To feed light in an incident light beam path into the optical axis, a device is required that can simultaneously direct illumination onto the sample and pass the emission to the detector. The simplest approach would be a gray mirror reflecting e.g. 50 % of the light and transmitting 50 %. Although this arrangement would do the task, it is very inefficient. Half of the excitation energy is wasted, and also half of the emission from the sample. As light intensity is just a matter of technical investment, illumination losses are not a serious issue. On the other hand, the fluorescence emission is a precious signal that should be collected as completely as possible, a loss of 50% is not acceptable. Therefore, semitransparent mirrors with higher ratios may be used. Often, 80 % vs 20 % split is accepted, which implements a waste of 80% of the excitation light and collection of 80 % of the emission. Also, 95/5 splitters are sometimes discussed.
A significant improvement in the energy balance is the introduction of chromatic mirrors. These mirrors have delicate coatings that cause interference effects. Depending on the design of these multi-layer coatings, it is possible to reflect over 90 % of the desired excitation light (short wavelength) and transmit over 90 % of the emission light (longer wavelength). This is the typical approach when "dichromatic" or "dichroic" splitting mirrors are implemented. The simplest design reflects short wavelength and transmits long wavelength. The position of 50 % reflection and transmission is used to characterize the dichroic mirror (e.g. "dichroic 580"). Depending on the fluorochrome and the appropriate excitation color, a pool of various dichroic mirrors must be available if the system is to suit more than one single application.
A classic dichroic mirror is not sufficient for contemporary multiparameter fluorescence measurements and recordings. Here, typically 2 or 3 (or more) colors are simultaneously applied, and the corresponding emissions must be collected between the excitation bands. Still, the mirror technology was able to meet these requirements. Complicated coating protocols allow the production of mirrors with alternating reflection bands and transmission bands – tagged double, triple or quadruple dichroics.
For a long time, this solution was also the common approach for confocal fluorescence microscopy.
Fig. 1 left side: The incident light fluorescence beam path requires a device B that feeds the light from the light source L onto the sample S. The light emitted from the sample must pass through the same device in order to be recorded by a detector D. Such a device is called a "beam splitter". X and M refer to excitation-filter and emission-filter, respectively. Right side: Transmission/Reflexion of a dichroic mirror (long-pass version). The 50/50 position λo specifies the characteristics of this mirror.
A completely different solution is possible by employing acousto optical devices . As described in the tutorial about AOTF , the acousto optical crystal can be “excited” by a mechanical wave. The consequence of this mechanical excitation is the deflection of a very narrow spectral band (in the range of 1 ... 3 nm) to a different direction (1st order) compared to the non-refracted beam (0th order). How can we use this feature to generate a device for injection of excitation light in an incident-light fluorescence beam path?
The trick is to use the acousto-optical crystal in "reverse" mode. Upon applying a mechanical wave that would deflect the desired excitation color (for example the 488 nm Ar-line), this laser line is then in an unusual mode fed into the 1st order – and will consequently exit the crystal along the principal direction. The light must then pass through the objective lens and interact with the sample. Here, the fluorescence emission is generated and then collected by the objective lens. The emission will travel through the acousto optical crystal along the principal direction without losses, as the emission is Stokes-shifted and consequently has a different wavelength as compared to the used laser line. The emission is then directed to a detector, which is typically a multichannel (spectral) detection device.
As pointed out in the AOTF tutorial, the acousto-optical device is easily programmed to perform multiple lines for 1st order deflection simultaneously. For AOBS purposes, this immediately offers to simultaneously feed various combinations of laser lines onto the sample. With 8 electronic channels, it is immediately possible to create an "octuple dichroic" device – with all the freedom to decide which combinations of laser lines should be applied. For 8 lines, 256 different combinations are possible.
What an AOBS can do better
The invention of the AOBS solves many of the problems connected with mirror-type beam splitters. Firstly, there are no mechanical movements of parts. Consequently, there is no need for complicated co-alignment of mirrors on wheels or sliders. Also, there is no mechanical disturbance that might cause vibrations or other mechanical influences on the scanned image. And thirdly, the reprogramming is done within a matter of microseconds as compared to seconds in mechanical devices. The fast programming allows fast sequential scan modes to be improved, e.g. line-by-line changes of the illumination regime, without losses on the emission side due to fixed-feature mirrors. It also allows region-of-interest illumination with dedicated beam-splitting performance for each region and fast ratiometric imaging.
The emission collection by an AOBS is close to maximum. The average transmission is at about 96%, which is close enough to the absolute maximum. The bands required for injection of the excitation light are in the range of a few nanometers. This is not possible with mirror coatings, even when used in low-angle operation (a method where the beam is not fed at 45°, but smaller angles). Consequently, the efficiency in photon collection is best with acousto optical beam splitters. This is always an important requirement, but especially beneficial in low-light applications, live sample microscopy and similar situations.
As the AOBS is programmable, it serves for any desired wavelength or combination. It is not necessary to design a series of devices that fit for the requested combinations, in fact, it is even ready for any combination of new lines that may be available in the future. If the researcher’s application changes and requires other excitation regimes, it is not necessary to modifiy the dichroics wheel or slider (which usually needs a service engineer to work on-site). By a simple mouse-click, the desired feature is readily available in an AOBS.
For practical considerations, it is also worth noting that the programming of the AOBS is executed automatically by the software upon calling a laser line or any combination of available laser lines. There is no risk of having the wrong beam splitter in the emission path, which may cause unnecessarily noisy images or wrong spectral recordings.
The flexibility of the AOBS is virtual infinity. The tunability is stepless (usually by digital control 1 nm) over the full visible spectrum 400 … 800 nm. When assuming a minimum distance of 10 nm to the next excitation line (which is even a very conservative assumption), and a total of any number of lines between 1 and 8, the number of programmable designs exceeds the million mark. This is the precondition for coupling a white light laser source into a confocal microscope.
|AOBS||Dichroic 45°||Dichroic low angle|
|Switching speed||10 μs||≈ 1,000,000 μs||≈ 1,000,000 μs|
|Transmission (peak)||96%, flat white||90%, color modified||98%, color modified|
|Bandgap (loss)||2–6 nm||40–80 nm||20–40 nm|
|No. of bands simultaneously||8||4||4|
|Flexibility (8 laser lines)||256||≈ 8||≈ 8|
|Flexibility (White Light Laser)||› 100,000||≈ 8||≈ 8|
|Minimal distance of bands||‹ 10||50||50|
Tab. 1: Comparison of beam splitting devices. Clearly, the AOBS is the method of choice for all applications. Even the transmission, which is shown here as peak transmission over the full spectrum, is a very conservative 96 %, where modern dichromatic setups may locally feature a somewhat better transmission (the gain is marginal from 96 % to 98 %). Still, the AOBS has no fixed gaps, like any multiple-dichroic.
- V. Seyfried, H. Birk, R. Storz and H. Ulrich: Advances in multispectral confocal imaging. Progress in Biomedical optics and imaging. Vol 5139, 22 - 23 June, pp 146 ... 157
- Confocal Excitation - From Filter Wheels to AOTF
- Borlinghaus RT, Colors count: how the Challenge of Fluorescence was solved in Confocal Microscopy, Modern Research and Educational Topics in Microscopy. Editors: Méndez A and Díaz J, Formatex Vol 2, 890-899 (2007)