I want to equip the microscope to be able to make ratiometric measurements of fluorescence in living cells. I have searched several articles, but the information is ambiguous, some papers mention a shutter ..
Hi,
Which ratiometric probe?
Set up can be quite different depending on the probe
Philippe Hulin its for FRET (YFP/BFP) genetically encoded biosensor in living cells
First you need a good excitation source with same power for YFP and BFP or CFP.
LED are good but not perfect. The best is xenon lamp. Xenon has the same power for all excitations (in visible).
1) Expensive solution :
Dual bands emission filter with dual bands dichroïc mirror, with filters wheel in front of the source (xenon). You need a very high quality filter.
It's slower than with LED's because LED do not need filters. You can add a DualView system and acquire the image pair in the same time.
2) cheaper solution :
2 classics dichroïcs cubes. Good but slower.
Others solutions dear colleagues??
You have two answer a few questions first...
(i) What time resolution is needed?
If seconds is enough, 3 Filter sets are enough to get the signal and all controls: 1. BFP Excitation&emission, 2. BFP excitation/YFP emission. 3. YFP Excitation& Emission. If you know you have a fixed stoichiometry you might omit Nr. 3. With this you record all channels after each other, however changing filter-sets add some 100 ms for each filter-change.
If you need it faster, or the mechanical vibration is an issue for your sample,you will need either
(A) 2 cameras. Zeiss has their own solution, otherwise there is e.g. Cairn Twincam (affordable, one image is mirrored and must be mirrored in the software or in post) or hamamatsu w-view gemini 2C. or
(B) If a subset of the image is sufficient there are also solutions available to project two images with different colors on one chip, e.g. Cairn optosplit (Brands are not an endorsement, just implementations I know about.).
(II)Do you want to image or are you want to read out the average of the field of view?
Especially in the GPCR field often cell are picked and super-fused, and the whole cell signal is read out as signal. This is usually faster than imaging.
Idea: Signal form a microscopy port is spit by a dichroic and projected on two point detectors, e.g. PMTs, Signal than needs to be digitalized, often patch-clamp electronics is used for this.
There used to be a Photometry cystem by Till photonics, including a mask for selecting cell, which is unfortunately off the market. Similar concept follows, but without the mask is the DAGAN Photomax-200.
Once you figured your requirements out in more detail things like the excitation source can be discussed.
best,
Ralf
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