SPE3 Microscope Information
SPE3 microscope components
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. |