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Hello all,

I´m about to add TIRF to my home-built scope but due to lack of experience 
with TIRF I´d rather ask a few questions in advance.
The goal is to do multi-colour TIRF with 488nm and 650nm through a 100x1.65 
objective.

Due to the high difference in RI, i think the critical angle is not the 
issue here. But I´m wondering whether the differences in penetration depths 
between the two lines is critical when doing 'ordinary' live imaging of 
yeasts, hela cells etc. Assuming both lines have an angle of ~65°, the 
resulting penetration difference is ~25nm. From my perspective, 25nm 
doesn´t sound much when normally dealing with diffraction limited 
distances. On the other hand the nominal penetration of the 488nm would be 
around 75nm and then 25nm difference is quite a lot. The question I like to 
adress: Do you generally correct the difference in penetration depths in 
multi-colour TIRF? If so, do you alter the inclination of the laser in 
relation to the ojective or do you rather enter the objective in a parallel 
manner but at distinct lateral distances from the objective´s center?

Second question aside. Does someone happen to know about a method how to 
clean and re-use the extremely expensive and fragile Sapphire coverslips? 
Is there maybe another, cheaper source than Olympus?

Many thanks in advance for any hints and best regards,

Steffen Steinert, Dipl.-Ing.

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Universität Stuttgart
3. Physikalisches Institut
Pfaffenwaldring 57
70550 Stuttgart

Tel.: 49/711/68569832

http://www.pi3.uni-stuttgart.de/en/
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