Connecting the Dots: Laser Scanning in Confocal and Multiphoton Microscopes

par | Fév 7, 2023

Taking the time to understand the inner workings of an imaging system can help you design more robust experiments and acquire higher-quality data. It can also help you trouble-shoot when a system is not performing as expected. Over the next few posts, we will highlight some key examples, starting with comparing the illumination and detection systems of wide-field and scanning microscopes. As the name implies, wide-field microscopes illuminate the entire field of view at once. When using a wide-field microscope, we can view or capture the image in one go, much as when we snap a picture with a smartphone. By contrast, scanning microscopes detect fluorescence point by point, so that the image is built up over time (brief summary of relevance of wide-field vs. confocal for a biologist). 

When using a standard wide-field fluorescence microscope, the images tend to look bright, but on closer inspection, they also contain substantial blur and haze. This occurs because the illumination light excites fluorescence from a volume that is much greater than the volume that is in focus. The image acquired then contains the focused image, but overlaid with the fluorescence emission from other regions within the illuminated volume that are not in focus (see tutorial here).

Confocal microscopes minimize out of focus haze by placing a physical barrier known as a pinhole before a detector. The pinhole rejects the out of focus light while allowing the focused light to reach the detector. Multiphoton imaging also reduces out-of-focus light through the underlying photophysics.  The result is that multiphoton fluorescence emission, unlike the single-photon process, is confined to a small volume as we discussed in a previous post. So we don’t detect much haze and blur because there is little there in the first place. Although both confocal and multiphoton microscopes reduce out-of-focus light in different ways, they both lead to similar results- clearer thin images of a sample that can be used to create 3D visualizations of objects. We call this “optical sectioning,” as we use light to create thin views of our specimens. 

Laser scanning confocal and almost all multiphoton microscopes use scanning systems that excite and record fluorescence in sequence rather than all at once. There are many ways to scan over the sample, yet the most common approach is to move the excitation laser beam across the sample sequentially in X and Y by using precise and electrically controlled mirrors. These electromechanical optical devices are known as “galvo mirrors”, where galvo is shorthand for galvanometer. Sometimes you might come across references to “resonant scanners” which refer to specialized high-speed galvos that are often used in combination with a standard galvo unit. These can be less flexible than slower standard galvo mirrors, but can offer advantages due to their higher speeds. 

The galvo mirrors tilt and deflect the laser so that it can move across the sample in X and Y within the field of view.  As the laser spot moves and excites fluorescence, the detectors record the fluorescence for each location. Each point represents a separate reading that can be viewed as an individual pixel in the captured image.

As shown in the first figure, one of the mirrors, called the “X” mirror, moves the laser from left to right along a line in the field of view (left side). When the laser spot reaches the end of the line, the “X” mirror snaps back to the start position, while the “Y” mirror steps the laser down a small amount at the same time (middle). The “X” mirror then begins sweeping the laser from left to right again, this time along the second line (right). This pattern repeats, with the “X” mirror moving left-to-right and the “Y” mirror stepping down each line, until the laser is scanned over the entire field of view, as shown in the next figure.

It is possible to tell the galvo mirrors to go faster or slower by controlling the speed at which they sweep the sample. This is often described as “pixel dwell time” and it is the length of time the laser rests on each spot in the field of view. Standard galvo mirrors can sweep across a field of view about 500-1000 times per second, so an image 512 pixels across by 512 lines down can be acquired in about 0.5 to 1 second.

A laser scanning confocal microscope typically allows the operator to adjust the pixel dwell time (and by extension, the frame rate). Note pixel dwell is inversely related to the frame rate. Frame rate is how many frames the microscope can scan in a second (this is the convention). When you increase pixel dwell, each frame takes longer to scan as the laser is moving slower. Thus, increasing pixel dwell decreases frame rate, but increases signal. 

Often you can select the pixel dwell time and/or frame rate in the acquisition software. By slowing the motion of the laser spot down as it moves across the sample the pixel dwell is increased and the detector gathers more signal from each spot. With most shot-limited detectors such as PMTs, often it is better to average multiple lines or images from a faster scan rather than to increase dwell time. Yet there are applications where the converse, increasing dwell time, is the better choice, such as when you are using the laser to photoconvert or photobleach your fluorescent probe. 

Pixel dwell time and frame rate are important when carrying out live-cell imaging, especially when the changes you want to track are fast relative to the image acquisition. Some pointers for speeding up image acquisition can be found here.

The addition of laser scanning galvo mirrors require more complicated design, manufacture, and alignment. Unsurprisingly, these instruments are more expensive to purchase and operate compared to standard wide-field microscopes. The additional features and settings also take more time and effort to learn how to use compared to a standard wide-field microscope. Yet by informing yourself, you can unlock the potential of these flexible and sophisticated instruments. We will highlight some more examples in future posts.

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