Image analysis. Intensity measurements Size measurements Organelle localization Colocalization Cell mobility Distance measurements FRAP, FLIP, FRET
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1 Dr. Kees Straatman
2 Image analysis Imaris Volocity ImageJ/Fiji Huygens deconvolution NIS-Elements (incl. deconvolution in RKCSB) ScanR analysis CellR analysis Cell Profiler FV1000/LAS
3 Image analysis Intensity measurements Size measurements Organelle localization Colocalization Cell mobility Distance measurements FRAP, FLIP, FRET ImageJ workshop Thursday 21 and Friday 22 July
4 Intensity measurements Absolute measurements of fluorophore concentration is almost impossible but relative measurements are possible. Comparable and reproducible imaging conditions are needed.
5 Be aware Sample problems: Uneven dye loading/labelling Leakage of dye Photo bleaching Photo conversion Unequal cell thickness Microscope set up: Lamp Life Lamp alignment Objectives used Filters used Camera issues: Gain Offsets, Exposure times Efficiency of converting Photons to electrons
6 Noise 3 types of noise in imaging system: Dark Current: noise from heat and cosmic noise (exposure dependent) Read Noise: noise of reading the signal (fixed) Photon shot noise: detection of photons is a statistical process depending on the number of photons counted. This uncertainty has a Poisson distribution and is:
7 Reduce noise E-cad myo2a
8 Mean filter Can be 3x3, 5x5, 7x7 Can be 3D ( )/25 =
9 Reduce noise Mean filter (pixel 2 (5x5 kernel))
10 Median filter Can be 3x3, 5x5, 7x7 Can be 3D
11 Reduce noise Mean filter (pixel 2 (5x5 kernel)) Median filter (pixel 2 (5x5 kernel))
12 Overview Ch Raw Decon Decon Nikon 3D_median 2D_median Raw Decon Decon Nikon 3D_median 2D_median 2D_Mean
13 Overview Ch Raw 100 Raw Decon Decon Nikon 3D_median Decon Decon Nikon 3D_median D_median 40 2D_median
14 Overview Ch Raw Decon Decon Nikon 3D_median 2D_median Raw Decon Decon Nikon 3D_median 2D_median
15 Reduce noise
16 Complete Z-series Background raw decon 3D-median 2DMedian 2D-Mean
17 Background correction (fluorescence) Rolling ball background subtraction (ImageJ/scanR). The subtraction is depending on surrounding. The radius of the ball should be at least the size of the largest object that is not part of the background. ROI in the same image. Don t make the ROI too small. The subtraction is a constant. You can choose what is background. Set an offset. Sliders in software.
18 Background correction (fluorescence) raw rollingbal back -59 decon
19 Background correction (fluorescence) Take an image with the light source blocked (Dark). Take an image without sample but with the light source on (Light). Take your images (Image) Signal = (Image Dark)/(Light Dark) * bit factor bit factor = 255 (8 bit) 4095 (12 bit) (16 bit)
20 Background correction (Bright field) Take your images (Image) Take an image without sample but with the light source on (Background)). Subtract Background from Image Background Image Background corrected
21 FRAP controls Leica: Widefield Super-resolution with Ground State Depletion Leica SR GSD Maximum resolution down to 20 nm Standard fluorochromes can be used no need to change your protocols Ground state depletion (GSD) can be achieved by using high power lasers to transfer fluorophores into longlived dark states a non-fluorescent molecule state. Stochastically, as soon as single fluorophores return from the dark state, new bursts of emitted photons are recorded...
22 FRAP controls Fixed material Ch1 Region2 (Intensity Average) Ch2 Region2 (Intensity Average) 0
23 Materials and Methods Make, model and type of microscope used Type, magnification, and numerical aperture of the objective lens used Light source used Fluorochromes used Peak transmission and bandwidth of filter sets used (or the manufacturer part number) Camera make and model Enviromental conditions during live cell imaging: temperature, CO 2, buffer, chamber, media, etc. Other motorized components used Acquisition software Name and version of image processing and analysis software used Details about image enhancement during image processing and analysis: type of filters, background subtraction, gamma adjustments, type of deconvolution, 3D reconstructions, surface or volume rendering, etc.
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