Paulsson:Pmalkus microscopy2

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misc

  • someone (Dann or Dirk?) oscillations in image brightness (background?) with a period of approx. 6 minutes: due to fluctuations in lamp output? benefit from a line conditioner?

thoughts after JW's Digital Imaging Course

How to evaluate camera noise in the lab... JW offered a protocol, she says it's very doable, but not simple.

Some light sources produce diffuse, unfocused light, which is delivered through optic fiber. Laser/fiber set-up can be thrown out of alignment too (eg by jostling optic fiber), which then needs to be realigned by laser technician.

Lara P. says that coverslip thickness is important even when immersing in oil.

Low fluor media for live cell imaging.

Dirty coverslips scatter light... JW offered her cleaning protocol, and said that buying "pre-cleaned" doesn't cut it.

If signal is strong enough, and you can afford to bleach a little... always look through the eyepiece before acquiring image. 1) Your eyes have a broader dynamic range than the camera, and your eyes may see objects of intermediate signal strength clearly, that are difficult to see in the acquired image. The image can be gamma adjusted to improve the appearance of such intermediate objects.

2) Your eyes will see if there's bleed through from other channels (eg orange in a YFP/RFP double label), all of which will only appear grey in the acquired greyscale image.

Method to evaluate our microscope

Using fluorescent beads of uniform size and intensity?

Tricks for detection

Binning: Pools pixels prior to readout of CCD, thus increase signal and reducing readout noise. However, resolution is lost.


Fixatives

Sink disposal:
formaldehyde - 9mg/liter/day

0.5g paraformaldehyde
45ml dQ
4ul 10N NaOH
warm to 65 deg (stir approx. 30min)
add 5ml 10x PBS
check pH, want 7.4
filter through 0.2um
make fresh every time!

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