All of the applications of the FluidFM system can be easily followed through fluorescence or bright-field 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 emitting it at another one. A molecule that absorbs light at one wavelength (excitation) and emits it at another (emission) is called a 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:
Figure 1. 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 separate from the one used for excitation.
Fluorescence filters
To collect the light from 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. (a) Scheme of a fluorescence filter set (cube). (b) Available filters in the FluidFM BIO Series. Images adapted from Evident 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 OMNIUM, the filter sets available are the following ones:
Filter | Excitation Filter | Emission Filter | Color in the visible spectra |
U-FUNA | 360 – 370 nm | 420 – 460 nm | Blue |
U-F39002 (GFP) | 480 nm | 535 nm | Green |
U-F39004 | 540 nm (528 - 552) | 605 nm (578 - 632) | Red |
U-F49004 | 545 nm (533 - 557) | 605 nm (579 - 640) | Red |
What should be considered when selecting a fluorophore and a filter set?
To obtain a better resolution of our samples and adapt the fluorescence intensity to our needs, it is important to consider the following concepts when selecting the fluorescence presets and fluorophores for the experiment:
- 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.
- Photostability. The fluorophore must be able to maintain its properties after repeated exposures to illumination light.
- Excitation and emission properties of the fluorophore. The filter set should be compatible with the range of wavelengths of the fluorophore (and vice versa).
- Environmental stability. Some fluorophores can be sensitive to air, light or temperature.
The table below lists common fluorophores used in FluidFM experiments and their compatible filter sets:
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) |
Imaging Presets Definition
Define and test imaging parameters for each sample type and experimental condition to ensure optimal image quality.
To adjust imaging parameters: open the Settings menu in the right panel of ARYA and select View.
Figure 3. List of imaging parameters available in ARYA.
- Illumination: The relative output level of the light source setting determining the amount of transmitted light directed into the optical path.
- Brightfield illumination is delivered through the probe's optical path, so changes in probe height (Z-position) will affect image brightness. Ensure the probe is at a representative working height when defining presets.
- Target Lens: Magnification used.
- Target Mirror: Fluorescence filter used.
- Shutter: Controls UV light path (open or closed).
- Attenuator: Controls excitation light transmission to the sample via motorized neutral density filters (higher value = higher irradiance).
- Light paths (for left or right): determines how light is directed through excitation, objective, and detection components to the camera or observation port (eye piece).
- Autoexposure: Automatic adjustment of exposure time to reach a defined target image brightness without manual intervention.
- When off, the duration for which the camera sensor collects light during image acquisition (exposure time), typically expressed in milliseconds (ms). Longer exposure times increase signal intensity but may increase noise and risk of saturation.
- Autogain: Automatic adjustment of the Electronic amplification applied to the camera signal (gain).
- When off, gain value needs to be defined. Higher gain increases brightness but also increases noise.
Imaging parameters are critical for cell survival and should be optimized to minimize phototoxicity.
After optimizing your imaging parameters, save them as a preset using the Preset tool in the left panel. Keep in mind that brightfield presets are optimized for a specific probe height, so if the probe Z-position changes significantly, brightfield illumination may need to be re-adjusted.
Imaging presets can be used for live experiments (e.g., to follow an injection or an extraction). Defined presets can also be chosen for long term observation (observe workflow) or for tracking during your Live-Seq experiments (Observation and Cell Tracking sections in Clean Biopsy Collection).
How can you use the data from an observe workflow?
When using the Observe workflow in ARYA, we can get time-lapse images of selected points (e.g. cells) with selected presets. Together with ImageJ a time-lapse video can be generated for any observed point. In the example below, we generate a video of CHO cells injected with Lucifer Yellow and Alexa 568. In the experiment, a picture of all injected cells was taken every hour for 24h after the injection, in ausing presets for bright field and two fluorescence channels (green and red). A merged image of the 3 channels is also generated by default in ARYA.
- EXPORTING DATA OF A SPECIFIC POINT WITH ARYA
Once the Observe workflow is complete, click on Results history (folder icon, on the bottom left corner of ARYA, indicated in red):
Figure 4. In red, results history in the left side of ARYA.
From the different display modes, select the one on the right (Point filter), which will group the images according to points. Select a point of interest: images will appear chronologically in the menu below. Click then in "Export point data...".
Figure 5. How to export data from specific points in ARYA.
The export menu for images will appear ([Linkplaceholder: Results export section]). The final folder where the pictures will be stored must be specified: this folder must contain ONLY the exported pictures (make sure not to create subdirectories nor export meta data as CSV). Click on "Export".
- CREATING A TIME-LAPSE WITH IMAGE J
Download and install ImageJ (http://imagej.net/ij/download.html).
Open ImageJ and import the images of interest by selecting File>Import>Image Sequence:
Figure 6. Importing the observation pictures to ImageJ.
Select the folder with the images and then, one image of it. Click "Open".
Specify the sequence options for image importing:
Figure 7. Importing the image sequence into ImageJ.
- Number of images: Enter the total number of images in the folder.
- Starting image: Enter the position number of the first image in your sequence. For example, if your merged images begin at position 4 in the folder, enter 4.
- Increment: Enter the step size between images. For example, enter 4 to import every fourth image (e.g., to select only merged images from a multi-channel series).
Click "Ok".
ImageJ generates a stacked image file:
Figure 8. Stacked file generated after introducing the importing sequence.
To save the video, select File > Save As > AVI. Choose the compression format (JPEG or PNG) and set the frame rate.
Figure 9. Video properties definition.