User:Pranav Rathi/Notebook/OT/2010/12/10/Olympus Water Immersion Specs

From OpenWetWare
< User:Pranav Rathi‎ | Notebook‎ | OT‎ | 2010‎ | 12‎ | 10
Revision as of 19:03, 18 December 2010 by Pranav Rathi (talk | contribs) (Resolution and achievable spot size)

Water immersion objective details

WE are using Olympus UPLANSAPO (UIS 2) water immersion IR objective for DNA stretching and unzipping. The detail specifications of the objective can be found in the link:[1] Other specification are as follows;

Mag 60X
Wavelength 1064
NA 1.2
medium water
Max ray angle 64.5degrees
f # 26.5
Effective FL in water 1.5 to 1.6mm (distance between the focal spot and the exit aperture surface)
Entrance aperture diameter 8.5mm
Exit aperture diameter 6.6mm
Working distance .28mm
Cover glass correction .13 to .21 (we use .15)

Resolution and achievable spot size

The resolution and the spotsize (beam waist) presented here is in the theoretical limits; we cannot achieve better than this. Resolution and spotsize are diffraction limited and to reach these limits our optics has to be perfect; no aberrations and other artifacts. Since our optics is not perfect and very clean we can hardly reach these limits in real life; definitely the resolution and spotsize in real is worse than the numbers presented here. A good way to do a quick estimation of the resolution (diameter of the airy disk) is that its 1/3 of a wavelength λ=.580 μm; λ/3*n= 145 nm. Since we do all our experiments in water we will have take index of water in account (n=1.33). I am ignoring the NA of the condenser in the calculations.

  • Wavelength of the visible light λv = .590μm.
  • Wavelength of the IR λIR = 1.064μm.
  • Diameter of the incident beam at the exit pupil (D=2ω'o.)=6500μm. (ω'o is the incident beam waist)
  • Focal length of the objective f=1500μm.
    • Angular resolution inside water:
[math]\mathrm{\theta} = \sin^{-1}\frac{1.22\lambda_v}{nD}= 8.1e^{-5}rad[/math]
  • Spatial resolution in water;
[math]\mathrm{\Delta l} = \frac{1.22f\lambda_v}{nD}= 122nm[/math]

Since we are not too sure of the focal length of the objective, so i derived the resolution formula in terms of the numerical aperture NA (the math can be seen through this link[2]).

  • Resolution in terms of NA:
[math]\mathrm{\Delta l} = \frac{1.22\lambda_v}{2n}\sqrt{(\frac{n}{NA})^{2}-1}= 127nm[/math]
  • Now the minimum spotsize (beam waist ωo) can be calculated using the same formula where Δl=2ωo. But this time for infrared wavelength
    • Minimum beam waist;
[math]\mathrm{\omega_o} = \frac{1.22f\lambda_{IR}}{2nD}= 112nm[/math]

and the beam diameter 224nm.

  • In terms of NA:
[math]\mathrm{\omega_o} = \frac{1.22\lambda_{IR}}{4n}\sqrt{(\frac{n}{NA})^{2}-1}= 116nm[/math]

and the beam diameter 232nm.

I also used Gaussian approach to calculate the spot size and the results are not much different, which proves that either approach is right.

  • With Gaussian approach;
[math]\mathrm{\omega_o} = \frac{\lambda_{IR}}{\pi n}\sqrt{(\frac{n}{NA})^{2}-1}= 121nm[/math]

and the beam diameter 242nm.

Results are not much different and either approach is right. First order Gaussian approximation is not correct for high NA (for high convergent beams) objective lens. Thus the best theoretical value for the spotsize is needed to be multiplied by pi (121 X pi = 380 nm). This value is more correct. The results of the beam waist can be experimentally verified by directly measuring the spot size, but the process is rather cumbersome and hardly interesting. The results given here are the best in terms of the theoretical limits of the aberration free optics. Experimentally we suffer on number of bases: one of them is experimental-setup itself; because of aberrations introduces by index mismath among water, oil and glass interfaces, which also introduce the multiple reflection. And we should also not forget that the focal plane of the objective is not infinitely thin in z-direction, which implies the out of focus rays degrading the overall image reducing the resolution and degrading the spotsize.