Fluorescence Images
Figure 1: In WF microscopy (left) the captured image contains light from the focus area as well as significant background signal from out-of-focus areas above and below the focal plane. Confocal techniques (right) can employ various means to avoid this background contribution and thereby generate an image with better contrast.
sample is excluded during image acquisition; the maximum number of photons are collected and later reassigned to their real places of origin. Tis reveals many structures in the result- ing 3D volume that were not visible before and so were lost in the out-of-focus light. Deconvolution microscopy is therefore also known as a signal-inclusive method. Notably, it might take a bit longer to acquire a single opti-
cal section with SIM or CLSM than it does with a WF image. For example, SIM requires at least three images per section to be acquired, and a single point scanning confocal laser scanning approach requires point-by-point scanning, usu- ally making it slower than simply snapping a camera image. It is important to note that the deconvolution of WF
images must not be regarded as a replacement for an optical sectioning system. Instead, any micrograph collected with a light microscope will benefit from deconvolution, including optical sections (for example, from laser scanning confocals, spinning disk confocals, or structured illumination images).
So long as the right mathematical model is used, deconvolu- tion will always help restore the photon signal to the exact place where it belongs (Figure 4), and this will increase con- trast and resolution. Of course, one side effect in the case of WF images is that the out-of-focus blur disappears. When deciding which method to choose, there are many aspects to consider if a 3D volume is ultimately needed. Tese range from the desired time and spatial resolution to spectral flex- ibility, penetration depth, and various other factors (Table 1).
Sharp Images in 2D What do you do when all you need is a crisp 2D image;
whether in the form of a time series or as a large tiled image of a sample? As illustrated in Figure 2, very flat objects usu- ally continue to reveal their biology quite well with a WF image. Still, there will be a certain background fluorescence that negatively influences the aesthetic aspects and contrast. Te human eye likes crisp details along with sharp contrast and colors. A first and really quite simple way to make a fluo- rescence image look more appealing and reveal some addi- tional details is to adjust the display curve in the soſtware. So long as an image is not exported as a TIF or JPG image, this will not in itself change the image data but will only influence the look of the image on the screen. As out-of-focus haze in WF images is typically less intense than the in-focus struc- tures, this approach can work to a certain extent. However, it fails where in- and out-of-focus structures are lying on top of each other, and it is also quite an arbitrary procedure that depends ultimately on the operator’s preferences. Terefore, more powerful methods are usually called for. Over the past few decades, numerous processing meth- ods have been developed for delivering crisper, sharper
Figure 2: Figure 2A shows a WF image of cultured flat epithelial cells (actin filaments in green, mitochondria in purple) of a rat kidney. This comparison depicts the challenges posed to a WF instrument by a thick and scattering sample like the rat kidney (2B).
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