I have read that upright microscopes are normally used for slices recordings. But I need the set up for whole cell experiments. My problem comes when I try to approach the pipette to the cell. I have to patch keratinocytes and they are very flat cells so I need more or less a steep angle to be able to make the seal. Due to the objectives wheel I don't have enough space and I cannot get that steep angle because the manipulator hits it. Besides, the objectives are immersion.
Has anybody have the same problem? Any ideas?
Thank you in advance.
Hi Lucia. You can try various angles ranging from 30 to 40 degree. Moreover if the sample is slice then you can place the area of your interest near the periphery of perfusion chamber i.e little away from the center. I had also same problem initially however later with a lot of angles and distance trials it worked out. If you have not done patching before, you can run a dummy exp and place the manupulator at appropriate distance and angle so that it can touch all the areas of coverslip and move up and down without touching the objective. I can suggest you some objectives with higher working distance if you let me know which objective lens you are using.
Good Luck.
Hi Lucía,
You can set an upright microscope setup by using perfusion chambers like the one in the link I attach (very long link), by growing the cells in circular coverslips. If you are using an upright microscope similar to Olympus IX70, then you can try to patch the cells from an angle of 45 degrees. I managed to patch some COS7 cells, which also are very flat.
http://www.google.de/imgres?imgurl=https%3A%2F%2Fwww.warneronline.com%2Fimg_md%2F640232_RC-25%2CRC-25F.jpg&imgrefurl=https%3A%2F%2Fwww.warneronline.com%2Fproduct_info.cfm%3Fid%3D735&h=115&w=200&tbnid=uXmd5_dexb0XfM%3A&zoom=1&docid=-SxeYNRTyya5OM&ei=wiVjVIXtKo2rOuqHgKAH&tbm=isch&client=firefox-a&iact=rc&uact=3&dur=856&page=1&start=0&ndsp=52&ved=0CCMQrQMwAQ
Hope that helped!!
As people have said, you need an objective with as much working distance as you can get. Preferably above 3 mm, but you can make it work with as little as 2mm of working distance. However, in my experience of patching flat cells in culture, you do not need to come in especially steeply. Just get the pipette close to the surface of the cover slip, and move horizontally until you push up against the membrane..
If you grow cells attached on Petri dishes, try to cut the upper part of the dish, so it is lower and you can get closer with the electrode. We use a heated scalpel to mold plastic and cut.
Maybe a swing nosepiece like the WI-SRE3 from Olympus could help.
Thanks everybody for the advices. They all were really helpful.
All are great suggestions thus far. I have used many of them myself with success. There is one trick, however, I don't see it mentioned and that is changing the taper length. I wouldn't normally play with this parameter first as it changes the cable properties of the pipette, such as axial resistance and fast capacitance (something you have to be careful with and may have to compensate for). If you still experience problems after trying out suggestions above, perhaps you can give this a try. It will give you a little more lateral clearance at extreme angles. You can also forge one sided or uneven taper using a trough heating element in the puller to give yourself more angles to play with.
I used to bend the end of my pipettes to increase the angle using a flame. with a tweezer, bring the end of the pipette close to the flame, then with another tweezer, push gently to bend just few degrees (maybe about 10 to 15 degrees). When you patch, bring the tip of the pipette pointing down (I hope the picture is clear enough...I did my best).
Hi, Lucia: We use an upright microscope with immersion objectives; the working distance is small, but we can get to the cells without much problem. All suggestions above are very good, and I have used any combination of them in the past, including bending the pipettes (neat trick, Fouad). What work best for us is the low borders of the chamber, which is the simplest costume made device: we use the cap of small cell culture dishes with a ~20mm perforation in the center over which we glue (with Vaseline) a 25mm glass cover slip with our cultured cells. This may not work for you if you have to warm your solution over 25C degrees, as the Vaseline will melt, but otherwise it's a good setup.
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