Frequently Asked Questions about the equipment:
Q: Does it make a big difference if my microscope is equipped with IR770nm vs. IR900nm DIC optics?
A: Yes! Emphatically. The IR900nm filter set-up is far superior for clear visualization into the adult slices. It is hard to explain if you haven’t seen this first hand. Once you see the high magnification brain slice image from the properly aligned IR900nm set-up you can’t go back. It is not simply an incremental improvement. With IR770 nm optics you may be able to see about 40 microns deep, depending on animal age and brain region, but the visualization of the electrode tip touching the targeted cell membrane might be hazy. With IR900 nm optics (and proper alignment for Kohler illumination) you would improve visibility to up to 100 microns or more with much better clarity of the electrode tip touching the target cell membrane. In juvenile brain slices for brain areas with low myelination such as mPFC it is possible to see all the way to the damaged cells on the opposite slice surface in a 300 micron thick section.
Parts list for IR900nm-DIC upgrades on an Olympus BX51WI upright microscope:
1) 1050nm hot mirror, 45mm (#45HM1050IR). This replaces the 850nm hot mirror
2) 900nm band pass filter (#32IR900). This replaces the 775nm/100 band pass filter
3) IR analyzer for transmitted light, 900nm (#WI-ANIR). This replaces U-AN analyzer/slider
4) 32mm round IR polarizer for 900nm (#32POIR). This replaces the round polarizer for visible to 775nm light.
5) DIC prism for WI-SRE3/WI-SNPXLU2 (#WI-DICTHRA2). This replaces part WI-DICT2.
The DIC prism and the IR polarizer are the least obvious things to change, but it is important to be matched to the IR900 parts. Check with your local rep to see if you can get credit for the returned IR770 parts. The total cost may be around $2200 for the switch. Without credits for the returned parts it would be around $5000.
The microscopes on our rigs have the condenser aligned to the bottom edge of the stage platform, the iris all the way open, and have been optimized for Kohler illumination. With this particular setting and the IR900 filters in at 40X objective power (black level about halfway and gain on auto), it is necessary to turn the transmitted light control almost all the way up to see the clear IR-DIC image on the monitor. This may vary from setup to setup, but the new IR900 filters will block out more of the light, so you should notice that you need to use much higher light to see. This can translate into more heating of the specimen, which should not be an issue in most applications, but the correct hot mirror is critical to cut out the higher wavelengths.
For detailed notes on the optimization of optics for Kohler illumination, see Davie et al, 2006 Nature Protocols (Box 1).