having been involved in the setup of a number of systems, I
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Hi Liang,
having been involved in the setup of a number of systems, I can confirm that it's fairly easy, although there are a few questions you need to ask before you begin. The most important of these are:
- What are you going to be using the system for? If you are going to try and measure depth using the evanescent decay, you need a well controlled angle and good beam quality. In practice this tends to mean spatial filtering (e.g. by using a single mode fibre) and illuminating a large field of view so as to get a reasonably uniform illumination in the bit you're interested in. If you're doing PALM/STORM or related work, you want to maximise the intensity you get, which means illuminating only the area you need too and probably skipping the spatial filter (the method is surprisingly robust against beam quality). Because you're only illuminating a small area, however, you will need reasonably easy to use controls to centre it on your field of view. Single particle tracking will be somewhere between these two extremes.
- Do you need computer control of the TIRF angle? This will influence how you couple the light in - the easiest approach to computer control is probably to put either a mirror, or the end of a fibre on a motorised stage at one of the systems fourier planes.
In general I like to build a folded 4-f system with mirrors at both the conjugate image and fourier planes. This allows you to set the illumination position and angle (mostly) independently. The other trick I use is to use a polarising beam splitter to combine the laser based TIRF illumination with the arc lamp. You lose 50% on the arc lamp, but the lasers pass without loss and the alignment is much more stable than if using a flip mirror.
cheers,
David
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From: cail <[log in to unmask]>
To: [log in to unmask]
Sent: Saturday, 14 September 2013 8:37 AM
Subject: Anyone has suggestion on setting up an objective based TIRF system
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Dear all,
Due to the fund limitation, we cannot afford a commercial system. We would like to setup one by purchasing lasers, lens, mirrors and holders. Is there any instruction available? Anyone has a successful experience to share?
Thank you so much for your time.
Sincerely,
Liang
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