Homogeneous field acquisition. Zooming helps decreasing intensity range between corner (min) and center (max) of the image. {F5837353, size =full} |Zoom | 0.6 | 1 | 1.5 | 2 | | -- | -- | -- | --| -- | | Intensity variation range (max - min) | 70% | 50% | 30% | 20 % | = Consequences when measuring intensities in samples = Acquired on LSM710: 3 channels, 1AU pinhole for each channel, pixel size 210nm/px and zoom 0.5. First the cell in the corner was acquired, then the cell in the center, to show the trend goes beyond photobleaching. {F5884771, size=full} Values on the right represent rounded average intensity for the same cell at the bottom right of the field of view (top row) at the center of the field of view (bottom row) The loss of intensity is represented by dimming the color of the boxes around the measurements. == Loss == |Channel| Intensity Cell @ Edge| Intensity Cell @ Center | Loss | |---------|--------------------------|-------------------------|------| | Phalloidin| 4 | 28 | 85% | | DAPI | 46 | 143 | 68% | | Mitochondria (TOM20)| 20 | 60 | 67%|

Homogeneous field acquisition. Zooming helps decreasing intensity range between corner (min) and center (max) of the image. {F5837353, size =full} |Zoom | 0.6 | 1 | 1.5 | 2 | | -- | -- | -- | --| -- | | Intensity variation range (max - min) | 70% | 50% | 30% | 20 % | = Consequences when measuring intensities in samples = Acquired on LSM710: 3 channels, 1AU pinhole for each channel, pixel size 210nm/px and zoom 0.5. First the cell in the corner was acquired, then the cell in the center, to show the trend goes beyond photobleaching. {F5884771, size=full} Values on the right represent rounded average intensity for the same cell at the bottom right of the field of view (top row) at the center of the field of view (bottom row) The loss of intensity is represented by dimming the color of the boxes around the measurements. == Loss == |Channel| Intensity Cell @ Edge| Intensity Cell @ Center | Loss | |---------|--------------------------|-------------------------|------| | Phalloidin| 4 | 28 | 85% | | DAPI | 46 | 143 | 68% | | Mitochondria (TOM20)| 20 | 60 | 67%| = Compensating Uneven Illumination = One way to compensate is to estimate the effect of the illumination using homogeneous samples. Chroma slides, like in the experiment on top, are simple. Other methods involving saturated dye solutions yield better results, especially if the dyes are the same fluorophores as the ones used in imaging. See [[https://www.ncbi.nlm.nih.gov/pubmed/18173639|the associated article]] M. A. MODEL, J. L. BLANK, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope Journal of Microscopy, Vol. 229, Pt 1 2008, pp. 12–16 {F6068550} == Applying FlatField illumination correction at the BIOP == We developed an ImageJ Groovy script to process this data, which can be downloaded below. It works on tiles LSM, CZI and LIF files. rBIOPFFSTITCH

Homogeneous field acquisition. Zooming helps decreasing intensity range between corner (min) and center (max) of the image. {F5837353, size =full} |Zoom | 0.6 | 1 | 1.5 | 2 | | -- | -- | -- | --| -- | | Intensity variation range (max - min) | 70% | 50% | 30% | 20 % | = Consequences when measuring intensities in samples = Acquired on LSM710: 3 channels, 1AU pinhole for each channel, pixel size 210nm/px and zoom 0.5. First the cell in the corner was acquired, then the cell in the center, to show the trend goes beyond photobleaching. {F5884771, size=full} Values on the right represent rounded average intensity for the same cell at the bottom right of the field of view (top row) at the center of the field of view (bottom row) The loss of intensity is represented by dimming the color of the boxes around the measurements. == Loss == |Channel| Intensity Cell @ Edge| Intensity Cell @ Center | Loss | |---------|--------------------------|-------------------------|------| | Phalloidin| 4 | 28 | 85% | | DAPI | 46 | 143 | 68% | | Mitochondria (TOM20)| 20 | 60 | 67%| = Compensating Uneven Illumination = One way to compensate is to estimate the effect of the illumination using homogeneous samples. Chroma slides, like in the experiment on top, are simple. Other methods involving saturated dye solutions yield better results, especially if the dyes are the same fluorophores as the ones used in imaging. See [[https://www.ncbi.nlm.nih.gov/pubmed/18173639|the associated article]] M. A. MODEL, J. L. BLANK, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope Journal of Microscopy, Vol. 229, Pt 1 2008, pp. 12–16 {F6068550} == Applying FlatField illumination correction at the BIOP == We developed an ImageJ Groovy script to process this data, which can be downloaded below. It works on tiles LSM, CZI and LIF files. rBIOPFFSTITCH