Fluorescence Confocal Microscopy

In the following subsection, we discuss the advantages and drawbacks of several fluorescence confocal microscopy methodologies for acquiring three-dimensional images of the helical col- loidal packings.

2.3.3.1 Background on Confocal Microscopy

Confocal microscopy has existed for nearly a half century, though decades of engineering and analysis have yielded many improvements with respect to speed and, to a lesser extent, resolu- tion [111, 124]. The distinguishing feature of a confocal microscope is the presence of a pinhole in front of the detector. This pinhole is located at a conjugate focal point, or “confocal” point, of the focal point located in the sample. This pinhole blocks light emitted from an out-of-focus plane, thereby improving the axial resolution of images and blocking background fluorescence. In addition, the existence of the confocal pinhole greatly improves the in-plane (lateral) resolu- tion of the image, making confocal microscopy ideal for a variety of high-resolution scanning techniques.

The original confocal setup used a stage-scanning mechanism to create complete images; the sample was moved to expose its entire volume to a single illumination point. This mechanism not only hindered the speed of scanning, but the rapid motion of the stage can interfere with the sample. Since confocal microscopy techniques are extensively used to image fragile biological samples, most modern commercial confocal systems use techniques which involve scanning the illumination point/apertures, rather than the sample. These can be generally categorized as either spinning disk/tandem scanning confocal microscopes (TSM), or confocal laser-scanning microscopes (CLSM).

Tandem-scanning confocal microscopy was first developed by Egger and Petran in 1967. This technique was based off a variation of an early video-formation technique developed by Paul Nipkow in 1884, the Nipkow disk. This disk contains a series of pinholes distributed at uniform angular and radial spacing; when placed in front of an image, a single rotation of this

disk allows a full scan of the image, converting the original two-dimensional image into a one dimensional series of intensities. The revised Nipkow/Petran disk used in TSM is employed in place of the single confocal aperture in the confocal microscope. Though the confocal image is taken only in discrete points, the spinning of the disk allows a full𝑥𝑦scan of the image, and the distribution of the pinholes enables multiple points to be imaged in tandem. The development of this scanning technique led to an exponential increase in the scanning speed compared to other confocal microscopes at the time, providing video-rate image sampling (30 frames/sec).

Confocal laser-scanning microscopy (CLSM) is a more recent technology that utilizes a single confocal pinhole (or, in some cases, a slit), but employs mirrors to shift the lateral position of the focused beam within the sample. Though this seems like a more sophisticated technology than TSM, CLSM has both relative advantages and disadvantages compared to spinning-disk microscopies. CLSM is often preferred in biological applications, since it has a much higher lateral and axial resolution than spinning-disk methods due to its use of a single pinhole. In addition, since an entire beam is focused to a single point on a sample, CLSM has much more efficient illumination than TSM. Historically, most CLSM setups provided much slower scans than TSM, but increased innovations have given CLSM setups faster scanning speeds over the past decade or so. However, the increased intensity of the excitation beam can result in dye saturation, which decreases the relative fluorescence of the in-focus plane. In addition, since they require high-quality lasers, CLSM setups typically cost several orders of magnitude more than spinning-disk optical systems (which don’t require laser sources, though they are often required for quality fluorescence images).

epoxy glass glass pNIPAm suspension glass

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Figure 2.8: Imaging artifacts from confocal scans of helical packings. (a) Illustration of an𝑥𝑦

cross-section of the experimental configuration (not to scale). (b) Brightness of a confocal scan of a (2, 3, 5) packing averaged along the tube length (𝑧direction) Inset: expected𝑥𝑦brightness distribution with perfect imaging, given placement of particles in helical packing outlined by dotted lines. (c)𝑥𝑧slices of a confocal scan of the same (2, 3, 5) packing close to the objective (bottom), at the middle of the packing (middle) and far from the objective (top).

2.3.3.2 Advantages and Disadvantages of TSM and CLSM

Though both TSM and CLSM setups were available in our laboratory, our TSM setup (QLC- 100, Visitech Int.) provided slightly higher quality images and video compared to our CLSM system (VT-Eye, Visitech Int.) for this particular geometry of microgel packings in cylinders. This difference may seem counterintuitive, considering the increased sophistication and newness of the CLSM system, but there are a few potential explanations. As previously mentioned, the CLSM setup requires higher intensity sample illumination, and it was thus more prone to cause photobleaching in our samples, which was problematic when trying to track particle dynamics in real-time videos that lasted for at least several minutes.

Additionally, aberrations in the three-dimensional structure apparent in all confocal images were amplified in the CLSM scans. Though the microcapillary was immersed in index-matched UV epoxy, the index mismatched, highly curved boundary between the glass tube (𝑛= 1.52) and the interior aqueous suspension (𝑛 = 1.33), as well as a slight index mismatch between

pNIPAm particles and the surrounding aqueous media, caused significant distortions in scans for three-dimensional structure. As mentioned previously, TSM utilizes multiple pinpoints of light passing straight though the sample, while CLSM utilizes a single point, or line of light, coming into the sample at an angle. In an index-matched and/or isotropic geometry, there should not be a difference in these imaging techniques. However, as shown in the imaging of the fluorescent surface of a polystyrene bead in Fig. 2.9 [87], differences in the imaging of curved, index mismatched interfaces are apparent.

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Figure 2.9: Confocal scans of a 6 𝜇m polystyrene bead with a fluorescent shell, comparing TSM (a-b) and CLSM (c-d) scanning methods (taken from [87]). Note the slightly increased deformation from the expected circular shape in the CLSM images.