Confocal Excitation

From Filter Wheels to AOTF

December 09, 2011

Fluorescence excitation needs specifically colored light: it should excite the probe efficiently (close to the excitation maximum) but leave room to collect the emission without spilling into the detection path. In confocal microscopy, multiline lasers or laser “batteries” are classically used. This arrangement requires devices that pick the requested lines which fit the currently employed fluorochromes. Intensity control is another task that must be accomplished – most lasers will show increased noise when intensity is controlled directly at the source. The introduction of acousto-optical tunable filters has simplified filtering and at the same time improved the flexibility significantly. For coupling white light laser sources, the AOTF is only sensible device.

Filter wheels – the early approach

The light source for fluorescence excitation in confocal microscopes is a laser or a combination of lasers with varying color. Lasers are used for technical reasons [2]. Unfortunately, traditional lasers emit usually only a single wavelength, or a few lines only (a modern solution is the white light laser [3]). However, the fluorochromes used in biomedical imaging show excitation bands anywhere in the spectrum between near UV and near IR. In most cases, it is necessary to record emission from several different fluorochromes at the same time. To accomplish this task, a series of laser beams are combined by dichroic mirrors. In this case, the mirror is used in an inverse mode as compared to "beam splitters" that separate excitation and emission light in fluorescence illumination.

As lasers in general have a comparably narrow band of low-noise operation, it is not advisable to control the intensity by the laser power directly. Consequently, a device is needed that allows the laser intensity to be attenuated separately. Similarly, laser lifetime will decrease if the source is switched off and on too many times. Gas lasers in particular do not tolerate power on when still at high temperature.

A typical implementation of all these requirements is a "laser battery", consisting of a series of lasers (usually 3 to 5). All lasers are equipped with light shutters that are driven electromechanically and controlled by software. All lasers need a servo-controlled device – a wheel or slider with a sequence of grey filters – for attenuation. Multiline lasers require in addition a wheel or slider that contains a series of edge and band filters in order to pick a single line or combinations of lines.

Obviously, the whole setup can become slightly byzantine, it is prone to misalignments and drift problems, it may introduce mechanical vibrations during filter selection. It is also quite slow when illumination regimes need to be modified, and due to the limited number of filters that fit on a wheel or slider, the device is also very inflexible.

Laser battery, as introduced in early confocal microscopes, e.g. Leica CLSM and Leica TCS 4D. The system is rather inflexible as compared to an AOTF solution and is very elegantly replaced by recent developments that employ supercontinuum generation devic

Fig. 1: Classical "laser battery" with 5 laser tubes, 5 mechanical light shutters, 8 beam combination mirrors, 5 attenuation filter wheels and 1 line selection filter wheel for multiparameter confocal microscopy.

Acousto-optical tunable filter – method of operation

A significant step for simplifying the mechanical setup and increasing the flexibility by orders of magnitude is the implementation of an acousto optical filter (AOTF). Basically, an AOTF is a device that allows desired colors to be controlled to point in a different direction, whereas the rest of the colors pass straight through the AOTF [1]. AOTFs are made of a crystal, typically TeO2 or SiO2 or other compounds with similar properties. The crystal is excited by a mechanical wave in the range of a few hundred MHz by a mechanical transducer (the term "acoustic" refers to the mechanical waves, although the frequency does not meet what we would tag as audible acoustics). Most importantly, the direction in which the desired beam is deflected is fixed and does not depend on the color of that light. And as an additional benefit, the amount of light that should point in the requested direction is controllable by the amplitude of the mechanical wave.

Upon irradiation with (collimated) laser lines of various wavelengths, as provided by a "laser battery", the AOTF allows any line to be picked and directed at any desired fraction of the full intensity in the requested direction. It is possible to select a multitude of lines truly simultaneously. The number depends mainly on the amount of electronic equipment one is prepared to invest. Decent systems offer 8 lines simultaneously.

In total, the AOTF completely replaces all shutters, attenuation filters and line selection filters described above by a single crystal that is mechanically fixed in place. Any combination of lines is easily selectable; when the laser battery offers a total of 8 lines, the number of combinations reaches 256 – not a problem with the AOTF. Plus: each of the selected lines is steplessly controllable in intensity. In essence, the AOTF is an "8-channel laser dimmer".

Engineers may argue that the AOTF directs the two polarization modes in different directions. This is completely true, but does not harm the concept. Lasers are polarized anyway, and consequently the light will always point in the desired direction. The perpendicular direction will stay empty without any consequences.

The AOTF in combination with classical laser combinations fully replaces all light shutters, attenuation filters and line selection filters. Leica first introduced the AOTF for confocal microscope systems in 1992. The first system equipped with AOTF was t

Fig. 2: AOTF (box on the right) replaces all shutters, attenuation filters and line selection filters in a classical laser battery for confocal illumination. Any of the lines that enter the AOTF may be suppressed, attenuated or fully transmitted for use in the confocal microscope. Any combination of the lines emitted by the laser may be combined (a very high, though finite number of combinations).

AOTF – free additional benefits

Fig. 3: Scanning regions of interest with different laser lines or different intensities of the same line. During line scanning, the illumination regime changes at preset points according to the predefined patterns. Any combination is possible, in virtually any pattern, which is drawn by hand with a graphical curser onto a prescanned image. Of course, geometrical patterns may be applied as well (rectangles, circles, ellipses…). “Zero lines” is also a pattern, and often used to protect the background cells in cell cultures.
The region-of-interest scan was first made possible by the introduction of the AOTF for confocal microscopes. The Leica TCS 4D was the first AOBS confocal. In region-of-interest scan mode, the lasers are switched quickly (within a few pixels, depending on

Besides the obvious benefit that the AOBS allows any laser combination that fits the composition of fluorochromes in the particular sample, it has further benefits. Let us assume that the specimen is stained with two different fluorochromes, one very strong and the other quite weak. Here, the stepless intensity tuning allows the fluorescence emission to be balanced in order to record both fluorescences at similar intensities by dimming the excitation for the bright dye. This is very easily possible online, during imaging. The control of the AOTF is fast and does not require the scan process to stop.

Balancing the excitation by stepless intensity control is not only beneficial for comparability of the fluorescence channels, it is also a valuable tool to improve separation of fluorochromes. Many dyes show long "tails" in their emission spectrum that cause the wrong dye to be picked up in the right channel. By reducing the excitation to a minimum, separation can be improved (crosstalk reduction).

Finally, the switch between sets of selected lines (and intensities) is very fast – within a few microseconds; compared to about a second for mechanical standard filter wheels. This fast switch allows the illumination regime (which lines at what intensities) to be changed during the scan of a single line. This feature is used to paint patterns of different illumination colors onto the sample, so called "regions of interest", where different lasers are active. It is also used to "blacken" the area where no signal has to be recorded. This keeps these regions of the sample alive for later research. The picture at the beginning of this article shows such regions of interest, where 5 different parameter settings have been applied in a single scan.

Finally, if a white light laser is chosen as light source, the AOTF offers to pick any of the colors that are available in the white spectrum. In this case, the tunable acousto optical filter turns the white light laser source into a tunable light source.

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