Copy of Leica
- John Gallagher
Leica
Location: MRC Building room 3.17
This is a Leica DMIRB inverted research fluorescence microscope suitable for observing cells on slides or in glass-bottomed or plastic bottomed vessels using either phase contrast or fluorescence microscopy. The microscope has a Hamamatsu C4742-95 Orca greyscale camera suitable for acquiring images of fluorescent specimens but not suitable for imaging histologically or histochemically stained specimens.
The microscope has 20x and 40x long working distance objective lenses that have adjustable correction collars so the objectives can be corrected to focus through different thicknesses. For example, coverslips are usually only 170 microns thick but the growth surface of standard cell culture dishes is approximately 1 mm thick, so a correction collar would have to be set differently for specimens mounted on one or other of these surfaces.
Objective lenses
Magnification | Immersion | Designation | Contrast Technique | Coverslip Correction (mm) | WD (mm) | |
|---|---|---|---|---|---|---|
10x | Air | N Plan | 0.25 | Ph1 | None | 17.6 |
20x | Air | N Plan LWD | 0.4 | Ph1 | 0 - 2 | 3.2 - 1.9 |
40x | Air | N Plan LWD | 0.55 | Ph2 | 0 - 2 | 3.3 - 1.9 |
The Long Working Distance (LWD) objectives have optical correction collars that must be adjusted for the varying thicknesses of plastic culture vessels, cover slips and other surfaces. Generally the collars will be set correctly for plastic tissue culture dishes, but if you find it difficult to satisfactorily focus on your sample it may be because the correction collar has been adjusted for a different material.
Fluorescence filter sets
Filter Position | Filter Set | Excitation Filter | Dichroic Mirror | Emission Filter | Fluorophores |
|---|---|---|---|---|---|
1 | Leica A4 | 360/40 | 400 | 470/40 | DAPI, Hoechst |
2 | Leica GFP | 470/40 | 500 | 525/50 | FITC, GFP |
3 | Leica N2.1 | 515 - 560 | 580 | LP 590 | Cy3, TRITC |
4 | Blank | N/A | N/A | N/A | Transmitted Light |
Leica's filter set web page.
Camera Calibrations
The magnification calibrations for images taken using the Hamamatsu Orca camera with 0.63X de-magnifying C-mount are as follows:
Magnification | 100 pixels = | 1 pixel = | |
|---|---|---|---|
10x | 1 | 106.34 µm | 1063.4 nm |
20x | 1 | 53.17 µm | 531.7 nm |
40x | 1 | 26.59 µm | 265.9 nm |
10x | 2 | 212.68 µm | 2126.8 nm |
20x | 2 | 106.34 µm | 1063.4 nm |
40x | 2 | 53.17 µm | 531.7 nm |
Eyepiece Reticle Calibrations
For those of you who wish to count cells while looking down the eyepieces, the reticle dimensions (taking into account differences in magnification) are as follows:
Magnification | Dimensions of a small square in the reticle |
|---|---|
10x | 100 x 100 |
20x | 50 x 50 |
40x | 25 x 25 |
On a related note, the Field Number (FN) of the eyepieces is 22 millimetres. The diameter of the view field is given by the following equation: Diameter = FN / Objective Magnification x Tube magnification. The tube magnification in the Leica is 1 X, so the diameter of the view field at 10X, for example, is: 22/(10 x 1) = 2.2 millimetres.
You can make the camera parfocal with the eyepieces by using the eyepiece focus to get a sharp image of the reticle.