The main advantage of two-photon microscopy is the ability to image deeper into scattering samples, such as tissue and live specimens. One way that two-photon objectives assist deep imaging is by providing extended working distances compared to lenses for other imaging modalities. However, when imaging deep into complex tissues, it is often necessary to focus through many layers of cells or tissue with variable optical properties. These heterogeneous layers can induce spherical and chromatic optical aberrations that can significantly degrade image quality. To tackle this issue, some lenses feature correction collars (see figure 1) that can be adjusted so that aberrations are minimized.
© Joel Glover
In confocal microscopy, correction collars are sometimes used for correcting minor deviations in coverslip and media thickness. The two-photon equivalent, however, typically features a larger adjustment range to account for the greater sample thickness imaged with two-photon systems. When adjusting a correction collar, optical elements in the lens move up and down to tweak the focus. A long working distance two-photon lens will have larger optics (see previous blog post) and also require moving them over longer distances. This large internal travel range requires a complex adjustment mechanism which increases the size, weight, and cost of such an objective.
When imaging deep into the tissue, however, a correctly adjusted collar can greatly improve focus and overall image quality, often making the extra size and cost worthwhile. Figure 2 illustrates a chase where the correction collar is used to minimize the aberrations in the image- this will be seen as a crisper and less hazy image.
Unless the collar is motorized, the collar is adjusted manually while the sample is on the stage. This requires a gentle touch to rotate the collar without disturbing the sample. For an upright microscope, the adjustment is straightforward, but accessing the collar can be challenging on an inverted microscope. Adjusting the collar is an iterative process:
1. First, focus on the sample and capture an image.
2. Next make a small adjustment to the collar, refocus, and acquire a new image.
3. Compare to the images acquired before and after the collar adjustment.
4. If the image quality improves, keep rotating the collar slightly in the same direction while refocusing after each adjustment.
5. Repeat until you observe the image quality degrade and move back to the optimized “sweet spot” for the collar setting and proceed with imaging.
6. This process must be repeated if you move to a new region of interest within your sample, especially if you move to a deeper or shallower focal plane, but generally, only small tweaks to the collar will be required for small lateral (x,y) movements.
The adjustment of the correction collar for optimal image quality demands patience and practice. While the process may seem intimidating when the collar is set optimally for a given position the tightness of the laser focus is improved which drastically impacts signal strength. As mentioned in our previous post, the efficiency of two-photon fluorescence and second harmonic depends on a tight focus: Not only will your image be sharper due to the reduction in laser spot size, but the concentration of photons into a smaller volume also increases signal production resulting in a brighter image. This can allow you to image much deeper into the sample, possibly allowing access to structures that would otherwise literally be beyond the reach of the uncorrected lens.