Archive for the 'leica SP2' Category

Application Letters Leica TCS SP2 and TCS SP2 AOBS/ confocal express

http://www.leica-microsystems.com/website/download.nsf?opendatabase&language=english

leica SP2 confocal microscopy

Leica AOBS SP2 confocal microscopy

instrument_photo.jpg (22275 bytes)

http://www.aecom.yu.edu/aif/instructions/aobs/index.htm

The Leica confocal is one of a few laser scanning confocal microscopes in the AIF.  The  Leica may be programmed to scan many different wavelength ranges.   The Leica AOBS uses variable spectral detection instead of traditional emission filters.  There are a few laser lines for excitation and the dyes are limited by these.  Any dye that is excited at one of these following wavelengths can be used:   405; 458; 476; 488; 514; 561; and 633 nm.   According to the sales literature, the lasers are:  diode 20 mW 405 nm; Ar 100 mW 457 nm, 488 nm, 514 nm; diode 10mW 561 nm; HeNe 10 mW 633 nm.

Objectives:
63X N.A. 1.4-0.60 Oil lBL HCX PL APO
40X N.A.  1.25-0.75 Oil CS HCX PL APO
20X N.A.  0.70 1mm corr lBL HC PL APO

Automated stage for tiling XY, XYZ or XYT  volumes with 166.6 mm of travel in Z.

Here’s a picture of the new system as installed at the AIF on November 6, 2002.

Contents:

Leica Application Notes:


Sample method section for a paper:

Images were collected with a Leica TCS SP2 AOBS confocal microscope (Mannheim, Germany) with 25X and 60X oil immersion optics. Laser lines at 488nm and 543nm for excitation of Cy2 and Cy3 were provided by an Ar laser and a HeNe laser. Detection ranges were set to eliminate crosstalk between fluorophores.


Selected Bibliography

  1. Azios, NG, Krishnamoorthy, L, Harris, M., Cubano, LA, Cammer, M, Dharmawardhane, SF.   Estrogen and Resveratrol Regulate Rac and Cdc42 Signaling to the Actin Cytoskeleton of Metastatic Breast Cancer Cells. Neoplasia. 2007 Feb; 9(2):147-158.
  2. Bhatia S, Edidin M, Almo SC, Nathenson SG. (2005) Different cell surface oligomeric states of B7-1 and B7-2: Implications for signaling. Proc Natl Acad Sci U S A. 102(43):15569-15574. PMID: 16221763
  3. Eugenin EA, Berman JW.  Gap junctions mediate human immunodeficiency virus-bystander killing in astrocytes. J Neurosci. 2007 Nov 21;27(47):12844-50. PMID: 18032656
  4. Herskovits AZ, Davies P. (2006) The regulation of tau phosphorylation by PCTAIRE 3: Implications for the pathogenesis of Alzheimer’s disease. Neurobiol Dis. 2006 Jun 9; [Epub ahead of print] PMID: 16766195
  5. Lazar-Molnar E, Almo SC, Nathenson SG.  The interchain disulfide linkage is not a prerequisite but enhances CD28 costimulatory function. Cell Immunol. 2007 Apr 27; [Epub ahead of print] PMID: 17467674
  6. Maxson, ME, Cook, E, Casadevall, A, Zaragoza, O.  The volume and hydration of the Cryptococcus neoformans polysaccharide capsule. Fungal Genetics and Biology. March 2007; 44(3)180-186
  7. Shav-Tal Y, Darzacq X, Shenoy SM, Fusco D, Janicki SM, Spector DL, Singer RH. (2004) Dynamics of Single mRNPs in Nuclei of Living Cells. Science 304(5678):1797-1800.
  8. Zhang X, Schwartz JC, Guo X, Bhatia S, Cao E, Lorenz M, Cammer M, Chen L, Zhang ZY, Edidin MA, Nathenson SG, Almo SC. (2004) Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity. 20(3):337-47.

cryptococcus.gif (139047 bytes)

tearsheet-from-Wassim-paper.gif (93937 bytes)

imageJ import export file format

2. Importing Image Files

ImageJ primarily uses TIFF as the image file format. The menu command “File/Save” will save in TIFF format. The menu command “File/Open” will open TIFF files and import a number of other common file formats (e.g. JPEG, GIF, BMP, PGM, PNG). These natively supported files can also be opened by drag-and-dropping the file on to the ImageJ toolbar. MetaMorph *.STK files can also be opened directly.

