Speed Running Your Sample: Faster Laser Scanning in Confocal and Multiphoton Microscopes

par | Mar 22, 2023

In our previous post, we discussed galvo scanning systems used in laser scanning confocal and multiphoton microscopes. Galvo scanning is standard, yet for some applications, such as live-cell and large-format image acquisitions, systems that offer increased acquisition speeds are desirable or even required. Although we can speed up galvo scanning by reducing our field-of-view (FOV), such as by imaging a thin horizontal stripe, sometimes reducing the FOV is not feasible, such as when trying to capture rare and fast events and/or when stitching images to create large panoramas

When higher scanning speeds are required, it is important to consider different types of scanning systems. Higher scanning speeds can be achieved using faster mechanisms such as resonant and polygonal scanning mirrors. These systems allow the laser to sweep over the sample 10-20x faster than a standard galvo scanner allowing the user to capture full fields of view at “video rate” (30 frames per second, fps) or better. In practical terms, it is possible to acquire many 10’s of frames a second with a video rate system as compared to the single frame per second of a conventional galvo scanner. 

Fig.1 shows a diagram of a resonant scanner. The term “resonant” refers to the fundamental physical property known as resonance. Physical objects have a unique “natural frequency” of vibration. When applying an external vibration such as a sound wave that matches the natural frequency, the object will vibrate at its unique frequency in response. A dramatic example is provided by applying sound waves to a wine glass. When a singer hits just the right note that matches the natural frequency of the wine glass, the glass can resonate or “sing” or even shatter if the applied sound is loud enough. 

In the case of the mirror, an electrical or magnetic drive signal is applied at the resonant frequency of the mirror and its holder, such that the overall vibration causes the mirror to rotate back and forth. The rotation angle is shown as in Fig.1. Now, if a laser beam is directed onto the mirror, it will reflect back (the law of reflection).  As the mirror rotates back and forth, the reflected laser beam will also move from P1 to P2, tracing a line across the sample with the laser.

 

Fig.1 A resonant scanning mirror draws a path from P1 to P2 as it rotates (different positions shown as light and dark grey). Not shown: second galvo mirror used for Y-axis deflection.

 

One drawback to resonant scanning is that the rotation speed of the mirror changes as it rotates around the angle (). Figure 2 shows the position of the mirror as a function of time. Ideally, the mirror could instantaneously flip direction without any change in speed (the ideal response is shown as a hatched blue line), yet as a physical object when the mirror reaches the end of its sweep, it slows down, very briefly stops, then has to reverse and accelerate back in the other direction. The true behavior is shown by the solid red line, as the system needs time to settle and resume motion.

The consequence of the variable speed is that the pixel dwell on the sample must slow down as the mirror changes direction at the end of each sweep to go back the other way. This causes longer laser exposure at the edges of the frame compared to the middle. Commercial systems minimize the effects of variable speeds by cropping out the edges of the scan where the direction reverses or by applying more sophisticated software/hardware corrections.

 

Fig.2 Scanning profile of a resonant galvo mirror. Inset: Solid red line showing actual scan path is offset from blue dashed line showing ideal trajectory. Beam only travels at a relatively constant speed halfway between Position 1 and 2, and must slow down and reverse at Position 1 and 2.

 

Another high-speed scanning system, known as a polygonal mirror, is shown in Figure 3. To draw from a real-life example, a polygonal scanner can be compared to a disco ball. They are also used in some barcode scanners. A polygonal mirror is a prism with many sides which are covered in a reflective coating, much like a disco ball is a sphere covered in many small flat mirrors. 

As shown in Figure 3, when a laser beam hits a mirrored facet on the polygon it is reflected at an angle equal and opposite to the incoming beam. By spinning the polygonal mirror, the laser beam can be deflected at different angles as the facet angle shifts. The overall scan cycle is shown in Figure 4: (A-C) the mirror rotates and the beam moves. As the facet edge rotates past the beam (D), the laser snaps back to the beginning of its arc and begins to sweep again (back to A). Unlike the resonant galvo, the polygon mirror speed is not fixed (does not need to resonate at a particular frequency) so the wheel can be spun at a variety of speeds, allowing the user to adjust the scan rate.

A polygonal mirror also offers constant pixel dwell time as it spins, as long as it rotates at a constant rate. This rate is maintained by electronics which control the electric motor spinning the polygonal mirror, holding it at a velocity set by the user. Figure 5 shows the scan pattern of a polygonal mirror. Compare this to the scan pattern of the resonant mirror shown previously in Figure 2: The polygonal mirror will give a constant rate (straight line in position and time Fig.5) as it traverses from P1 to P2, whereas the resonant scanner follows a sinusoidal path (Fig.2) with variable speed (sinusoidal behavior at endpoints). Polygonal mirrors can also be affected by other factors, yet as with resonant mirrors, precision engineering minimizes the effects on imaging quality.

 

Fig.3 A polygonal scanning mirror rotating around center point.

 

Fig.4 Single scanning sweep as one facet is rolled through the laser beam. The beam will sweep from P1 to P2 as many times as the polygon has facets for each complete rotation of the polygon. I.e. if the polygon has 10 sides, the laser will sweep from P1 to P2 ten times for each complete rotation of the polygon.

 

Fig.5 Scanning behavior of a polygonal mirror. Mirror moves from position P1 to P2 at a constant rate along the straight line labeled “Scan One Line”, then snaps immediately back to P1 and begins the next sweep.

 

The higher frame rates provided by these scanners compared to standard systems can be useful for capturing fast fluorescence events, such as calcium imaging in neuronal populations, or tracking dynamic events in cell populations.

It is important to recognize that high speed means that the time the laser spends at each point in the sample is reduced. In other words, the pixel dwell time is reduced and thus proportionally less light is detected. As an example, if you capture 20 frames in one second, each frame will have about 1/20th the light compared to a frame that took an entire second to capture. In terms of pixel dwell times, a standard galvanometer scanner provides pixel dwells of 0.5 to tens of microseconds, while a polygonal or resonant system has pixel dwells on the order of 10’s to 100’s of nanoseconds (0.1 to 0.01 microseconds!).

When viewing images from high-speed scanning for the first time, users may even think something is wrong with their system if their only prior experience was on a widefield or a standard laser scanning confocal. This is because these techniques acquire images relatively slowly, with plenty of time to accumulate high signal. 

When working with fixed samples this works very well and gives a very clean-looking image. In comparison, video rate systems with their shorter acquisition times gather much less light and so each individual frame is dimmer. So when comparing images, it is also important to compare the time required to image the sample. While each individual frame of a video-rate acquisition can be “noisy” on its own, it is possible to easily improve imaging performance by applying a technique known as averaging. In our next blog post, we will discuss metrics of imaging noise, explore how averaging reduces it, and illustrate with some example images.

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