I am starting on a project doing live cell imaging using a widefield fluorescent microscope. I am transfecting a fluorescent protein and imaging with two channels at each time point. Do you know where can I find good guidelines for this type of experiments? I need to know what are the typical exposure times, ND filters used, objectives, any tips to avoid focus drift over time and any other useful suggestions to run a successful experiment.
Hi Virginia,
since you are asking which hardware to use, this means for me, you are at the planing stage of your experiment. Here some general advices:
- if possible use a LED light source to avoid phototoxicisity
- avoid colors in the blue range (like BFP, CFP) to avoid phototoxicisity
- thing about the size of the structures you want to see, this leads you (most probably) to the objective magnification (eg nuclues vs cell organelles)
- thing about the intensity of the signal you want follow (huge signal intensities in combination which a large structure --> smaller magnification nessecary, low signals (even in large structures) higher magnification nessecary)
- use objective with a high numerical apperture (if opossible NA>1)
- use objectives made for fluorescence live-cell imaging
- set your exposure time as small as possible (to avoid phototoxicity
- use filters which fit well for your fluorecent protein in respect to excitation and emmission bands. Here it is important to have a clear separation of your two colors (then you do not need to perform spectral unmixing of your data). If possible use broad filters (this means you will be able to detect more light). Check your filter setting with each color alone (to see if there is crosstalk between your channals)
- try the autofocus feature of your microscope if it contains one
Hope that helps you,
best GAD
Hi Virginia,
since you are asking which hardware to use, this means for me, you are at the planing stage of your experiment. Here some general advices:
- if possible use a LED light source to avoid phototoxicisity
- avoid colors in the blue range (like BFP, CFP) to avoid phototoxicisity
- thing about the size of the structures you want to see, this leads you (most probably) to the objective magnification (eg nuclues vs cell organelles)
- thing about the intensity of the signal you want follow (huge signal intensities in combination which a large structure --> smaller magnification nessecary, low signals (even in large structures) higher magnification nessecary)
- use objective with a high numerical apperture (if opossible NA>1)
- use objectives made for fluorescence live-cell imaging
- set your exposure time as small as possible (to avoid phototoxicity
- use filters which fit well for your fluorecent protein in respect to excitation and emmission bands. Here it is important to have a clear separation of your two colors (then you do not need to perform spectral unmixing of your data). If possible use broad filters (this means you will be able to detect more light). Check your filter setting with each color alone (to see if there is crosstalk between your channals)
- try the autofocus feature of your microscope if it contains one
Hope that helps you,
best GAD
Hi Virginia
I use stable cell lines for time lapse, for example H2B tomato stable transfected cells. For longer time lapse a transient transfection leads to an decrease in fluorescense.
Best Kerstin
In general, as mentioned, keep fluorescence excitation short to limit phototoxicity.
Check your medium for ingredients which may become toxic under your light source.
As Mayan mentioned, we'll need details in order to offer better advice.
1) Is it an inverted scope?
2) Are you culturing in a petri dish?
3) I presume you are using monolayers/adherent cells?
4) Are you using transmitted light or phase? Or just fluorescence?
5) Do you have z stack capability in the microscope stage and the software?
6) How long do you need to record for, and at what kind of frequency? 48 h @ 5 mins?
7) Are you regulating temperature, CO2, evaporation? Medium perfusion?
Focal drift can be due to evaporation, physical movement of the sample/scope...
Good luck,
Stuart
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