Hi Pedro,

for my opinion, the only refractive index you should play with is that of the embedding media.

Changing the refractive index of the immersion oil will negatively affect the objectives point spread function.

Biological samples are usually embedded in aqueous media with a refractive index of 1.33.

That is the NA of the objective needs at least to be a bit bigger than 1.33, to be used as TIRF objective.

I would strongly recommend to use a high NA TIRF objective (NA > = 1.45)

The only way to use a NA 1.25 Objective in TIRF mode is to lower the embedding refractive index below 1.2, for example by embedding in air but this makes the use of TIRF illumination some kind of obsolete...

Best

Thorge

In Total Internal Reflection Fluorescence microscopy, it is the excitation beam that does the TIR. The TIR condition is an inequality of incident angle of the excitation beam and refractive indices, at the excitation-beam wavelength, of two light-transmitting material above and below the interface you observe. To maximize the intensity of the fluorescent emission, you should have a closer look at Fresnel equations. The microscope objective has nothing to do with the TIR condition because it is the fluorescence-collection lens as I imagine your optical system. The higher NA is desirable because fluorescent emission diffuses. I think NA 1.25 objective should see some signals if a NA 1.5 does at the same incident angle, because the two light collection efficiencies do not have an order-of-magnitude difference. If I were developing the instrument and facing a problem, I would first check the terminal spot size of the collected fluoresce on the spectrometer slit. This should generate requirements for the excitation beam such as the spot size on the observing interface and focusing conditions, accordingly.

I did not know about the objective based TIRF.  Thank you for your enlightening me.

Thanks both for your contributions !!!

Actually, I agree and appreciate all the info that Hiroki Fujiwuara gave about experimental tricks to get an optimized alignment of the TIRF microscope .

However, I actaually was refering to what Peter Kapusta understood. Before I join to my post doc position, the group bought a 1.25 NA , with a 100 X Obective , cause they were thinking on a biggest magnification , MISTAKEN !!!

Any ways I am here , it is a  beautiful objective, and I need to use it , so ...

What do you think of, fixing the difference on the refractive indexes between the 2 media, in order to reduce the required angle for reach the TRF condition ?

I am afraid of lossing to much fluorescence , due to a high refractive index missmatch ... but I dont see another option, Do youo ?

And thanks for youro contributions colleges

Dr Pit Navarro  

Hi Pedro,

for my opinion, the only refractive index you should play with is that of the embedding media.

Changing the refractive index of the immersion oil will negatively affect the objectives point spread function.

Biological samples are usually embedded in aqueous media with a refractive index of 1.33.

That is the NA of the objective needs at least to be a bit bigger than 1.33, to be used as TIRF objective.

I would strongly recommend to use a high NA TIRF objective (NA > = 1.45)

The only way to use a NA 1.25 Objective in TIRF mode is to lower the embedding refractive index below 1.2, for example by embedding in air but this makes the use of TIRF illumination some kind of obsolete...

Best

Thorge