Leica

Owned by Andrew Vaughan
Last updated: Oct 28, 2020 by 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

NA

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

Binning Factor

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.

Fluorescent Illumination

The DMIRB stand is fitted with an LED pE-300 white light source enabling excitation of all commonly used fluorophores.  The state of the light source is displayed as either OFF or ON on the manual control pod.  Switch on the LED by pressing the 'on/off'' button.  The user has control of three main peaks of emissions: UV, BLU (blue) and GYR (green, yellow, red).  Engage the Select button to activate a light source.  Intensity levels are displayed from 0-100%, and can be adjusted using the + or - buttons.  

Further Information