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3D Optical Reconstruction of the Nervous System of the Whole-Body Marine Invertebrates
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Chemical & Biomedical Imaging

Cite this: Chem. Biomed. Imaging 2023, 1, 9, 852–863
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https://doi.org/10.1021/cbmi.3c00087
Published November 10, 2023

Copyright © 2023 The Authors. Co-published by Nanjing University and American Chemical Society. This publication is licensed under

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Abstract

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Optical clearing of invertebrates, the number of species of which is 20 times greater than that of vertebrates, is of fundamental and applied interest for neuroscience in general. Herein, the optical clearing of invertebrates to identify their morphology and neurostructure remains unrealized as of yet. Here, we report on fast (from a few seconds to minutes) and uniform whole-body optical clearing of invertebrates (bivalves, nemertines, annelids, and anomura) of any age and thickness (up to 2 cm) possessing complicated structures and integuments. We developed the protocol unifying dimethyl sulfoxide (DMSO)-based immunostaining of the animals followed by their optical clearing with benzyl alcohol/benzyl benzoate (BABB). Confocal microspectroscopy revealed that the protocol provides an increase of the fluorescence signal by 2 orders of magnitude and decrease of the light scattering by 2 orders of magnitude, thereby accelerating the confocal bioimaging of the whole body. Moreover, by tracking the optical clearing over time with 0.3 s resolution, we revealed that the clearing process is described by the Gompertz growth function, allowing us to determine the physical mechanism of the clearing and its optical parameters. Thereby, we were able to identify in detail and to describe previously unknown neurostructures of different invertebrate animals, paving the way to discovery in neuroscience.

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Introduction

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Optical clearing of animals (1) and plants (2) is one of the powerful tools today for high-resolution confocal optical microscopy, which makes it possible to investigate the fundamental biological processes and complex morphology of animals despite their high degree of heterogeneity at the micro and macro scales. (3) The diverse chemical and optical protocols for the optical clearing (4,5) are aimed at matching of the refractive indices (RI), (6) hyperhydration, and the transformation of the body to achieve its optical transparency. The latter is possible through minimizing the scattering (by lipids, cytoskeleton, collagen, elastin fibers, and tissue inhomogeneity) and absorption (by heme, melanin, lipofuscin, etc.) of light by the body. As a result, fluorescent (FL) confocal imaging of the whole body allows one to reveal new or hidden structures and mechanisms of functioning of animals. (7,8)
Nevertheless, achieving a high degree of optical transparency of the whole body (regardless of tissue type) of animals for any age, with a high speed and uniform clearing, and avoiding the morphology distortion and a decrease in the FL signal remains a challenge. Indeed, with rare exceptions, (9) chemical protocols for the optical clearing take a long time (from hours to days); (4) while the older the animal, the more difficult it is to achieve a high quality clearing. (10) Moreover, the chemical and morphological heterogeneity of tissues and organs significantly complicate the process of uniform optical clearing of the whole body. (3) In addition, with rare exceptions, (11) the chemicals utilized for the optical clearing can also decrease the immunostaining FL signal, which raises a series of fundamental and technological challenges for biological imaging.
While most of the chemical protocols are aimed at the optical clearing of vertebrates (66 thousand species) and mammals in particular, 1.3 million species of invertebrates with a high complexity of development of their organ systems and varying cell organization remain a challenge for the optical clearing. This is partly due to the various integuments, calcifying tissues, pigments, and shells that protect animals from predators. Therefore, the development of a universal protocol for fast and uniform optical clearing, allowing visualization of tissues, organs, and whole bodies of invertebrates, regardless of their age, will stimulate biomedicine and neurobiology in general.
Here we report on a new protocol providing simultaneously fast (from a few seconds to minutes) and uniform whole-body optical clearing of marine animals belonging to different taxonomic groups (bivalves, nemertines, annelids, anomura, and separate tissues and organs of vertebral animals as a reference), possessing different structures, integuments, and organization of the body of any age and thickness (up to 2 cm). The protocol developed is based on the joint use of dimethyl sulfoxide (DMSO)-based immunostaining of the animals followed by their optical clearing with benzyl alcohol/benzyl benzoate (BABB). Confocal microspectroscopy confirmed that the protocol provides an increase of the FL signal by 2 orders of magnitude and a decrease of the light scattering by 2 orders of magnitude, thereby simplifying and accelerating the confocal bioimaging. Moreover, by tracking the optical clearing over time with 0.3 s resolution, we revealed that the Gompertz growth function optimally describes the clearing process, allowing one to reveal the hidden physical mechanisms of the clearing and its optical parameters such as clearing rate, light scattering, and absorption coefficients. Thus, we were able to identify in detail and to describe the neurostructures of different invertebrate animals (larvae and adults), paving the way to new discoveries in neuroscience.

