Hey everyone,
I read a lot about the theory of TIRF microscopy by now and started wondering wether or not it is possible for those of us with small financial background to set up a white light objective based TIRF microscope with the epifluorescence Scope I am using at the moment? Any experience or publication suggestions? The illumination system is the special point I am struggeling with, since the non-collimated light of my Arc lamp needs to be somehow focused with a lens or be "thinned" with a opaque disk (or something similar) for being defined enough to get it a a steep angle to the back focal plane of my objective.
Or maybe you tell me directly, forget it and buy one. Either way, I am very thankful for your answers!
PS: A review I found, written by Axelrod (link added), stated that it is possible. Did anyone of you try this? Are there specialists for a Olympus IX-50 stage (creating a back focal plane with two lenses)?
http://www.sciencedirect.com/science/article/pii/S0076687903610037
Hi Benni,
Cheap does not go very well with TIRF since it looks like you want to use the same objective to illuminate and image: you need a TIRF objective to start with. Otherwise prism based illumination is also possible.
Let say you have one of those TIRF objective, if in principle you can always get a straight beam with white light, you are likely to lose a very large amount of it on the way and then the excitation will be very (too?) low to achieve any fluorescence excitation. Lasers are definitely better suited... and you can get pretty cheap ones those days.
Hi Benni,
Dont forget that white light is composed of a spectrum of seven primary colors (wavelength), blended together. Different colors of light have different angles of refraction when passed through a lens and therefore are not brought into focus at the same focal point.
Hello Benni,
I don't know if a incoherent TIRF is actually possible to make; what i suggest is to be really aware of the fact that your source is an extended source.
That means that you will never have a "collimated" beam unless you use a pinhole, which i guess would limit your power output too much. The implication of an extended source is that the angle of the light path at the interface with the sample will have to be much steeper than in a system using a coherent source.
Do your calculation really well, i suggest simulating the system with a software like Zemax, if you have access to it. If you don't, try to calculate the angle of incidence with standard geometrical optics for three point sources, one at the center of your lightbulb, and two at the extremities. You want to find yourself in a situation in which all of the three angles are over the TIRF limit: in that situation, the central beam defines your light path.
As a suggestion, if you really want to try something similar, and have a bit of money to invest on it, try looking for an LED light source. There are relatively cheap ones which are much smaller than a standard white lamp, and will give you more power at your fluorescence excitation wavelenght.
Good Luck!!!
Wow thank you guys for these very helpful answers!!! I´ll try wether I can achive something with your tips, if so I will let you know! Maybe even if not, just to give other people the opportunity to not make my mistkaes :)
Hi Benni,
I am a year late with my answer, but indeed you could build a TIRF system if you choose not to illuminate through the objective, but rather create the evanescent field by using prisms or waveguuides coupled to the microscope slide or coverslip. (there are also commercial systems kile that, see tirf-labs.com).
It may be a little clumsier to set up but you get lower background/better contrast and can use any objective - you do not need a special TIRF objective
However, it is probably not possible or practical to use white-light system.
In this paper (see below), they used high power LEDs coupled to a high-index coverslip. It was a single color set-up, but one could probably have room for another set of LEDs of different colors. LEDS are really cheap these days, you can get the six LEDs and constant-current power supplies for under $200 if you shop wisely.
High performance, LED powered, waveguide based total internal reflection microscopy
Srinivasan Ramachandran,, Daniel A. Cohen,, Arjan P. Quist,
& Ratnesh Lal Scientific Reports 3, Article number: 2133 (2013)
http://www.nature.com/articles/srep02133
Stan
Hey Stanislav,
Uh this sounds indeed very interesting. I am working now with a conventional objective based/laser system in a different group. But having the mentioned possibility to do experiments on our Scope is very tempting. "Shopping wisely" sounds like you might have ideas where to get those LEDs (plus power supply) for a decent price? Maybe you could help me out here one more time.
Thanks in advance
Okay, maybe someone is still reading this:
The setup Stanislav mentioned does work! So if you got to this question because you wanted to build a wight light TIRF system, try the LED waveguide based one! Have fun
Dear Benjamin: Accidentally bumped into this thread. Incase you are still looking. Please see if this suits you
Article Compact 3D printed module for fluorescent and label-free ima...
If you are still looking for a solution for objective-type TIRF, a simple but well performing LED-based DIY setup can be found there:
Article Artifact‐free objective‐type multicolor TIRF microscopy with...
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