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imagestreamx [2018/06/08 13:00]
flowcytometry
imagestreamx [2021/03/31 21:08] (current)
flowcytometry
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-<a href="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=imagestreamx_wiki.jpg"><​img src="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=imagestreamx_wiki.jpg" width=300></​a>​+<a href="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=imagestreamx_wiki.png"><​img src="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=imagestreamx_wiki.png" width=300></​a>​
 <td style="​border:​0px solid white;">​ <p style="​line-height:​1.8">​ <td style="​border:​0px solid white;">​ <p style="​line-height:​1.8">​
 <​b>​Location</​b>:​ <a href="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=flow_cytometry_map.jpg">​Room P2-A-49</​a>​ (<img src="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=telephone_icon.gif"​ width=15>​ 47224) <​br> ​ <​b>​Location</​b>:​ <a href="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=flow_cytometry_map.jpg">​Room P2-A-49</​a>​ (<img src="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=telephone_icon.gif"​ width=15>​ 47224) <​br> ​
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-&rarr; <a href="​https://​imm.medicina.ulisboa.pt/​intranet/booking/145"><​img src="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=date.png"> ​ Amnis ImageStreamX Booking</​a>​+&rarr; <a href="​https://​my.agendo.science/calendar/?​id=MTBjYWxlbmRhcnM="><​img src="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=date.png"> ​ Amnis ImageStreamX Booking</​a>​
  <​br> ​  <​br> ​
 &rarr; <a href="/​facility/​flowcytometry/​doku.php?​id=amnis_imagestreamx_usage"><​img src="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=chart_line.png"> ​ Amnis ImageStreamX Usage Statistics</​a>​ &rarr; <a href="/​facility/​flowcytometry/​doku.php?​id=amnis_imagestreamx_usage"><​img src="/​facility/​flowcytometry/​lib/​exe/​fetch.php?​media=chart_line.png"> ​ Amnis ImageStreamX Usage Statistics</​a>​
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 ===== System overview ===== ===== System overview =====
  
-{{  :cell_analyzers.png?​0x300|}} The BD LSRFortessa 1 //cell analyzer// ​is a benchtop ​digital ​flow cytometer equipped with a fixed-alignment flow cell that is gel-coupled ​to the collection optics. It has three spatially separated ​lasers - 405 nm, 488 nm and 640 nm. Its primary function is to characterize complex populations of cellsIndividual cells pass single file through ​streamwhich is interrogated ​by the three lasers with the emitted ​light being detected ​by photomultiplier ​(PMTtubes. The system electronics calculates ​the time it takes single cell to pass through each laser intercept ​and assigns the signal ​of each PMT to the appropriate cell. Besides fluorescence, the physical properties of the cell are also analyzed ​with side scatter (SSC), which reflects ​the internal complexity ​of a cell and forward scatter (FSC), which is roughly proportional to cell size. The sample flow rate can be set to lowmedium and high values, which correspond approximately ​to 12, 35 and 60 μl/minute, respectively. If your cells are stained with PerCP-Cy5.5 or you need high-throughput samplercheck the [[bd_lsr_fortessa_2|BD LSRFortessa 2]] instead. If you need multicolor ​analysis of up to 16 parameterscheck the [[bd_lsr_fortessa_x20|BD LSRFortessa X-20]].+{{  :how_the_imagestream_works.png?​0x300|}} The Amnis ImageStreamX ​is a benchtop ​multispectral //​imaging ​flow cytometer// equipped with two CCD cameras ​that can acquire up to 12 images simultaneously of each cell or object in brightfield,​ scatter and multiple fluorescent channels at rates up to 5000 objects/sec. It has five lasers - 375 nm, 488 nm, 561 nm, 642 nm and 785 nm (dedicated ​to side scatter)Cells in suspension are hydrodynamically focused in flow cellwhere they are illuminated ​by a brightfield ​light source and by lasers. High numerical aperture ​(NAobjective lenses collect fluorescence emission, as well as scattered and transmitted light from the cells which then intersect ​spectral decomposition element that focus different spectral bands on different physical locations of two CCD cameras. By collecting large numbers of digital images per sample ​and providing numerical representation ​of image-based features, the ImageStreamX combines single ​cell standard microscopy information ​with the statistical significance ​of large sample ​sizes in standard ​flow cytometry. While you can use it to measure fluorescence intensity as in conventional flow cytometersthe best applications for the ImageStreamX take advantage of its imaging abilities ​to locate ​and quantify the distribution of signals within ​cells or even between cells in cell conjugatesThe system also has dedicated software application used for spectral compensationimage analysis and statistical ​analysis of acquired images (IDEAS) with predefined wizards for apoptosiscell cycle, co-localization,​ internalization,​ shape change, spot counting and others.
  
-  * Multicolor analysis of up to 10 parameters +  * **12 imaging channels** (2 brightfield channels + 10 channels for fluorescence detection) 
-  * Three lasers with violet ​(405 nm), blue (488 nm) and red (640 nm)+  * **5 lasers** with UV (375 nm), blue (488 nm), yellow-green (561 nm), red (642 nm) and far red (785 nm) for side scatter 
 +  * **Multimag**:​ 20x, 40x and 60x magnification 
 +  * **Extended depth of field**: projects all structures within a cell into a single plane of focus 
 + 
 +{{  ::​img-12channels.jpg ​ |}}
  
 ===== Configuration ===== ===== Configuration =====
  
-{{:bd_lsrfortessa_1_configuration_wiki.png?800|}}+{{::​imagestreamx_fluorochrome_chart.png?700|}} 
 + 
 +===== Objectives Information ​ ===== 
 + 
 +^  Magnification ​ ^  20x  ^  40x  ^  60x  ^ 
 +^  Numerical Aperture ​ |  0.50  |  0.75  |  0.90  | 
 +^  Field of View (μm)  |  120  |  60  |  40  | 
 +^  Pixel Size (μm)  |  1.0  |  0.5  |  0.33  | 
 +^  Depth of Field (μm)  |  8.0  |  4.0  |  2.5  | 
 + 
 +===== Image Acquisition Performance =====  
 + 
 +{{::​high_speed_imaging.png?​600|}} 
 + 
 +===== Additional information ===== 
 + 
 +  * **[[prepare_samples|How to prepare samples for Flow Cytometry]]** 
 +  * {{::​imagestreamx_sample_preparation_guide.pdf| Sample Preparation Guide}}. **Note**: Samples are run using 1.5 ml microfuge tubes (Sarstedt ref. 72706) {{::​microfuge_tube.jpg?​60|}} 
 +  * {{::​imagestreamx_brochure.pdf| ImageStreamX Brochure}}
  
 ===== Booking Rules ===== ===== Booking Rules =====
imagestreamx.1528455649.txt.gz · Last modified: 2018/06/08 13:00 by flowcytometry