Several more file formats can be imported via ImageJ plugins (e.g. Biorad, Noran, Zeiss, Leica). When you subsequently save these files within ImageJ they will no longer be in their native format. Bear this in mind; ensure you do not overwrite original data.

There are further file formats such as PNG, PSD (Photoshop), ICO (Windows icon), PICT, which can be imported via the menu command File/Import/*.PNGJimi Reader… .

2.1 Importing Zeiss LSM files

Files acquired on the Zeiss confocal are can be opened directly (with the “Handle Extra File Types” plugin installed) via the “File/Open” menu command, or by dropping them on the ImageJ toolbar. They can also be imported via the “Zeiss LSM Import Panel” which is activated by the menu command “File/Import/*.LSM”. This plugin has the advantage of being able to access extra image information stored with the LSM file, but it is an extra mouse click.

Images are opened as 8-bit colour images with the “no-palette” pseudocolour (!) from the LSM acquisition software. Each channel is imported as a separate image/stack. Lambda stacks are therefore imported as multiple images, not a single stack. They can be converted to a stack with the menu command: “Image/Stacks/Covert Images to stack”.

Once opened, the file information can be accessed and the z/t/lambda information can be irreversibly stamped in to the images or exported to a text file.

2.2 Importing Zeiss ZVI files

ZVI files can be imported via the menu command « File/Import/*.ZVI« . The files are opened as a single stack with the different channels interleaved. The channels can be separated with the « Plugins/Stacks-Shuffling/DeInterleave » plugin.

2.3 Importing Noran SGI file

Noran movies can be opened in several ways:

File/Import/Noran movie… opens the entire movie as an image stack.
File/Import/Noran Selection…
allows you to specify a range of frames to be opened as a stack.

The Noran SGI plugins are not bundled with the ImageJ package. To receive them, please contact tonyc@uhnresearch.ca or their author, Greg Joss, so he can keep track of users. Greg Joss gjoss AT bio.mq.edu.au is in the Dept of Biology, Macquarie University, Sydney, Australia.

2.4 Importing Biorad PIC files

Biorad PIC files can be now be imported directly via the menu command “File/Open”. Experimental information, calibration, and other useful information can be accessed via Image/Show Info. Biorad PIC files can also be opened by drag-and-dropping the file on to the ImageJ toolbar. The PIC file is opened with the same LUT with which it was saved in the original acquisition software.

2.5 Importing multiple files from folder

Each time point of an experiment acquired with software such as Perkin Elmer’s UltraVIEW or Scion Image’s time lapse macro is saved by the acquisition software as a single TIF file. The experimental sequence can be imported to ImageJ via the menu command “File/Import/Image Sequence…”.

Locate the directory, click on the first image in the sequence and OK all dialogs. (You may get a couple of error messages while ImageJ tries to open any non-image files in the experimental directory.) The stack will “interleave” the multiple channels you recorded, and can be de-interleaved via “Plugins/Stacks – Shuffling/Deinterleave.

Selected images that are not the same size can be imported as individual images windows using “File/Import/Selected files to open… ” or as a stack with the “File/Import/Selected files for stack… ”. Unlike the “File/Import/Image Sequence…” function, the images need not be of the same dimensions. If memory is limited, stacks can be opened as Virtual-Stacks with most of the stack remaining on the disk until it is required “File/Import/Disk based stack” .

2.6 Importing Multi-RAW sequence from folder

To form an image, ImageJ needs to know the image dimensions, bit-depth, slice number per file and any extraneous information in the file format (offset and header size). All you really need to tell it is the image dimension in x and y. These values should be obtainable from the software in which the images were acquired. Armed with this information follow these steps:

1. File/Import/Raw…

2. Select experimental directory.

3. Typical values for the dialog box are:

Image type = 16-bit unsigned            (or 8 bit typically)

width and height as determined earlier

offset = 0, number of image = 1, gap = 0, ‘white’ is zero = off

‘Little-endian byte order’ = on, ‘open all files in folder’ = on to open all files in folder.

Non-image files will also be opened and may appear as blank images and need deleting: “Image/Stacks/Delete slice”. The stack will “interleave” the multiple channels you recorded, and can be de-interleaved via “Plugins/Stacks – Shuffling/DeInterleave.

