In the last post, we reviewed how scattering and absorption attenuate the intensity of the laser used to excite fluorescence and reduce the amount of emission signal that can make it to the detector(s). Tissue is very complex, and fluorescence from different dyes, as well as the laser, will scatter and absorb differently in different samples. These effects become more pronounced as we try to image deeper into a biological specimen; one way to reduce the effects of scattering and absorption is to increase the power of the laser as you image deeper into the sample.
In practice, the increase in power with depth is determined experimentally by focusing to various depths in the sample, adjusting the laser power to maintain (approximately) consistent sample brightness as a function of focal depth. Note that the brightness of different colour fluorophores will be different at increasing depths. This is because scattering and absorption is wavelength dependent, so for instance a blue emitting fluorophore will undergo more absorption and scattering than a red fluorophore at the same depth.
In practice, you carry out power balancing by monitoring the signal intensity at one or more pixels (or a region of interest) directly or by observing the histogram, and recording the laser power needed to achieve consistent emission signal intensity for each z position. Keep careful track of your laser power while making these adjustments, as if you inadvertently focus to a shallower depth with a high power you may damage your sample. If your sample is stained with multiple fluorophores you may have to pick one or two colours to optimise on as each colour will experience different scattering so compensating for all colours equally will often be unfeasible.
Power compensation can be done manually but for convenience, some microscope systems are equipped for partially automated power adjustment of the laser when acquiring z-stacks. A typical power compensation curve is shown below, and the need for increased laser power as a function of depth is evident as the required power begins to increase rapidly as the focusing depth increases.
Figure 1: Adjusted laser power percentage with depth to maintain consistent signal in a four-colour stained liver tissue sample. See figure panel for corresponding images.
Some automated routines in acquisition software will automatically interpolate powers between two z positions. This simplifies the approach as fewer readings can be taken at representative depths, as an interpolation routine estimates the intervening values when you begin to actually acquire your z stack.
If your system does not have automated power compensation, you have to adjust the power manually for each z position. This can quickly become onerous as it means you acquire your z-stack one image at a time. Again we remind you to watch out for power settings that can damage your sample, particularly near the surface.
The figure panel shows a four-colour image of liver tissue (stained using immunofluorescence targeting the nuclei (blue), F4/80 (green), vimentin (red), and vasculature (magenta). Note here images were acquired at low spatial resolution and only the nuclei and vasculature are clearly defined.
On the right column, power and detector settings were adjusted for optimal dynamic range for each channel, and the pixel intensity distributions were kept constant as a z-stack was acquired 40 um into the tissue. On the left column, the same channel gains were used, but the laser power was adjusted in an attempt to keep the signal brightness roughly consistent for the blue, green and red channels.
| Depth | No power compensation | Power compensation |
| 5um |  |
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| 30um |  |
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Panel 1. Liver tissue stained with stained using immunofluorescence targeting the nuclei (blue using Hoechst), F4/80 (green using AF488), vimentin (red using DyLight550), and vasculature (shown as magenta using a far-red DyLight649). Credit: Liver tissue slides provided by OptiSlides, a division of Luxidea, Inc.
As shown, the far red dye (shown in magenta in the figure) maintains its brightness fairly well even in absence of compensation. This is because the far-red wavelengths are less affected by scattering and absorption. In the power-compensated stack on the right, where laser power is increased with imaging depth, the blue, red and green fluorescence signal are fairly consistent as imaging depth increases, but the far red dye actually gets a bit brighter as its emission is more efficient at escaping the tissue and the increased laser power yields higher signal.
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