The Sun has a surface temperature of nearly 6000 Kelvin (where the Kelvin temperature scale is the same as the familiar Centigrade scale except that the zero degrees C is about 273 degrees Kelvin). Its radiation peaks in the visible part of the spectrum at wavelengths of about half a micron, as shown by the yellow-green line in the graph above.

Just for the record (maybe other people will find this post interesting): CIDRE and BaSiC are giving very similar results for my data (just different treatment of the offset due to the particular CIDRE settings that I used): image2384×1478 1.06 MB

In fact, I do have more images. My naive approach was just to compute a median projection, thereby ideally removing the objects and only being left with the flat-field. This of course does not work very robustly if there are many objects in every image. Thus, thanks for pointing out CIDRE I will check it out!

there are a few documented methods. From these, I found the FFT bandpass filter to work nice and fast, but the result using your image posted above is not necessarily of better quality compared to what you already have.

Clouds of interstellar gas and dust that form stars are typically at temperatures of about 50 K (that’s about –220oC). They glow at far infrared wavelengths and are brightest at about 100 microns (red line in the graph above).  And the universe itself is filled with radiation corresponding to a temperature of just less than 3 K – very cold indeed – with peak emission in the millimetre wavelength range (blue line in the graph above).

The electromagnetic spectrum spans a wide range of wavelengths from very short wavelength and highly energetic gamma rays to very long wavelength and low-energy radio waves. The visible part of the spectrum is only a small portion. Infrared light is the same as the light that we can see except that the wavelength is longer and outside the range that our eyes can sense.

Infrared radiation was discovered by William Herschel in 1800. He was studying the heating effect of different colours of light by using a prism to produce a spectrum of colours and thermometers to measure their heating effect. He noticed that the heating effect got stronger as he went from the blue end of the spectrum to the red. In a moment of inspiration, he moved the thermometer beyond the visible red end and found that the heating effect was even greater.

Your new code (right?): https://github.com/saalfeldlab/stitching-spark/blob/42e01a6fe2751980102922513df309ba25b00f70/src/main/java/org/janelia/stitching/experimental/IlluminationCorrectionSliceParallel.java#L1452

If you’re only after a band pass, you could also try Plugins > Integral Image Filters > Normalize Local Contrast which is even faster than the FFT bandpass. Try various block-sizes with preview checked

Could you please test it? Update site: EMBL-CBA And then at the very bottom of Plugins > Restoration > Illumination > BaSiC (I am not sure it will work immediately because I had some strange dependency issues…)

the BioVoxxel Pseudoflatfield correction with a radius around 10 also doesn´t look too bad and it´s super easy to use. There are some artifacts in the lower left corner which I guess are due to the jpeg format of the sample image.

I was wondering whether there is an ImageJ workflow already for correcting brightfield illumination? I just tried an own attempt (median filter based) and I guess that would be OK but I also do not want to reinvent the wheel…

Infrared radiation (IR), also known as thermal radiation, is that band in the electromagnetic radiation spectrum with wavelengths above red visible light ...

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Clearly, depending on what it is that we want to observe, we need to look in different parts of the spectrum, and no one part will tell us everything. The Earth’s atmosphere transmits well in the visible and radio regions, but it blocks out everything from gamma rays to ultraviolet and most of the infrared.  So to study the Universe at those wavelengths we need to launch space-borne observatories.

It is interesting that the basic technique used by Herschel to discover infrared radiation is still used in modern instruments today, including instruments on board the Herschel satellite – the only real difference is a factor a billion or so in sensitivity.

Köhlerillumination

unfortunately you didn’t use the provided sample image for your experiments. Furthermore, the provided sample image is JPG-compressed …

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to me it seems you need a flat-field correction. [https://en.wikipedia.org/wiki/Flat-field_correction] This means, you should divide by the correction, not subtract it (also the Fourier filters do a subtraction, not a division). Otherwise the features in the dark (insufficiently illuminated) parts of the image get too weak. You could try, e.g., the following macro:

Itransmitted = I0all angles = ½I0. Problem: three polarizers A beam of unpolarized light of intensity I0 passes through a series of ideal polarizing ...

Criticalillumination

We humans, slightly warmer than room temperature, glow in the mid infrared and we’re brightest at about 10 microns wavelength (black line in the graph).   These days we are all familiar with infrared imaging, which allows us to see in the dark using electronic detectors that record infrared light emitted by warm objects such as people.  The pictures below show SPIRE team member Prof. Peter Ade in visible light (wavelength about 0.5 micron) and infrared light (about 10 microns).

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Widefield microscope

As an alternative to CIDRE there is also BaSiC. There is an update site available for it and you can find a bit of demo/documentation here. If I remember correctly it does work on stacks and was quite fast.

The short answer is that the sun has an illuminance of about 100k lux (lumens per square meter) on a perpendicular surface at sea level.

The whole region with wavelengths ranging from 1 micron to 1 mm is loosely called the “infrared”, but astronomers tend to break this up into sub-regions:  the “near infrared” (from 1 to 5 microns); the “mid infrared” (5 to 30 microns), the “far infrared” (from 30 to 300 microns) and the “submillimetre” (from 300 microns to 1 mm).  The exact boundaries are somewhat arbitrary, and the exact definitions can vary.

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As an alternative to CIDRE there is also BaSiC . There is an update site available for it and you can find a bit of demo/documentation here . If I remember correctly it does work on stacks and was quite fast.

If you have more images than CDRE can digest or you’re interested in 3D flatfields (I gues you’re not ;)), you can use our not yet published scale up of the CIDRE approach https://github.com/saalfeldlab/stitching-spark.

What magnification? If overall magnification is small, a normal Microscope may be sub-optimum and special illumination may be needed.

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If you have many images from the same microscope under similar settings, you can estimate the flatfield with CIDRE https://github.com/smithk/cidre.

I have now a similar dataset (a time-laps of 60 wells with 6x6 tiles per well in Brightfield) and it would be great if you would have code that I could use Thanks!

In the case in question they zero out harmonic spatial waves of large periods. Division is the way to go but improved image acquisition is superior!

PS: Here are again results obtained from the supplied sample image. Profiles are on the image diagonal from left bottom to right top.

I have now a similar dataset (a time-laps of 60 wells with 6x6 tiles per well in Brightfield) and it would be great if you would have code that I could use Thanks!

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In fact all objects glow (emit electromagnetic radiation), and they do this in the part of the electromagnetic spectrum that depends on their temperature. The diagram below shows how bright objects of different temperatures appear at difference wavelengths.

Darkfieldmicroscopy

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CIDRE works really well! However I would need to write an other UI that enables batch-processing of many experiments and also accepts Tiff stacks as input. I guess it would make to use your new code as a starting point, rather than the original one?