CyTRAK Orange™ imaging with eGFP (example)
CyTRAK Orange™ allows the direct segmentation of HCS images in LIVE cells
CyTRAK Orange™ has excellent compatibility with eGFP:
- Single step addition of probe to get labelling of nucleus (and cytoplasm)
- Simultaneous two channel emission, single excitation wavelength (488nm) is optimal for both probes
- Only one laser required
- Only single well-visit required to capture dual channel image
- Single illumination source (one laser line) avoiding instrument alignment
- Chromatic error or co-alignment of signal is not dependent on the excitation
- Pixel-pixel registration of channels is excitation independent
- Simpler instrument set-up enhancing the cross-compatibility of CyTRAK Orange™
The following workflow is an example of the image processing steps used with CyTRAK Orange™ and eGFP.
EXAMPLE
Live cells labeled with CyTRAK Orange™ (20 μM) for 20 minutes at 37ºC, no wash. Images captured using confocal microscopy 488 nm simultaneously (or can be termed as single excitation); (i) CyTRAK Orange™ channel: 598/40 nm (ii) GFP-cyclin B1 channel: 522/35 nm
| CyTRAK Orange: Red Channel |
CyTRAK Orange: Image processing 1 Apply median filter (3x3) array to remove punctate staining leaving nuclear compartment | eGFP, Green Chanel: Image processing 1 Apply median filter (3x3) array to remove noise in image |
CyTRAK Orange: Image processing 2 Apply erode filter (5x3) array to remove punctate staining leaving nuclear compartment | Recombine filtered images |
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