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While microscopes have been invented to investigate small details, they often need to be placed into context. This requires to see a larger region of their environment. The size of the visible area of a microscopic image is determined by the field of view (FOV) of the microscope. The FOV in a confocal instrument is influenced by the optical layout consisting of objective lenses, scan optics and scanner. In order to support a large FOV the technical challenge is to homogenously illuminate the area without the loss of resolution from center to edge.

Homogenous illumination in a confocal laser scanning microscope

In the simplest version of a laser scanning microscope we have an objective lens OL and a scan mirror MSc (Figure 1). In this design, if the scan mirror is tilted slightly, which is necessary for the scanning process, the pupil of the objective lens (shown as orange aperture) is no longer filled completely. Therefore, the edges of the image are not properly illuminated. This leads to a loss of intensity and resolution.

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).

Figure 3 X2Y scanner design to maximize field of view by introducing a second Y-galvanometer mirror. The X2Y-system ensures the pivot-plane of the scanning beam is not moving laterally. This avoids image degradation off-center that occurs, when the mirror is not placed in a plane conjugated to the back focal plane.

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