1.

Introduction

In the 1930s, Zernike developed an extremely powerful microscopy technique, phase contrast microscopy, which has made an enormous impact in biomedicine. In a phase contrast microscope, the sample is illuminated by spatially filtered white light. The component of light that passes through cellular structures undeviated is retarded in phase by $\pi /2$ making transparent objects visible in the final image.¹ In the 1940s, Gabor developed holography which enabled the recording of both the amplitude and phase information.² The combination of qualitative phase contrast imaging and quantitative recording from holography led to the development of quantitative phase imaging (QPI).³ QPI techniques image transparent structures and quantify the change in the path length of light as it passes through these structures. In constant thickness samples such as tissue sections, the phase value is only dependent on the refractive index. QPI techniques^4–14 are sensitive to path length changes at the nanoscale level. Previously, the QPI technique spatial light interference microscopy (SLIM) has been used for a variety of applications, such as measurement of cell growth,^15,16 testing blood,¹⁷ characterization of intracellular movement¹⁸ and diagnosis as well as prognosis of disease in tissue.^19,20 However, QPI has not yet been applied to whole organism imaging.

Structural imaging of arthropods is important for their classification and studying their evolution. In addition, the push to expand the availability to the wider public of information contained in biological collections has been focused on the digitization of collections records and high-quality imaging of specimens. High-quality imaging of collection holdings would allow systematists or ecologists to query a database from remote locations for specific taxa. Instead of relying on collections managers and curators unfamiliar with the groups of interest to make correct identifications, the investigator at a remote location would be able to identify and pinpoint specific individuals to borrow. Several systems have been developed and implemented in the imaging of pinned insects and other arthropods (e.g., Invertnet²¹), but imaging of slide mounted specimens lags behind. Problems related to the resolution of diagnostically important characters, speed of scanning and proper stitching of individual images, and lack of image contrast have slowed down the progress in the field.

Fixation of arthropod tissue can be challenging since the process of killing or fixation often alters the structure of the exoskeleton and tissue, which makes a detailed study of the organism difficult.²² It is, therefore, critical to have an imaging technique that enables the study of an entire arthropod as opposed to serial sections. In the past, different groups have used transmission electron microscopy (TEM), staining-based histology, confocal laser scanning microscopy (CLSM), and synchrotron-based tomography to study arthropods. TEM has been used to study chitin crystals in arthropod cuticles, surface, and ultrastructure of crustacean larvae and drosophila testes.^23–25 While TEM has high spatial resolution, the sample has to be thinly sectioned and coated with compounds of heavy metals to enhance contrast, making the technique invasive. Stain-based histology provides high contrast images of tissue at the expense of invasive staining and sectioning of samples. CLSM, which uses autofluorescence from different structures, has been used to study various parts of Drosophila and cuticles of crustaceans.^25–27 The disadvantage of CLSM is the long scanning time and reliance on autofluorescent structures. Synchotron-based x-ray microtomography tomography uses a phase contrast principle to obtain three-dimensional images of unstained arthropods.^28,29 However, the process of obtaining quantitative information using this method is extremely tedious.^28,30 Multicellular organisms have been previously studied using optical techniques, but have been limited to studying the superficial structure and motion of the organism due to limited resolution, or have required physical sectioning of the sample for improved resolution of smaller structures.^31–34

We propose using QPI as a label-free method for studying the arthropod external morphology and subcuticular structures. In this study, using both SLIM and the Hamamatsu Nanozoomer, we imaged a whole unstained springtail (Arthropod, Collembola) Ceratophysella denticulata mounted on a slide. The images that were obtained using SLIM have better contrast and provide information about the orientation of hair and cells on the arthropod and have levels of morphological character resolution that allow the identification of individuals to genus or subfamily. To our knowledge, this is the first time QPI has been used to image an entire multicellular organism with submicron resolution. We also imaged whole unstained springtails representing the genera Willowsia and Dicranocentrus to evaluate the utility of QPI to provide fast, quality images with adequate resolution of morphological traits that can be incorporated in next generation natural history collection.

2.

Materials and Methods

2.2.

SLIM Setup

SLIM uses the phase contrast microscopy principle where the scattered and unscattered components of light are separated by a $\pi /2$ phase shift, and adds three additional phase shifts to obtain four intensity images from which the final phase is calculated.³⁶ SLIM is sensitive to path length fluctuations of 0.3 nm. SLIM is an add-on module to a commercial phase contrast microscope (Zeiss Axio Observer Z1). The SLIM module projects the back focal plane of the objective to the liquid crystal phase modulator, which introduces three additional phase shifts. The four recorded intensity images are used to calculate the final phase image.

