A common approach to imaging specimens labeled with multiple fluorochromes is to capture individual images for each fluorochrome with a high-resolution monochrome CCD camera. These images are then pseudo-colored and layered to create a single composite image.
To achieve this, each fluorochrome is imaged using a dedicated filter cube installed in a multi-position slider or turret, allowing easy switching between matched filter sets.
For applications like co-localization analysis, which require precise overlay accuracy, the individual images must be aligned to within one pixel. Filter sets designed to meet this exacting standard are known as sub-pixel registration (SPR) filter sets.
The main factor affecting image registration is the “wedge” or slight angular variation in the dichroic beamsplitter and emission filter. To maintain SPR, beamsplitters are typically manufactured with a wedge specification of around 2 arc-seconds, while emission filters have a slightly broader tolerance of 10 arc-seconds.
Beamsplitters generally have stricter wedge requirements for two reasons: first, the angular deviation created by a substrate at a 45-degree angle of incidence (AOI) is greater than that at normal incidence; second, beamsplitters use a single substrate, which is easier to control for wedge accuracy.
Secondary factors, such as filter thickness, can also influence image alignment. Epifluorescence microscopes with standard tube-length optics—where the imaging path light beam is uncollimated—are more sensitive to thickness variations than microscopes with infinity-corrected optics, which provide a more collimated light path.
Maintaining thickness variations of 0.2 mm or less between emission filters and between beamsplitters within a set generally prevents thickness-related shifts.
Another potential source of misalignment is the flatness of the dichroic beamsplitter. Certain thin-film coating processes, like magnetron and ion-beam sputter deposition, can produce coatings with significant mechanical stress, which can warp thin substrates.
This warping effect is more pronounced when the light beam in the imaging path is uncollimated, leading to possible image shifts if the dichroic beamsplitters are produced using different deposition processes.
Ensuring that all dichroic beamsplitters in a multi-position slider or turret are fabricated by the same process helps minimize this effect, thus preserving image alignment across multiple fluorochrome channels.