Methods

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Before starting the experiments with different groups of marine invertebrates (bivalves, nemertines, annelids, and anomura), we checked in advance the immunostainings and optical clearing protocols for vertebrate embryos (Figure S1): mice Mus musculus (embryonic day 10, E10) with 2H3 antibodies (Neurofilament-M) and wild-type fish Danio rerio with antibodies (alpha acetylated tubulin/TH/CHAT) (44 days of development after fertilization). For immunostainings, we have used dimethyl sulfoxide (DMSO, 20%)-containing solutions, while for the optical clearing of the stained animals, we have used a benzyl alcohol/benzyl benzoate-(BABB, 1:2) based protocol. (12,13) Intriguingly, we have found that existing optical clearing protocols work successfully for vertebrate animals, while they are inapplicable for invertebrates. Therefore, we have modified the protocols as follows (all steps are summarized in Table 1).
Table 1. Benzyl Alcohol/Benzyl Benzoate (BABB) Tissue-Clearing Protocol for Marine Invertebrates (Common)a
FixationDecalcification (optional)Permeabilization (obligatory)Blocking solution (obligatory)Clearing (key components)Time of clearingImmunostating compatibilityClearing effect (+/++/+++)Detection of neurostructures (poor/good/excellent)
4% PFA7.5% EDTA5% Triton X-10010% NDS; 1% BSA; 1% Triton X-100; 10% DMSOBABB1–12 hYES+++excellent
2% PFAwithout5% Triton X-10010% NDS; 1% BSA; 1% Triton X-100; 10% DMSOBABB1–12 hYES+++good
1% gluteraldehyde/2% PFA7.5% EDTA5% Triton X-10010% NDS; 1% BSA; 1% Triton X-100; 10% DMSOBABB1–12 hNO+++poor
4% PFA7.5% EDTA1% Triton X-10010% NDS; 1% BSA; 1% Triton X-100; 10% DMSOBABB1–12 hYES+++good
4% PFA7.5% EDTA3% Triton X-10010% NDS; 1% BSA; 1% Triton X-100; 10% DMSOBABB1–12 hYES+++good
10% PFA7.5% EDTA5% Triton X-10010% NDS; 1% BSA; 1% Triton X-100; 10% DMSOBABB1–12 hNO+++poor
4% PFA7.5% EDTA5% Triton X-1001% Triton X-1001-PrOH1–12 hYES+++good
4% PFA7.5% EDTA5% TritonX-1001% Triton X-100; 10% DMSO1-PrOH, or t-BuOH1–12 hYES+++good
a

Abbreviations: PFA: paraformaldehyde; EDTA: ethylenediaminetetraacetic acid; NDS: normal donkey serum; BSA: bovine serum albumin; DMSO: dimethyl sulfoxide; PrOH: propanol; BuOH: butanol.

Immunostainings

After 4% PFA (paraformaldehyde) fixation for 3–5 h at room temperature, the larvae of bivalves and anomura and adult animals (bivalves, annelids, and nemertines) in each stage have been transferred from 100% methanol to 0.1 M phosphate buffered saline (PBS). The samples of bivalves and anomura were additionally incubated overnight in 7.5% ethylenediaminetetraacetic acid (EDTA) in PBS at room temperature for decalcification. All of the samples have been rinsed in PBS supplemented with 5% Triton X-100 (PBST) for 4.5 h with agitation. Then, the samples have been incubated overnight in a blocking solution (10% normal donkey serum (NDS), 1% bovine serum albumin (BSA), and 1% Triton X-100, 0.003% NaN3 in 0.1 M PBS).
For detection of neurostructures, the larvae have been incubated with primary antibodies at 4 °C in several combinations: serotonin rabbit Abs (ImmunoStar, 20080) with tyrosine hydroxylase (TH) rabbit polyclonal Abs (Millipore, Burlington, MA, United States, AB152) and with monoclonal mouse antisynapsin antibody (clone 3C11; Developmental Studies Hybridoma Bank, Iowa City, IA, United States) in the blocking solution at a dilution of 1:1000 for 5 days at 4 °C.
After being washed in PBS (4 times, 20 min each), the samples were incubated overnight at 4 °C in donkey antigoat (Invitrogen, A32814), donkey antirabbit (Invitrogen A32794), and donkey antimouse (Invitrogen, A32787) antibodies at a dilution of 1:1000 with 0.1 μg/mL Dapi dye (4′,6-diamidino-2-phenylindole). Then, the larvae of bivalves and anomura and adults (bivalves, annelids, and nemertines) were washed again in PBS with 0.1% tween 20 (PBST) (5 times, 20 min each).
As a control, we have shown that preincubation of the 5-HT antibody with the same conjugate (10 μg/mL, ImmunoStar, Cat. No. 20081) at 4 °C overnight eliminated all immunolabeling of serotonin in the tissues. The preadsorption of the diluted rabbit/goat antiserum with 10 mg/mL BSA overnight at 4 °C did not influence this staining; i.e., these antibodies recognized only serotonin rather than BSA. Also, only secondary antibodies (without treatment of primary antibodies) have been used for control antibodies on larvae and adult animals.

