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The beam homogenizer uses two square micro-lens arrays that consist of a periodic structure of micro-lenses, with pitch PMLA (Fig. 2A). The first array (MLA1) focuses the beam on the second array (MLA2), resulting in multiple point sources. The light from all these separate sources is collected by the microscope objective BTK inhibitor and overlaid in the focal plane where they form a quadratic illumination profile with a homogeneous intensity (flat top profile) with a size given by (18): When all components are positioned according to the above described distances, the resulting illumination profile is approximately square with a FWHM of 161 ��m. However, the actual width of the illumination profile, with a CV of <5%, is then about 130 ��m. This results in the need to scan 21 ��strips�� to cover an entire cartridge. To reduce this number and still <a href=""> be able to see the edges of the illumination profile for focus determination purposes, a12 may be slightly increased. To avoid large aspect ratios of the images and reduce the number of events that are located at the border of an image, the images are stitched together in groups of four. The accuracy of overlay of fluorescent channels is limited by the stage accuracy, which is 0.2 ��m. The analysis surface is never perfectly flat, resulting in a variation in focus position in the Z direction when scanning the surface. Prior to a scan, the focus positions are determined automatically at a grid of 6 by 5 positions on the surface. An automatic focusing algorithm uses the reflection of the red laser profile from the glass-sample interface. It integrates the reflection over the 400-��m range of the pi?zo positioner using the TDI camera. This generates an image that contains the profile of the illumination spot for every part of the 400-��m range. A custom made algorithm is then used to find the position were the illumination profile has the sharpest defined edges, indicating the focus position. Transducin These positions are then fitted by a 3D spline that estimates the correct focus position with an accuracy of 0.5 ��m (smaller than the depth of field of the microscope objective, which is 1.5 ��m for light with a wavelength of 550 nm) at each point on the surface. During a scan, the 3D spline fit is used to focus the objective with the MIPOS operating in feed forward mode. The bandwidth of the pi?zo system was determined by measuring the response at increasing frequencies. The ?3 dB point was found to be at 4.5 Hz, resulting in a ��bandwidth�� of 1.8 mm s?1. At a default scan speed of 1 mm s?1, this translates to 1.8 mm mm?1, which is more than enough to follow the variations in height of a typical cartridge, which are in the order of 5 ��m mm?1. The cartridge is scanned through the 180 �� 180 ��m2 illumination profile, see Figure 3A.
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