Some aspects of the image quality for the Radiance2000 MP

Introduction

The Radiance 2000 MP LSM uses a static set of lenses and mirrors to bring the Mai Tai laser from the Beam Conditioning Unit into the scan head. Currently, the Mai Tai is only aligned to the 488 line of the Kr/Ar laser using a 60× 1.40 NA oil immersion objective at 2.6 zoom. In addition, there are artifacts due to the chromatic and spherical aberrations of the optical system. Using a preparation of multiple fluorescent microspheres, we can demonstrate the co-alignment or deviation from such for these 2 lasers.

Preparation

We mounted the 500 nm Tetraspeck microspheres (Molecular Probe, T-7281) in glycerol with a #1.5 coverslip sealed with fingernail polish. The XYZ stacks were acquired using a 60× 1.40 NA oil immersion objective at 2.6 zoom, 1024×1024 pixels (78×78 µm), 500 Hz, and an optimized iris setting in a sequential manner for the blue, green, and red channels. The XYZ position of 5 microspheres were taken from the 4 corner regions and the center using ImageJ and the sync measurement 3D plugin.

Table 1: Acquisition parameter for the 500 nm microspheres (filter: Kalman 2)
laser	power	iris	gain
maitai (blue)	21.0	12.0	25
Kr/Ar 488 (green)	16.6	1.5	25
Kr/Ar 568 (red)	3.8	1.8	25

Table 2: Acquisition parameter for the 4 µm microspheres
laser	power	iris	gain	offset
Kr/Ar 488 (green)	7.2	1.5	15	-1.9

Result

Table 3: Average image shift* between channels ± standard deviation (µm), n=5
	X	Y	Z
blue vs green	0.2±0.07	0.4±0.26	0.9±0.06
blue vs red	0.1±0.14	0.2±0.21	0.9±0.06
green vs red	0.1±0.06	0.2±0.05	0.1±0.02

Note: *The theoretical resolution of the 60× 1.40 NA objective is 0.16 µm in the XY plane and 0.54 µm in the Z plane. Zeiss did a quality check during the preventive maintenance on 1/19/2006 and found the Z-resolution for the 60× oil immersion objective was 0.38 µm, and the power output at the turret for 488, 568, and 637 nm lines were 0.34, 0.55, and 0.30 mW, respectively.

Figure 1: A 3-channel merge projection of 500 nm tetraspeck microsphere at 60x, frame size is 78×78 µm.

500 nm tetraspeck microsphere at 60x, blue-green-red merged projection

Figure 2: Projections of a microsphere in the green channel from 3 orientations (clockwise:front, left, bottom) of the XYZ stack. The axial resolution (and spherical aberration) is better demonstrated using a 4 µm microsphere instead of the 500 nm ones.

Projections of a 4 µm microsphere from 3 orientations of the XYZ stack