Light Sheet vs. Confocal Microscopy

It is no exaggeration to say that life sciences bench research is heavily reliant on fluorescent probes—whether endogenously expressed or added exogenously—to localize various structures, proteins, and processes within cells, tissues, and sometimes even whole organisms, both at a given time and over the course of time. While these have traditionally been viewed on widefield microscopes, scientists are increasingly taking advantage of the benefits of confocal microcopy, and more recently light sheet microcopy, such as improved resolution and optical sectioning. Here we compare and contrast these technologies and how they address the biological questions faced.

How they do what they do

The main principle of confocal microscopy is that while the entire z-axis of a sample is illuminated, light that is out of the focal is rejected. (Thus the term “confocal,” meaning conjugate focal plane, points out Gary Laevsky, Director, Confocal Imaging Facility, Princeton University.) The excitation beam (most often a scanned laser) illuminates the sample in a Gaussian distribution (bell curve) along the z-axis, so there is illumination from above and below the focal plane. By placing a pinole in the emission light path “you only collect the data that’s coming from the waist of the Gaussian beam,” essentially throwing away what is not in focus, he explains. Opening or closing the pinhole, or using multiple pinholes, allows the user to attain the best resolution or confocality.

As with widefield microscopy, adjusting the plane of focus allows for the sample to be “optically sectioned,” only to a much more profound degree as there is a much greater contrast between what is in focus and what is not.

Among the driving factors in developing light sheet microscopes (LSFM, for light sheet fluorescent microscopes) was to allow long-term imaging studies in fields like developmental and cell biology, where a goal is to minimize disturbing the sample by the process of imaging itself.

A typical LSFM uses a Gaussian sheet of light, rather than a single point, to illuminate the sample from the side. The sheet is positioned at the focal plane, at a 90°angle to the detection lens. It illuminates a 2D array, which is mapped to a camera chip, describes Kurt Weiss, Director of Biochemistry Optical Core, University of Wisconsin-Madison. The chip is simultaneously detecting about 2000x2000=4 million pixels, versus one at a time to a PMT [photomultiplier tube] in a point scanning confocal.

This leads to three distinct advantages to the LSFM over the traditional point scanning confocal, he continues:

• You can image much faster.
• You can increase the pixel dwell time and reduce the light intensity.
• Perhaps most importantly, you only illuminate the plane which you image, so no illumination light needs to be “thrown away.”

The result is that the effects of illumination—photobleaching and phototoxicity—are significantly reduced.

The principal drawback to a LSFM, Weiss points out, is the need to fit the lenses quite close together and very near the sample, drastically constraining the available sample mounting and lens choices.

What they’re good for

In general, a confocal microscope will offer higher resolution imaging, making it a good choice for studying cellular structures, but at a lower speed and more light exposure than a LSFM. Weiss considers a thin layer of adherent cells, “preferably fixed and stained with a bright dye,” to be an optimal sample for a confocal. The optimal sample for a conventional LSFM “is something that needs moderate magnification (actually moderate NA owing to long working distances to fit lenses and what can fit between lenses),” citing an entire Zebrafish embryo as an example, with cleared tissue being another oft-cited example. The benefit is greatest in thicker samples where a full 3D acquisition is required, with diminishing returns as the sample thickness is reduced, Weiss says.

Of course, there are trade-offs to be reckoned with when looking for the best system to meet your research needs. For example, a Nature Reviews article suggests several factors to consider when selecting a microscope to answer a project’s key biological questions: sample viability, speed, resolution, contrast, and depth penetration.

There are other considerations as well, adds Weiss: “data processing complexity, mounting options, sample transparency, system cost, commercial vs. DIY, system complexity (alignment), and range of magnification/sample types.”

Laevsky explains that while a big confocal data set might approach a few 100 megabytes, “data sets from light sheets in the terabyte realm are totally run of the mill.”

Blurred choices

While consideration of many of these variables may make the choice between a traditional confocal and LSFM relatively straightforward, “things get more blurred as you consider different methods of forming confocal pinholes (spinning disc and line-scanning confocal) and different ways of generating and scanning a light sheet (DSLM [digital scanned laser light-sheet fluorescence microscopy], ASLM [axially scanned light-sheet microscopy], single objective),” explains Weiss. “Here confocal becomes more efficient and light sheet becomes less efficient in terms of parallelization and light dose, with trade-offs to get better speed in confocal and better sectioning and favorable sample and lens geometries in LSFM.”

There are other variations on and relatives of confocals and LSFMs that may supplement or supplant their capabilities as well. Multi-photon imaging, for example, can reduce light scattering and thus allow for increased depth penetration of thicker samples. Total internal reflection fluorescence (TIRF) microscopy affords a high contrast, high-resolution look at “stuff that’s within 100 nm of the coverslip,” notes Laevsky.

And Zeiss’ new Lightfield 4D module uses a multi-lens array to capture 37 angular perspectives of a sample simultaneously from a single camera, enabling reconstruction of a full 3D volume from a single snapshot. With acquisition speeds of up to 80 volumes per second, "the Lightfield 4D opens up new possibilities for capturing fast biological processes in real time,” points out Vimal Gangadharan, Head of Growth Marketing & Apps Dev, ZEISS Research Microscopy Solutions. “For instance, researchers can now study cardiomyocyte contraction dynamics in developing heart organoids with high temporal and spatial resolution—previously a major challenge due to motion artifacts and limited imaging speed.”

The bottom line: know what questions you’re trying to answer, and talk to your core director or expert. “We generally see it as part of our mission to not only guide them to the right instrument, but then train them on it,” Weiss noted.