated upon as a series of individual standard tiff files or bitmap files. The PC windows command prompt program tiffsplit4.exe can perform this process rapidly. The generated individual images can be manually overlayed in red/green/blue and processed ad lib. in Photoshop or processed by other general purpose image programs. Tiffsplit4.exe can also be used to generate extended focus and/or maximum projection images and red-green-blue overlays.
Last updated 10-24-2006
|© 1998-2003 Michael Chua, Cell & Molecular Physiology, UNC. All rights reserved.|
|To Confocal Homepage||To Operating the confocal||To Booking the confocal||Go back a page||Comments: Dr. Michael Chua|