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Application Note

LIGHT INTENSITY DISTRIBUTION IN THE EM ICE LS

related instruments: EM ICE

2

LIGHT INTENSITY DISTRIBUTION

IN THE EM ICE LS

Application Note for High Pressure Freezer EM ICE

Paul Wurzinger & Julia König, Leica Microsystems, Vienna, Austria

In the EM ICE LS the light is coupled into the high pressure chamber via a light fibre and a sapphire window. The axis of the sapphire window is directed towards the centre of the sample and tilted by 60° to the sample plane, as is represented in Figure 1.

Figure 2 shows photographs taken from the backside of a semitransparent foil mounted on the bottom side of a 6mm middle plate and illuminated from the top. The inclination of the light path is towards the right-hand side in these images. Note that there are shades caused by milled features on the cartridge which are necessary to guide the LN2 flow correctly (“cartridge shades”). The shades are shifted relative to the center of the sample recess. This shift is caused by the inclined illumination and varies depending on the vertical sample position (influenced by spacer rings, carriers, etc.).

Figure 1: Schematic view of the light stimulation geometry; viewing direction is the cartridge axis

Figure 2: Photographs of light intensity distribution on a semitransparent foil on the bottom of a 6mm middle plate taken from the bottom side and illuminated from the top. The light path inclination is towards the right-hand side in the images; a: green module; b: amber module

Cartridge shadesa b

LNT Application Note - LIGHT INTENSITY DISTRIBUTION 3

There is a quite homogeneously illuminated area of roughly 2x2 mm2 beween these cartridge shades. This area is extended towards the left-hand side (opposite to the illumination entrance) probably because of reflections from the half cylinder and the inner surface of the high pressure chamber.

While the photographs of Figure 2 show the qualitative illumination pattern very well, intensity measurements with a calibrated instrument are necessary to determine the absolute illumination intensity. These were done using an OPHIR Nova II meter assembly with an PD300-TP sensor. A foil with a 1 mm aperture was mounted in front of the sensor and the aperture was scanned in 0.5 mm steps in x/y-direction about 0.5 mm beneath the upper half cylinder. The result is represented by a graph created with Micorsoft Excel in Figure 3. This graph is centered around the maximum intensity measured and the shades give the relative intensity (in %) to this point. The size of the aperture causes the cartridge shades to grow in width so that the maximum intensity here seems restricted to a 1x1 mm2 area.

The absolute intensity for sample illumination was determined using the setup described for Figure 3. As there are small variations depending on the actual LED in a module the modules have the following intensity ranges:Blue (λ = 460 nm): 5.5 - 8.0 mW/mm2UV (λ = 385 nm): 5.0 - 7.5 mW/mm2Green (λ = 520 nm): 1.5 - 2.5 mW/mm2Red (λ = 660 nm): 2.3 - 3.3 mW/mm2Amber (λ = 590 nm): 0.8 - 1.1 mW/mm2

Figure 3: Excel-Representation of the Intensity distribution (UV module) measured with a 1mm aperture in steps of 0,5 mm in x and y in the central part of the sample position; The axis are centered around the point with maximum intensity.

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Leica Mikrosysteme GmbH | Vienna, AustriaT +43 1 486 8050-0 | F +43 1 486 8050-30

www.leica-microsystems.com

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