2.7 Importing AVI and MOV files

There are two plugins which can open uncompressed AVIs and some types of MOV file.

For opening (and writing) QuickTime you need a custom installation of QuickTime to include QT for Java (see section 1.3). QuickTime movies are then opened via “File/Import/*.MOV ”.

Uncompressed AVIs can be opened via “File/Import/*.AVI ”.

2.8 Importing Scanalytics IPLab IPL files

IPLab files can be imported directly by ImageJ. File/Import/*.IPL . Allows Windows IPLab files to be opened directly with the “File/Open” menu command, or drag-and-drop.

The spatial calibration should be imported correctly from your IPLab software.

2.9 Importing Leica SP2 LEI series

Leica SP2 experiments are saved as multiple tiff files in a single folder. This can contain many different series acquired during the one experiment. Along with many tiffs, the folder also contains a text description in the *.TXT files and a Leica proprietary file *.LEI.

Double clicking, drag/dropping of « File/Open« ‘ing the *.LEI files should run the Leica TIFF Sequence plugin. Alternatively, run the menu command « File/Import/*.TXT Leica SP2 series » and select the experiment’s TXT file from the open dialog.

A second dialog will open listing the names of the series in the folder. The user can then select those that are to be opened. The appropriate spatial calibration should be read form the txt file and applied to the image. Leica ‘Snapshots’ do not have spatial calibration saved with them. The entry in the TXT file for the series is written to the ‘Notes’ for the image and can be access by the menu command « Image/Show Info…« .

Folders with large numbers of series in could potentially generate a dialog so large that some names are « off screen ». The maximum number of series names per column can be set by running the plugin with the alt-key down.

2.10 Other Import functions

These import plugins import the image data as well as meta-data.

Leica SP- Leica multi-colour images are tiffs. They can be opened as multiple files to a a single stack. Each channel can be imported separately by adding ‘c1′ or c2’ etc. as the import string. Alternatively, they can be all imported to the one stack then separated by de-interleaving them (« Plugins/Stack – shuffling/Deinterleave« ).

Olympus Fluoview – available from http://rsb.info.nih.gov/ij/plugins/ucsd.html. Not bundled with the current download.

Animated GI F – This plugin opens an animated GIF file as an RGB stack. Also opens single GIF images.

File/Import/ICS,IDS Image Cytometry Standard file format from Nico Sturman.

File/Import/*.DV *.DV files generated on DeltaVision system format from Fabrice Cordelieres

File/Import/*.ND *.ND files created with MetaMorph’s ‘Multidimensional acquisition”. From Fabrice Cordelieres

leica SP2 tiff sequence – ressources

formats:

  • tiff
  • volume tiff
  • raw
  • avi

———————————–

plug-in imageJ

This plugin opens multi-TIFF series acquired with Leica SP2 confocal.

Run the plugin; select the TXT file associated with the TIFF series and select the series to be opened. This information is then passed to the native « Import Sequence » function.

Optional ability to split the channels.

The plugin should apply the spatial calibration found in the TXT file.

ImageJ primarily uses TIFF as the image file format. The menu command “File/Save” will save in TIFF format. The menu command “File/Open” will open TIFF files and import a number of other common file formats (e.g. JPEG, GIF, BMP, PGM, PNG). These natively supported files can also be opened by drag-and-dropping the file on to the ImageJ toolbar. MetaMorph *.STK files can also be opened directly.

Several more file formats can be imported via ImageJ plugins (e.g. Biorad, Noran, Zeiss, Leica). When you subsequently save these files within ImageJ they will no longer be in their native format. Bear this in mind; ensure you do not overwrite original data.

There are further file formats such as PNG, PSD (Photoshop), ICO (Windows icon), PICT, which can be imported via the menu command File/Import/*.PNGJimi Reader…<!–[if supportFields]> XE « Jimi Reader…:Wayne Rasband and Ulf Dittmer (udittmer at mac.com) » <![endif]–> .

exemple pour Zeiss LSM:

Importing Leica SP2 LEI series

Leica SP2 experiments are saved as multiple tiff files in a single folder. This can contain many different series acquired during the one experiment. Along with many tiffs, the folder also contains a text description in the *.TXT files and a Leica proprietary file *.LEI.

