Sequential and simultaneous
When imaging a single fluorescent dye it is usually only necessary to (1) select the laser which most efficiently excites the fluorophore and (2) select the emission filter that most efficiently allows the emitted fluorescent light to reach the detector. However, when attempting to image multiple fluorescent probes in the same sample, it is essential that excitation laser wavelengths and emission filters are carefully chosen. In order to do this, researchers must have a more thorough understanding of the excitation and emission spectra of the fluorescent dyes they are using.
There are a range of wavelengths (colours) of light that can excite a fluorescent probe, with some wavelengths being more efficient at excitation than other wavelengths. A graph of all excitation wavelengths (and the efficiency of excitation) represents the excitation spectrum for that particular probe. The excitation spectrum for the DNA stain DAPI is shown below in the dotted line.
When DAPI is excited by the appropriate wavelength of light, the molecule enters a transient excited state for a short period of time (the "lifetime" which, for many probes used in confocal microscopy, is usually between 1 and 10 nanoseconds), before it may decay back to some resting energy state and emitting the previously absorbed energy as a photon of light. The emitted light can have a range of wavelengths of varying intensities and a graph of these values is called the emission spectrum. The emission spectrum for DAPI (when bound to DNA) is shown as the shaded area in the graph below:
Fig 1: Excitation (dotted line) and emission (solid line) spectra for DAPI. Note the range of wavelengths emitted when DAPI fluoresces (from approximately 400nm (violet) to 600nm (red)) and the maximum value at approximately 440nm.
To image DAPI on a confocal microscope, it is best to excite DAPI with a laser with a wavelength close to the excitation peak (approximately 350nm (UV)). However, UV lasers are expensive and can easily alter biological molecules, so they are seldom used. Instead, since DAPI fluorescence is usually very strong, a cheaper 405nm (violet) laser (which excites DAPI with about only 10% efficiency) is more commonly used. DAPI fluorescence is usually detected through a 440nm longpass filter. However, if more than one fluorescent probe is used in the same sample, it is more common to detect DAPI fluorescence through a 450/50nm bandpass filter.
It is well worth visiting the animated tutorials on fluorescence, fluorescence spectra, filters and lasers that can be found on the Life Technologies website: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
Fluorescence Spectral Overlap
When two (or more) fluorescent probes are used within the same sample, it is possible that the emission spectra of the two probes overlap (Fig 2).
Fig 2: Emission spectra for DAPI (blue) and AF488 (green). Note the overlap in the spectra between approximately 500nm and 600nm.
Since the fluorescent signal is usually only detected as an intensity of photons passing through an emission filter, separating the signals from each probe can become particularly difficult.
Imaging more than one fluorescent probe at the same time (exciting with all required lasers and detecting the signal through all filters at the same time) is usually referred to as simultaneous image collection.
In a typical experiment simultaneously imaging the fluorescent probes DAPI and AlexaFluor 488 (AF488) involves exciting both fluorophores with both the 405nm and 488nm laser at the same time:
Fig 3: Simultaneous excitation of both DAPI and AF488.
The fluorescence signals resulting from dual excitation of these probes are usually collected in two separate detectors through two separate emission filters.
The DAPI signal is collected through a 450/50nm bandpass filter. This signal should have very little signal emitted by the AF488 fluorophore (since the AF488 emission will not pass through the 450/50nm filter).
Fig 4: Emission spectra for DAPI and AF488. DAPI fluorescence is detected through a 450/50nm bandpass filter usually into the first detector.
The AF488 signal is collected through a 525/50nm bandpass filter. However, as can be seen in figure 5, at least some of the fluorescence emission from both DAPI and AF488 will pass through the 525/50nm filter. In this example, almost 30% of the total signal detected in the second detector will originate from DAPI. Detection of more than one fluorescence signal in a single detector is usually referred to as spectral bleedthrough and it can be very difficult to separate the different fluorophore signals from each other.
Fig 5: Emission spectra for DAPI and AF488. DAPI fluorescence is detected through a 450/50nm bandpass filter (usually into the first detector) while AF488 fluorescence is detected through a 525/50nm bandpass filter (usually into the second detector). However, note that almost 30% of the signal seen in detector 2 originates from DAPI fluorescence.
Simultaneous imaging of more than one fluorescent probe has the advantages of more rapid image collection and, for live cell imaging, no temporal displacement between the two images. Good experimental design using adequately spectrally separated fluorophores can avoid (or at least minimise) spectral bleedthrough. An example of this would be imaging DAPI and AlexaFluor 647 (AF647) (Fig 6).
Fig 6: Simultaneous imaging of DAPI and AF647 in a dual labelled specimen is possible without spectral bleedthrough because of minimal cross-excitation or emission of the two fluorophores.
Sequential imaging involves exciting fluorophores on the specimen with only one laser at a time and collecting fluorescence photons emitted by the excited fluorophores. Then, by swapping to another laser wavelength and detecting photons emitted from another fluorophore, spectrally separated signals can be collected. Provided each laser line excites only one fluorophore, all emitted photons will be derived only from the relevant fluorophore with no spectral bleedthrough.
When sequentially imaged a specimen that has been dual labelled with both DAPI and AF488, a single laser is used for excitation and the emitted photons collected (Fig 7a). Then the other laser is used for excitation and the relevant emission photons collected (Fig 7b).
Fig 7a: Sequential imaging of DAPI and AF488 in a dual labelled specimen. In this example, DAPI is imaged first (and the 488nm laser is turned off during image collection).
Fig 7b: Sequential imaging of DAPI and AF488 in a dual labelled specimen. When imaging the second fluorophore (AF488), the 405nm laser is turned off during image collection and the only signal collected is derived from AF488.
Simultaneous imaging three or four fluorophores in the same specimen will almost always produce significant spectral bleedthrough (as can be seen in Fig 8). Careful selection of fluorophores combined with sequential imaging can usually eliminate (or at least minimise) spectral bleedthrough.
Fig 8: Sequential imaging of DAPI, AF488, AF568 and AF647 in a four fluorophore labelled specimen can minimise spectral bleedthrough.
Sequential imaging has the advantage of minimising spectral bleedthrough and, in most cases, should always be used when performing analyses of colocalisation of multiple fluorophores. However, sequential imaging of four fluorophores means image collection time will take at least four times longer. Additionally, the temporal separation between each fluorophore image using sequential imaging usually precludes using this technique to image rapidly moving live samples.
Some Possible Fluorophore Combinations
|DAPI + AF488||1|
|DAPI + AF568||2|
|DAPI + AF647|
|DAPI + AF488 + AF568||3|
|DAPI + AF488 + AF647||4|
|DAPI + AF568 + AF647||5|
|DAPI + AF488 + AF568 + AF647||6|
- DAPI bleedthrough into AF488 channel.
- DAPI bleedthrough into AF568 channel if DAPI signal is very strong.
- DAPI bleedthrough into AF488 channel and, if DAPI signal is very strong, also into AF568 channel
- DAPI bleedthrough into AF488 channel but not AF647 channel.
- Minimal DAPI bleedthrough into AF568 channel; significant AF568 bleedthrough into AF647 channel.
- Spectral bleedthrough into all channels except DAPI channel.
All graphs on this page were generated on the Fluorescence SpectraViewer from the Life Technologies website: