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A5.19. How to Setup MicroPoint for Uncaging

MicroPoint is an extremely flexible tool for photo-stimulation, delivering precisely positioned laser pulses over a wide wavelength and energy range. Depending on energy and wavelength setting, the device can be used in order of decreasing energy to perform the following:

ablate cells and organelles,

bleach fluorophores

activate photo-switchable fluorophores and

release caged compounds - breaking molecular bonds.

The Micropoint 1 system is driven by a pulsed nitrogen dye laser. This laser has some patented features that allow it to deliver about 25 different wavelengths determined by the contents of a dye cell resonator. The dye cell contains various mixtures of mainly coumarin dyes, which fluoresce strongly and in combination with the resonator provide a tunable pulse delivery system. The nitrogen laser provides driving energy in 70 uJ, 3ns pulses at 337 nm and at 10Hz. These pulses are converted in the various dye cells to wavelengths in the range 365-650 nm with duration of about 4ns. The energy delivered to the specimen can be varied from about 50 uJ down to pJ levels.

When using the system at visible wavelengths, where microscope optics are well corrected and transmit strongly, the selection of optical components is not too critical, though alignment and focus must be handled with care. But at

whole cells, organelles, individual filaments such as actin and microtubules and at even lower energy levels the system can be used for photo-bleaching,

I need to know what objectives you are using

– details of mag, NA and Nikon model number.

This will allow us to establish the UV transmission properties of the lenses. The fact that you can induce specimen damage proves there is enough light coming through the system.

For uncaging, focus is critical because it is typically done at energy levels below bleaching.

Using the first surface mirror for setup is only a first step, because you must account for the

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Andor iQ How To Documents cover slip and any medium in which the specimen exists.

One of our experienced engineers from PI has suggested a 3 or 4 step focus process.

1. Take a slide in which you can identify single microtubules or actin filaments or similar and with a DAPI cube in place and mercury lamp, illuminate the specimen

– the UV cube should be transmitting 365-400 nm

–mercury has a 365 and 405 nm line. Get an idea of how long the filaments take to bleach to 50% intensity

– a camera helps. This time will give you an idea of how long you may need to pulse the 365 nm MicroPoint for similar effect.

2. Now turn the 365 nm energy level down below the damage threshold and try the same thing with the MicroPoint

– pulsing continuously for several seconds if needed. Make note of how long this takes

– again it will depend on the energy setting, but also on the objective transmission and the focus of the MicroPoint.

3. Now spot one of the filaments and trigger the MicroPoint to output 5-10 pulses. You should see some bleaching if not increase the power a little until you do. Now continue this process, changing the focus in between bleaching actions, reducing energy when possible at each step. You should be able to get to the point of bleaching a single filament with a resultant spot in the order of the diffraction limit of the objective.

4. Now when you go to the cell culture, you may have to correct again by a similar approach to 3 above because you are now in “water” or thereabouts and focus will again be shifted.

Once you have setup MicroPoint for a given objective and specimen you should note the setting for later recall.

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