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Objectives on the Leica Confocal Microscope

 

The following objectives are on the Leica Confocal:

10X PL FL N.A. 0.30
20X PL APO N.A. 0.7
25X PL FL N.A. 0.75 Oil Immersion
40X PL APO N.A. 1.25 Oil Immersion
100X PL APO N.A. 1.40 Oil Immersion
63X PL APO N.A. 1.20 Water Immersion

A 5X PL FL N.A. 0.15 is also available -- ask Greg to put it on if you want to use it.

 

The 40X and 100X oil immersion objectives have a variable aperture. This should be fully open (turned fully clockwise) for maximum resolution and intensity.

The 63X water immersion objective has a cover slip correction collar. All cover slips vary but generally a #1 cover slip is .14 mm and a # 1 1/2 is .17 mm. Adjust the collar for the best image: while looking at the specimen by eye, adjust the collar in small increments, refocusing continuously with the microscope's fine focus.

 

The following table contains important information about the Leica's objectives:
LENS
N.A.
IMMERSION MEDIUM
WORKING DISTANCE
XY RESOLUTION
Z RESOLUTION

OPTICAL SECTION
w/ PINHOLE = 1 AU
5 X
0.15
DRY
12 mm
1.3 um
19.4 um
10X
0.3
DRY
11 mm
0.651 um
4.768 um
11.17 um
20 X
0.7
DRY
0.59 mm
0.279 um
0.768 um
3.92 um
25 X
0.75
OIL
180 um
0.260 um
1.108 um
2.29 um
40 X
1.25
OIL 
100 um
0.156 um
0.334 um
0.70 um
63 X
1.20
WATER
220 um
0.163 um
0.290 um
1.25 um
100 X
1.40
OIL
90 um
0.140 um
0.236 um
0.56 um

N.A. = Numerical Aperture

All values are calculated for an ideal lens and are provided by Leica, EXCEPT the optical section thickness which was measured with our lenses as full-width-half-max from an XZ image collected in reflection mode of a mirrored surface.

WORKING DISTANCE: The working distance of a lens is the space between the front element of the objective and the top of the cover slip Any space between the cover slip and the tissue adds to the working distance since a manufacturers figure for the working distance of a lens presumes that the tissue is just under the cover slip

 

Contact Greg Martin at gvm@u.washington.edu for additional information. Suggestions and comments are always welcome!

© 2007 Keck Microscopy Facility, University of Washington