[OpenSPIM] sharp widefield image, but out of focus FP emmission

[Weber, Michael]

Hey Jaap,

This does sound like light sheet alignment to me. It will be a lot more difficult with a 100x lens than it is with a 10-20x. When looking into your chamber from top, do you see a nicely focused beam close to the focal plane of your detection objective?
Next, just use water, remove cylindrical lens and emission filter and try to bring the focus of your laser beam into the field of view. Keep in mind that the vertical position of your laser (y in your FOV) might be off as well.

Best,
Michael

From: Jaap van Krugten<mailto:j.van.krugten at vu.nl>
Sent: Thursday, March 9, 2017 3:48 AM
To: openspim at openspim.org<mailto:openspim at openspim.org>
Subject: [OpenSPIM] sharp widefield image, but out of focus FP emmission


Dear OpenSPIMers,


We are building an openSPIM with a 100x detection objective as only major adaptation. Our light sheet is nicely shaped and thin, and if we look at our sample using a white light LED opposite of the detection objective, it is in focus. However, if we turn on the laser, we can not get the GFP emission light in focus. This seems odd, as our wide field image is in focus.

We are quite certain the laser is properly aligned, and we did try moving the light sheet through our sample. Any ideas what else we can try?


Thanks in advance,

Jaap van Krugten




Jaap van Krugten, MSc | PhD candidate | in vivo single-molecule fluorescence microscopy
Physics of Living Systems, Department of Physics and Astronomy | Vrije Universiteit Amsterdam

+31 (0)20 59 87904 | room U-030, W&N
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