Double clicking, drag/dropping of « File/Open« ‘ing the *.LEI files should run the Leica TIFF Sequence plugin. Alternatively, run the menu command « File/Import/*.TXT Leica SP2 series » and select the experiment’s TXT file from the open dialog.

A second dialog will open listing the names of the series in the folder. The user can then select those that are to be opened. The appropriate spatial calibration should be read form the txt file and applied to the image. Leica ‘Snapshots’ do not have spatial calibration saved with them. The entry in the TXT file for the series is written to the ‘Notes’ for the image and can be access by the menu command « Image/Show Info…« .

Folders with large numbers of series in could potentially generate a dialog so large that some names are « off screen ». The maximum number of series names per column can be set by running the plugin with the alt-key down.

plug-in download:

http://rsbweb.nih.gov/ij/plugins/leica-tiff.html

2006/02/16:First version
2006/03/02:Fixed error arising from series with similar names containing spaces; errors arising from images with Gray LUT.
2006/03/20:Filenames listed in multiple columns (max number of rows per column can be set by running the plugin with the alt-key down.).

—–author

Author: Tony Collins (tonyc at uhnresearch.ca)
Wright Cell Imaging Facility, Toronto, Canada

http://www.uhnresearch.ca/facilities/wcif/software/Plugins/LeicaTIFF.html

Save to plugins folder; compile and run plugin.
The compiled version is bundled with latest WCIF ImageJ bundle along with modified HandleExtraFileTypes.class (courtesy of Greg Jefferis) to allow double-clicking of the *.LEI file to open the sequence.

Code for HandleExtraFileTypes.java from Greg:

//  Leica SP confocal .lei file handler
        if (name.endsWith(".lei")) {
            int dotIndex = name.lastIndexOf(".");
            if (dotIndex>=0)
                name = name.substring(0, dotIndex);
            path = directory+name+".txt";
            File f = new File(path);
            if(!f.exists()){
                IJ.error("Cannot find the Leica information file: "+path);
                return null;
            }
            IJ.runPlugIn("Leica_TIFF_sequence", path);
            width = IMAGE_OPENED;
            return null;
        }

———————-Huygens Software reads and writes

The Huygens Software reads and writes (among other → File Formats) TIFF series with Leica style numbering if there are more channels (different wavelength), slices or frames (in a Time Series) than in a simple Numbered Tiff series.

An image of four slices and two frames is named with Leica style numbering as follows:

c_t00_z000.tif
c_t00_z001.tif
c_t00_z002.tif
c_t00_z003.tif
c_t01_z000.tif
c_t01_z001.tif
c_t01_z002.tif
c_t01_z003.tif

And an image sTCh of four slices, three frames and two channels:

sTCh_t00_z000_ch00.tif
sTCh_t00_z000_ch01.tif
sTCh_t00_z001_ch00.tif
sTCh_t00_z001_ch01.tif
sTCh_t00_z002_ch00.tif
sTCh_t00_z002_ch01.tif
sTCh_t00_z003_ch00.tif
sTCh_t00_z003_ch01.tif
sTCh_t01_z000_ch00.tif
sTCh_t01_z000_ch01.tif
sTCh_t01_z001_ch00.tif
sTCh_t01_z001_ch01.tif
sTCh_t01_z002_ch00.tif
sTCh_t01_z002_ch01.tif
sTCh_t01_z003_ch00.tif
sTCh_t01_z003_ch01.tif
sTCh_t02_z000_ch00.tif
sTCh_t02_z000_ch01.tif
sTCh_t02_z001_ch00.tif
sTCh_t02_z001_ch01.tif
sTCh_t02_z002_ch00.tif
sTCh_t02_z002_ch01.tif
sTCh_t02_z003_ch00.tif
sTCh_t02_z003_ch01.tif
------------

-----------------------quelques ressources des centres
http://www-ijpb.versailles.inra.fr/fr/lcc/fichiers/equip-sp2.htm
http://www-ijpb.versailles.inra.fr/fr/lcc/fichiers/pdf/instruction-utilisation-SP2.pdf

http://www.itg.uiuc.edu/ms/equipment/microscopes/lscm.htm
leica LCS lite

http://microscopy.unc.edu/How-to/leicasp2/default-viewing.html

http://ijm2.ijm.jussieu.fr/imagerie/fichiers

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