Z Stack

If you are trying to run a z stack in several xy positions and are experiencing the problem that your z stack seems to start in the middle of the z, think about this: - When you set your reference position in the xy tab, you can decide to include z and/or include the PFS offset before saving the position - When you set your z stack top and bottom, you should not have the PFS on. Remove the PFS, set the top and bottom using the microscope focus wheel then turn the PFS back on before you press run now. This is because the PFS will anyway turn itself off during the z stack acquisition. It is only used to find the reference position. Do not set your z stack using the PFS wheel.

CFP/YFP

This information only concerns those of you who image CFP/YFP together with a red fluorophore. There is no problem if one wants to image CFP/YFP only.

When imaging CFP/YFP and red, one needs to select the 457/514/561 dichroic mirror and choose appropriate emission filters in front of each detector.

Case 1: when the 540/30 emission filter is chosen for detector 2 (PMT2) then a 640 LP dichroic mirror is automatically selected above PMT2. This means that the red light goes to PMT2 and therefore no red goes to PMT3. The red image acquired by PMT3 is thus black.

Case 2: when the 525/50 emission filter is chosen for PMT2 then the dichroic mirror above PMT2 is automatically set to 560 LP. This way we get a red image on PMT3.

All of you choose the second solution to image CFP/YFP/red because this is the only one that gives a red image on PMT3.

We want to bring to your attention that the 525/50 emission filter is not optimal for CFP/YFP FRET because it does not give enough discrimination between CFP/YFP (as seen when doing the ratio of area under curve for CFP and YFP). The FRET ratio is artificially lower than it should be. The 540/30 filter is best for FRET ratio but it doesn’t allow combining with red imaging.

If you would like to image CFP/YFP/red with the best CFP/YFP ratio, come and talk to us. In this case we will need to order a 540/25 or/and change secondary dichroic mirror above PMT 2 to 590 or 600 LP.

Immunology and sectioning

Sectioning

  • Read the Leica sectioning handbook, it's full of tips and trouble-shooting.
  • At the microtome, orientate blocks so that the knife cuts the tissue at the softest part first, and at the broad face of the tissue section.
  • Cool the blocks before cutting, on ice. For hard samples letting the blocks sit on wet ice can soften the tissue and allow for better sections.
  • After sectioning, dry the slides at 37C overnight with slides in a vertical position, and then bake at 60C for one hour.

Immuno-staining

  • Bubbles in the mounting media: Apply the coverslip at an angle, seal the edges of the coverslip with nail polish, store the slides vertically, store the slides at -20C.
  • Non-specific speckles of signal: Ab aggregate, spin at max RPM, 4°C, 10'.
  • The first time using a new antibody, prepare slides with a serial dilution, and only try to do multiple-labelling with other antibodies after single labelled experiments have proven successful.
  • Be aware that some fluorescent agents (such as DAPI) have very broad emission spectrum and may cause problems, and others (such as FITC) bleach rapidly and may make imaging more difficult.
  • For each experiment make a slide without primaries, but with the secondaries, to check for non-specific staining.
  • For experiments with overlapping spectra or with auto-fluorescence in the tissue, make a slide with no primary, no secondary, and a slide with each primary and secondary pairing alone. These can be used with microscopes equipped with spectral detectors to get accurate results.
  • Background autofluoresence: Wavelength specific irradiation1): CuSO4 in ammonium acetate buffer or Sudan Black B in 70% ethanol2): NaBH43): Pontamine Sky Blue4). Or image in far-red instead of green/red.

Image acquisition

  • Avoid imaging at the edge of the image field (to avoid vignetting).
  • Keep the saturation controls on, and the auto-LUT off, but aim to have the image looking the same if the saturation controls are off and the auto-LUT is on.
1)
Simple method for reduction of autofluorescence in fluorescence microscopy.
Neumann M, Gabel D
J Histochem Cytochem50p437-9(2002 Mar)
2)
Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.
Schnell SA, Staines WA, Wessendorf MW
J Histochem Cytochem47p719-30(1999 Jun)