Adjustable sensitivity

First generation scanners had much slower scanning speed, meaning the time over which each spot/pixel was excited was long enough to make it a concern. The Tecan scanner is much faster, meaning that photo-bleaching has for all intents and purposes become a 'non-issue'. In-house bench studies suggest 1% photo-bleaching per scan depending on default laser

Table 16.1

Small pinhole (0.3mm) Medium pinhole (1.0 mm) Large pinhole (3.0 mm)

70 |m = ± 35 |m around the focus 300 |m = ± 150 |m around the focus 800 |m = ± 400 |m around the focus

Figure 16.2.

Detection principle and optic scheme of LS for confocal or non-confocal scan.

power. Bleaching and stability in general strongly depends on oxygen, surface chemistry, temperature, humidity and light exposure. Only the latter can be influenced by the instrument.

Photon statistics dictate that the best signal/noise ratios are obtained by having maximum photon output (to a certain degree, of course), which is only achieved by having maximum laser power. Reducing the laser power therefore always reduces the achievable signal/noise ratio, whereas reducing the PMT gain for a wide range does not affect signal/noise. Therefore, the best way to adjust dynamic range is to maximize photon output and to adjust the gain of the detector accordingly. PMTs and electronics of the LS are working linear over the 16-bit dynamics. The software allows adjustment of the PMTs in LS systems over five logs to amplify the photon signal. Commonly array detectors offer two logs (1-100%) adjustable range.

In addition to using maximal laser power in order to increase photon collection, image data acquisition with LS offers several options for improvement:

(i) Use 'optimize integration time' (increase dwell time to maximum) and reduce scan width and resolution accordingly.

(ii) Average scan from two up to eight times.

(iii) Scan the sample through a glass matrix to increase photon collection by a factor of two.

(iv) Increase the pinhole size if required to achieve high uniformity on bulky and warped sample.

(v) Scan evanescence resonance glass slides from Novartis (NovaChip) and adjust the angle of incidence of laser beam according to the laser wavelength used (0° for 633 nm and 20° for 532 nm). The LS Reloaded can achieve more than 20-fold higher sensitivity for single and dual color scans.

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