Optical Clearing Protocol

We hypothesized that the BABB protocol, often used for the optical clearing of vertebrate animals, (14) could serve as a basis for the clearing (depigmentation) of other organisms such as marine invertebrates. Therefore, after immunostaining, all the samples were washed in PBST (5 times, 10 min each) at room temperature with constant shaking. Then, the samples have been dehydrated in a 50% methanol solution in PBS for 30 min with constant shaking and placed in pure methanol for incubation at room temperature at least 3 times for 10–30 min with shaking. Finally, the samples (larvae and adults) were rinsed in BABB solution for 15 min with shaking and placed in a pure BABB solution for at least 1 h.

Bioimaging

The samples of immunocytochemically stained animals were scanned on an LSM 780 confocal microscope (Zeiss, Germany) using the Zen software with the following laser wavelengths: 405, 488, 555, and 647 nm. All the images have been composed in Z-stack mode with an optical slice thickness of 1 μm along Z-axis. The obtained images were then transformed into projections in the maximum intensity mode. IMARIS 7.0 software (Bitplane, Switzerland) was used to process the resulting images. The converted images were saved in TIFF format and transferred to Photoshop CS software (Adobe, San Jose, CA, United States), where the contrast and brightness were adjusted for optimal clarity. The negative controls for each fluorochrome were scanned using the same settings. The final Figure panels have been edited in Adobe Illustrator CS2 (Adobe System, San Jose, CA, USA).

Confocal Optical Microspectroscopy

To reveal the dynamics of the optical clearing, confocal optical microspectroscopy has been utilized. (15−18) For this, a white light from the lamp (for the transmittance measurements, Ocean Optics, HL-2000 FHSA) or laser light (for FL measurements, supercontinuum Fianium laser source, 405 nm wavelength, 6 ps pulse duration, 60 MHz repetition rate, 10 nm line width, and 10 mW integral power) has been weakly focused on one side of the animal by an objective (Mitutoyo 10x M Plan APO, 0.28 NA), while the transmitted/FL light has been collected from the other side by an objective (Mitutoyo 10x M Plan APO NIR, 0.26 NA), and then analyzed by a confocal spectrometer (Horiba LabRam with a cooled Si-based CCD camera, Andor DU 420A-OE 325, and 600 g mm–1 diffraction grating), allowing one to measure the optical transmittance or FL spectra from 300 to 1600 nm with 0.3 s time resolution. The diameter of the exciting area of the animal was 1 mm (not in the focus of the objective), while the diameter (equal to 0.74 × λ / NA, where λ is an optical wavelength and NA is a numerical aperture of the objective) and the depth (equal to 1.28 × λ / (n – (n2 – NA2)0.5, where n = 1 is the RI of air) of the collecting area were 1.5 and 8 μm due to the confocal scheme of the spectrometer. (19)

RESULTS and DISCUSSION

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Optical Clearing

As a model marine animal, we have used oyster Crassostrea gigas, which have a solid shell, opaque soft body, abundant pigmentation of organs, and complex anatomy of organs (Figure 1A–D). After isolation and fixation of the whole oyster body (adult samples, 50 mm in length, 30 mm in width, and 10 mm in depth), we performed optical clearing by BABB (Figure 1 B–C). Intriguingly, we have distinguished individual organs (digestive gland, heart, gills, muscle adductor, and mantle) of an adult mollusk in 1 h, which had not previously been visible (Figure 1C). In addition to massive ganglia, we identified peripheral nerves in different types of tissues. For example, after using the protocol, the innervation of the highly autofluorescent digestive glands (Figure 1F1–F2), gills (Figure 1F3–F6) and mantle (Figure 1F3) became clearly visible, and the innervation of the dense muscle adductor (Figure 1F4–F5) of the mollusk has been easily observed.