The arthropod slide was imaged using the $40\times /0.75\mathrm{NA}$ objective on the SLIM setup. The imaging features are illustrated in Fig. 1. We used the mosaic feature on the microscope, such that 294 ($21\times 14$) images were stitched together using a plug-in built in-house. The arthropods in this study were imaged with prerecorded manual focusing of different points in the mosaic. The focus point was set every 5 frames, resulting in approximately 10 focal points per sample. The imaging system has an autofocusing feature, but due to the relatively small sample size, the computational cost of autofocusing was not justified. Thus, we rendered a final quantitative phase image of the arthropod of dimensions $7.04\mathrm{mm}\times 3.5\mathrm{mm}$. It took 10 min to align the microscope, set up the imaging mosaic, and focus the sample. The actual imaging time was 7 min for each sample.

Fig. 1

An unstained slide with the whole arthropod mounted on it is imaged using the mosaic feature and $40\times /0.75\mathrm{NA}$ objective of the spatial light interference microscopy (SLIM) system. Four intensity images corresponding to each $\pi /2$ phase shift is recorded and the final phase image is calculated from these four images. In postprocessing, with the use of ImageJ, the individual frames are stitched together to obtain the final quantitative phase image of the arthropod.

3.

Results

The images of C. denticulata that we obtained using the Nanozoomer and SLIM are shown side-by-side in Fig. 2. The Nanozoomer is only able to detect the highly absorbing structures such as the eye of the arthropod and the hard exoskeleton. On the SLIM image, in the highly absorbing regions, we see the transparent structures such as setae and cuticular ornamentation in the same region.

Fig. 2

(a) The unstained sample imaged using the Nanozoomer bright field microscope. While the Nanozoomer provides the advantage of speed, it can only capture highly absorbing structures such as the eye and the exoskeleton. (b) The SLIM image, which effectively captures the hair and cuticles on the external surface of the arthropod.

Figure 3 zooms in on a region of the arthropod. Clearly, in the SLIM image, we see individual hair (setae) and other cuticular structures on the body and limbs of the arthropod. On the bright field image, the setae and cuticular structures are transparent and not clearly visible. This is because the absorption in these structures versus the background and, as a result, the amplitude modulation is insignificant. The structure of the scales is clearly visible in SLIM images. The details in scale structure and shape are used to separate springtails into different genera. Figure 4 shows the ability of SLIM to differentiate between the scales characteristics of the genera Willowsia and Dicranocentrus.

Fig. 3

When we zoom into the bright field image of the arthropod (a), the structure of the cuticle and setae is not clearly visible. In (b), both are clearly visible.

Fig. 4

The physical properties of scales are used to categorize arthropods into various genera: (a) scale from a species of Willowsia which has a uniform phase pattern; and (b) scale from Dicranocentrus marias which shows greater discontinuity in fine structure, as seen in the phase distribution.

Figures 5 compares the ability of SLIM to image setae with greater contrast as compared with that of the Nanozoomer. In Fig. 6, we show the SLIM images of the different kinds of seta seen in Willowsia. The feathered seta is easily accessible in the quantitative phase image. Interestingly, on the microseta, the phase value appears relatively uniform; however, there is a nonuniformity in phase values along the length of the macroseta. In arthropods, setae serve as tactile receptors that can also detect vibrations in air or as chemosensory organs to detect smells and flavors.³⁷ Seta length has been shown to be an indicator of mechanical sensitivity in water and air, whereas the diameter is correlated with sensitivity in water.³⁸ Studies have also showed that arthropod setae can be categorized into different regions with specific mechanical properties based on diameter, wall thickness, and curvature.³⁹ Due to the clear visibility of individual setae strands, the length, diameter, and curvature of seta can be easily determined. In addition to the potential use as a tool to study seta function and their adaptive significance, SLIM provides information and long-term preservation of graphic comparative morphological data that may be analyzed in new and creative ways.

Fig. 5

The cuticle of springtail Ceratophysella denticulata is imaged with SLIM (a) and Nanozoomer (b). The contrast between the images produced by the two instruments is significant and the SLIM image allows a detailed study of the setae. In the bright field image, the contrast presented is the best to view the setae. The absorption in the exoskeletal region obscures the bright field image of the arthropod.

Fig. 6

The direction and thickness of individual seta are important in the categorization of arthropods. On SLIM images, this information is easily accessible along with the physical properties of the setae: (a) feathery setae (scales) from Willowsia; (b) macrosetae on Willowsia showing a nonuniform phase distribution; and (c) microsetae in Willowsia showing a more uniform phase distribution.

4.

Summary

This is the first time, to our knowledge, that QPI has been used to image a whole organism. QPI can be used to image arthropods that were fixed on slides many years ago without the need for invasive restoration or staining. With SLIM, one can quantitatively study the structure of setae and cuticular structures that are not visible with conventional bright field microscopy. In addition, the SLIM system provides levels of character resolution that allow the identification of individuals to genus in the case of Dicranocentrus and Willowsia. In qualitative phase contrast images or dark field images, the intensity depends on various external factors such as illumination intensity and the microscope used; therefore, the images are not replicable. The quantitative phase images are self-calibrated, so one would get the same phase values for the sample that is interrogated, irrespective of the illumination and microscope that were used. This makes SLIM a very valuable tool in the study of arthropods and would be an important addition to the natural history collections digitization effort.