Dr. Kees Straatman

Image analysis Imaris

Volocity

ImageJ/Fiji

Huygens deconvolution

NIS-Elements (incl. deconvolution in RKCSB)

ScanR analysis

CellR analysis

Cell Profiler

FV1000/LAS

Image analysis Intensity measurements

Size measurements

Organelle localization

Colocalization

Cell mobility

Distance measurements

FRAP, FLIP, FRET

ImageJ workshop Thursday 21 and Friday 22 July

Intensity measurements

Absolute measurements of fluorophore concentration is almost impossible but relative measurements are possible.

Comparable and reproducible imaging conditions are needed.

Be aware Sample problems:

Uneven dye loading/labellingLeakage of dyePhoto bleachingPhoto conversionUnequal cell thickness

Microscope set up: Lamp LifeLamp alignmentObjectives usedFilters used

Camera issues:Gain Offsets, Exposure timesEfficiency of converting Photons to electrons

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:

Reduce noise

E-cad myo2a

Mean filter Can be 3x3, 5x5, 7x7

Can be 3D

50 109 130 137 156

85 109 77 86 175

187 153 63 52 119

183 130 66 82 102

53 20 114 211 168

(50+109+130+ …… +114+211+168)/25 = 113

Reduce noise

Mean filter (pixel 2 (5x5 kernel))

Median filter Can be 3x3, 5x5, 7x7

Can be 3D

50 109 130 137 156

85 109 77 86 175

187 153 63 52 119

183 130 66 82 102

53 20 114 211 168

20-50-52-53-20-50-52-53-63-66-77-82-85-86-102-109-109-114-119-130-130-137-153-156-168-175-183-187-211

Mean filter (pixel 2 (5x5 kernel)) Median filter (pixel 2 (5x5 kernel))

Reduce noise

Overview Ch 1

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2D_median

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2D_Mean

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Reduce noise

Complete Z-series

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3D-median

2DMedian

2D-MeanBackground

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.

Background correction (fluorescence)

Background correction (fluorescence)

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back -59

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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)

65535 (16 bit)

Background correction (Bright field) Take your images (Image)

Take an image without sample but with the light source on (Background)).

Subtract Background from Image

ImageBackground Background corrected

FRAP controlsLeica: 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 long-lived 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 …...

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Ch1 Region2 (Intensity Average)

Ch2 Region2 (Intensity Average)

FRAP controls

Fixed material

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, CO2, 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.