Figure 1

Figure 1. Clearing (A–C) and Immunohistochemical whole mounting staining of adult oyster Crassostrea gigas. (A) Common view of oyster with shell. (B) Uncleared soft body of oyster (without shell). (C) Cleared soft body of oyster. (D) Schematic anatomy of inner organ of oyster. (E) 3D reconstruction of nervous systems of adult oyster. (F1–F6) Immunostainings with serotonin (red color) nervous system of oyster abbreviations: lp–labial palp, CG–cerebral ganglia, dg–digestive gland, vnc–ventral nerve cord, add–muscle adductor, VG–visceral ganglia, st–stomach. Scale bars, 50 μm.

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Nevertheless, we have faced a challenge of immunostaining the nervous system of the marine animals, since the protocols developed recently for vertebrates provided an unclear picture of the staining. To overcome this, we have gradually modified the vertebrate protocol (see Table 1). As a result, we have identified the key stages for high-quality immunodetection of the nervous system of oysters: (i) the combination of permeabilizing agents (DMSO/TritonX-100); (ii) an enhanced blocking solution (NDS/BSA) to reduce nonspecific binding of antibodies to immunoglobulin-binding agents of the animal; and (iii) BABB clearing from several minutes to a day with solution changes. It should be noted that all stages of immunostaining should be performed within 12 h or less (depending on the size, pigmentation, and animal integuments) after fixation, since the prolonged time of fixation leads to background and nonspecific immunostaining artifacts (see below).
As a result, Figure 1 shows that the immunostaining protocol modified for marine animals (adult bivalves and nemertines) provided a clear picture of tiny neurostructures of the central nervous system (main ganglia) and the elements of the peripheral system (like neurons and nerves in organs).

Challenges of the Marine Animal Clearing

Among bivalves, the oyster Crassostrea gigas is one of the most well-studied mollusks. (20,21) It is well known that the nervous systems of the oyster consist of three major, bilaterally symmetric pairs of ganglia (cerebral, pedal, and visceral) or fused ganglia (epiathroid state). For oyster and other bivalves, the paired cerebropleural ganglia are connected by commissures as well as being connected to the paired pedal ganglia via the cerebral–pleural–pedal connectives and to the paired visceral ganglia via long cerebral–visceral connectives. These ganglia innervate the labial palps, anterior adductor muscle, the anterior part of the mantle (via anterior pallial nerves), and sensory organs. The pedal ganglion innervates the foot, but in species with foot reduction (oysters), this ganglion is absent. Large paired visceral ganglion innervates the gills (via branchial nerves), heart, posterior adductor muscle, posterior part of the mantle (via posterior pallial nerves), and siphons. (22−26)
Also, the neurostructure of separate bivalve ganglia has been examined employing classical histochemical methods (22,23) and, later, the distribution of individual neurotransmitters in separate mollusk ganglia has been determined immunohistochemically. (27−32) However, the analysis of the nervous system of whole-body marine animals (avoiding the steps of cutting and surgery isolation) is still a challenge. In this sense, our results through the confocal scanning and reconstruction of the nervous system of the whole-body of mollusks proved an applicability of the modified protocol, which can be also applied to other species and taxa (Figure 1 and summarizing Table 2).
Table 2. Benzyl Alcohol/Benzyl Benzoate (BABB) Tissue-Clearing Protocol for Several Marine Invertebratesa
AnimalFixationDecalcification (optional)Permeabilization (obligatory)Blocking solution (obligatory)Clearing (key components)Time of clearingImmunostaining compatibilityClearing effect (+/++/+++)Detection of neurostructures (poor/good/excellent)
Molluscs (Gastropoda and Bivalves)4% PFA (3–5 h at RT for adults and 2–3 h for larvae)b7.5% EDTA (obligatory)5% Triton X-10010% NDS; 1% BSA; 1% Triton X-100; 10% DMSOBABB6–12 hYES+++Excellent
Nemertines4% PFA (2–3 h at RT)bWithout calcification5–10% Triton X-10010% NDS; 1% BSA; 2% Triton X-100; 10% DMSOBABB12–24 hYES+++Excellent
Annelids4% PFA (2–3 h at RT)bWithout calcification5–10% Triton X-10010% NDS; 1% BSA; 2% Triton X-100; 10% DMSOBABB12–24 hYES+++Excellent
Anomura (king crabs laervae)4% PFA (3–5 h at RT)b7.5% EDTA (obligatory)1% Triton X-10010% NDS; 1% BSA; 1% Triton X-100; 10% DMSOBABB12–24 hYES+++Excellent
a

Abbreviations: PFA: paraformaldehyde; EDTA: ethylenediaminetetraacetic acid; NDS: normal donkey serum; BSA: bovine serum albumin; DMSO: dimethyl sulfoxide; RT: room temperature.

b

The fixation time depends on the size of the biological object.

