Figure 1 In addition to the objective, confocal systems usually also use a tube lens TL positioned at the distance fTL to the backfocal plane of the objective and a scan lens ScL positioned at the distance fScL to the backfocal plane of the tube lens to achieve diffraction limited illumination (Figure 2). If the scan mirror is then positioned at distancefScL to the scan lens we speak of a “4 f design” since we are covering four focal lengths f between the objective and the scan mirror. Then, the pupil is always filled completely at all scan mirror positions, resulting in homogenous illumination of the whole field.
Figure 2: Avoiding vignetting or peripheral resolution loss by using a 4f scanner design. Scan lens (ScL) and tuble lens (TL) are positioned so that they conjugate the scan mirror (MSc) to the objective lens’s (OL) back pupil.
In a confocal system with two scan mirrors, one for the x and one for the y dimension, a compromise has to be made when positioning the scan mirrors. Either one scan mirror, or both, are moved out of the conjugated plane. This limits the usable field as a loss of intensity and resolution occurs towards the edges of the field.
Unlike all other confocal scanners, the Leica X2Y scanner built into the Leica TCS SP8 uses three scan mirrors, one for the x and two for the y dimension. The second Y mirror of the X2Y scanner guarantees that the pivot point of the light beam in the OL’s back pupil is always centered. By this arrangement, the optimal 4f design can be maintained to avoid vignetting and loss of resolution (Figure 3).
XY scanners are limited in the field size, but with the Leica X2Y scanning system the field number is increased up to 22. At 10x magnification this is equivalent to a square of 1,5 x 1,5 mm2. A larger field of view allows a larger specimen to be entirely fitted into one single image or to simply add more context to a complex topology such as in a tissue sample (Figure 4).
Figure 4: Platynereis dumerilii, 2 month. Blue: nuclei, DAPI. Green: tubulin, FITC. Grey: phalloidin, Rhodamin. Red: serotonin, Cy5. The whole organism fits into a single image (FOV). 3D maximum projection of image taken with a 10x objective fully zoomed out at 5000 x 5000 scan format. Sample: courtesy of Dr. Antje Fischer and Dr. Detlef Arendt, Heidelberg, Germany