Can anyone help with a fluorescence microscopy choose
Most of the applications of the FluidFM systems can be easily followed through fluorescence microscopy: from spotting a fluorescent solution in a glass surface to the injection of specific complexes into single adherent cells.
Fluorescence and fluorophores
Fluorescence refers to the physical property of an object absorbing light at one specific wavelength and then remitting it at another one. Then, when a molecule absorbs light (or it is excited) of one wavelength and emits in another one, that molecule is called fluorophore.
Each fluorophore has a characteristic excitation and emission spectra. Maximum values are the peaks of excitation and emission, respectively. However, most fluorophores absorb and emit in a range of wavelengths; for imaging, it is useful to think not just about their peaks, but also about the full spectrum of absorption (or excitation) and emission:
Excitation (blue) and emission (orange) spectra for Lucifer Yellow; peaks for excitation and emission are 428 and 544 nm, respectively.
The properties in the excitation and emission spectra can also determine the visualization under the fluorescence microscopy: the closer the spectra are, the more difficult it will be to see the emitted light from the labeled object as a separate from the one used for excitation.
Fluorescence filters
optical filters bandpass In order to collect the light form the specific fluorophore, a set of filters is introduced in the microscope: exciter filter, dichroic mirror and emission filter (or barrier filter). In our system, the three filters are located into a cube (figure 2).
Scheme of a fluorescence filter set (cube). (b) Available filters in the FluidFM BIO Series. Images adapted from
www.optolongfilter.com
website
The exciter filter has generally a defined band of wavelengths that it allows through. After going through this filter, it reflects onto the sample. Fluorophores in the sample will then become excited and, thus, emit light at a different wavelength. The dichroic mirror and, then, the emission filter will filter this emitted light.
In the FluidFM BIO Series, the filter sets available are the following ones:
Filter
Excitation Filter
Emission Filter
Corresponding color in the visible spectrum
U-FUNA
360-370 nm
420-460 nm
Blue
U-F39002
480 nm
535 nm
Green
U-F39004
540 nm
605 nm
Red
What should be considered when selecting a fluorophore and a filter set?
In order to obtain a better resolution of our samples and adapt the fluorescence intensity to our needs, it is important to take into account the following concepts when selecting the fluorescence presets and fluorophores for the experiment: fluorescent light filter
1. Selectivity of the fluorescent label. The fluorophore must be specific for a target: a molecule, a biological activity or a cellular location, without nonspecific background signal.
2. Photostability. The fluorophore must be able to maintain its properties after repeated exposures to illumination light.
3. Excitation and emission properties of the fluorophore. The filter set should be compatible with the range of wavelengths of the fluorophore (and vice versa).
4. Environmental stability. Some fluorophores can be sensitive to air, light or temperature.
Fluorophore
Excitation wavelength
Emission wavelength
Fluorescence filter
Lucifer Yellow
428 nm
544 nm
U-F39002 (green)
DAPI
359 nm
461 nm
U-FUNA (blue)
Hoechst
352 nm
455 nm
U-FUNA (blue)
Propidium Iodide
300 nm
610 nm
U-F39004 (red)
Alexa Fluor 555 dextran
555 nm
585 nm
U-F39004 (red)
Alexa Fluor 568
580 nm
600 nm
U-F39004 (red)
mCherry
587 nm
610 nm
U-F39004 (red)
GFP
480 nm
500 nm
U-F39002 (green)
Here, there is a list of the most common fluorescence filters used in FluidFM experiments, together with the filter sets used for its observation:
