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DMI4000 microscope


The SPE confocal system is attached to a DMI4000 microscope stand. The fluorescence filter turret and objective lens nose-piece are manually rotated but the positions are encoded so the software knows which objective lens is in place. The microscope stand focus in manual so confocal sectioning is achieved via a galvanometer driven stage insert (Z Galvo). Eyepiece or confocal light path selection is automated.

Objective lenses

Leica ACS objective lenses have been designed specifically for SPE confocal microscopes with 405 nm lasers to correct for colour registration errors between the violet and visible lines. They do not have a flat field so Leica recommends that the confocal zoom is set to ≥1.6.

Magnification

Immersion Medium

Designation

Numerical Aperture

Coverslip Correction

DIC Prism

10X

Air

ACS APO

0.3

None

C

20X

Multi Imm

ACS APO

0.6

0 - 0.17


40X

Oil

ACS APO

1.15

0.17

E

63X

Oil

ACS APO

1.3

0.17

E

Fluorescence filter sets (for observation down the eyepieces)

These fluorescence filters are only used when you're focusing on your specimen down the eyepieces. The filter turret is automatically rotated to the empty Position 4 (BF) when a confocal scan is initiated.

Turret Position

Filter Set

Excitation Filter

Dichroic Mirror

Emission Filter

Example Fluorophores

1

A

BP340-380

RKP400

LP425

Hoechst/DAPI

2

I3

BP450-490

RKP510

LP515

FITC/GFP

3

N2.1

BP515-560

RKP580

LP590

Texas Red

4

BF

N/A

N/A

N/A

Transmitted Light

5

N/A

N/A

N/A

N/A

Empty

6

Pol

N/A

N/A

N/A

Polariser for DIC and polarised light microscopy

Positions 4 to 6 are irrelevant for fluorescence.

Lasers and dichroic mirrors

The SPE3 has the following lasers. Note that it differs from the other SPEs in that it has a 561 nm laser instead of a 532 nm laser.

Laser Line

Example Fluorophores

405 nm

DAPI, Hoechst

488 nm

FITC, GFP

561 nm

Texas Red, mCherry

635 nm

Cy5, Alexa 647

Dichroic mirrors separate the excitation light from the emitted fluorescence. Without dichroic mirrors or other beam splitting technologies your image would be contaminated with laser light.

Dichroic Mirror

Description

QD 405/488/561/635

Quad band that reflects all four laser lines. This is the one you're most likely to use

DD 488/561

Double dichroic. Only reflects the 488 and 561 lines, which means the other two lines will not be blocked

RT 30/70

A beam splitter for back scatter or reflection imaging. Not useful for most fluorescence.

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