Dynamics

Generally, the dynamics of the optical clearing of animals remains unknown. With the exception of a number of studies, (33−39) where time evolution of the optical transmittance is observed, the math describing the process itself and the associated changes in the optical parameters (scattering and absorption coefficients) of a complex heterogeneous medium are still underexplored. Therefore, an adult bivalve (oyster Crassostrea gigas) has been utilized again as a model marine animal to monitor the dynamics of the optical clearing. Figure 2 shows the evolution of the optical transmission spectra (400–1000 nm) over time for different areas of an adult oyster (5 cm in length, 2 cm in width) which have been measured in transmittance geometry.

Figure 2

Figure 2. Optical transmittance spectra for the bivalve oyster Crassostrea gigas during the optical clearing. (A) Optical transmittance spectra for the mantle, adductor, gills, ganglia of oyster with 1 cm thickness of the samples except the mantle (0.2 cm), and corresponding organic solvents (methanol and BABB). (B) Evolution of the normalized optical transmittance for the mantle and adductor over time. Crosses are experimental points; dotted lines are the Gompeltz growth fit. (C–F) Evolution of the optical transmittance over time for the mantle and adductor with corresponding evolution in log scale of the normalized transmittance at Dapi and Alexa 555 excitation wavelengths (402 and 545 nm, respectively).

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In detail, Figure 2A demonstrates the optical transmittance spectra for a small area (inset in Figure 2B) of oyster organs (mantle, adductor, gills, and ganglia) recorded in 10 min after the optical clearing started. The differences in shape and intensity of the optical transmittance (40) come from absorption and scattering of light by organs and tissues. (3) Intriguingly, the evolution of the optical transmittance over time is different for these areas (Figure 2B): the thinner and more homogeneous the tissue (mantle), the faster clearing occurs. Indeed, soft mantle tissue can be optically cleared up to 90% of transmittance for 20 min, while adductor muscle becomes transparent (80% of transmittance) in 200 min. Since the process of the optical clearing by the proposed protocol is quite fast, we have managed to track it in detail for a dense tissue like adductor: Figure 2F shows 3 orders of transmittance increase for adductor, while we have observed only 1 order of transmittance increase for the mantle because of 1 min technological gap between the clearing start and spectral measurements.
Next, we have fitted the time dependence, t, of transmittance for tissues (Figure 2B) using a series of math functions, which are well known in biophysics. (41) It turned out that the most optimal function describing the optical clearing process is Gompertz growth (42,43) (Figure S2):
T=exp{−exp{−v(t−t0)}}
where T is a normalized number of photons transmitted by tissue, v is optical clearing rate, and t0 is a lag time. According to the optical clearing protocol, we have expected that the replacement of water by methanol and BABB during the clearing should be a diffusion process. However, we have observed the pure Gompertz trend (Figure S2), which is usual for the growth of cells and animals in limited conditions. (43) From the fitting, one can also estimate the rate and lag time of the clearing: v equals 0.56 ± 0.25 s–1 and 0.015 ± 0.002 s–1 for mantle and adductor, respectively, while t0 equals 0.93 ± 0.17 and 122 ± 5 s for these tissues. It means that the optical clearing of dense tissues is ∼40 times slower and has a hundred-fold lag time. In our case, the latter can be explained by the light scattering, which obstructs the optical clearing (i.e., the replacement of water molecules by organic solvents raises the refractive index matching, (3,44,45) while the absorption of light by tissues is constant over time and depends only on the concentration of heme, melanin, lipofuscin, etc.).
Back on the problem of the light scattering by tissues, it is wellknown that this process is caused by the refractive index mismatch between the surrounding and the nanometer/micrometer-scale organelles, cells, and proteins. (3,4) Moreover, the efficiency of this process is proportional to the size of the light scatterrers and (m2 – 1) or (m – 1) parameter (depending on the Rayleigh or Mie type scattering, respectively), where m is a ratio of the RIs of the organs, organelles, tissues, and surrounding (water, BABB, etc.). Therefore, best matching of the RIs between the scatterers and the organic solvents (m ∼ 1) yields suppression of the light scattering. The latter effect can be expressed through the changes in intensity of light transmitted, T, through the tissue in diffusion regime:
T=const×exp{−μeffz}
where z is the thickness of the tissue, while μeff = (3 μa (μs + μa))−2 combines its scattering μs and absorption μa coefficients. Taking into account the experimental spectra in Figure 2C,E, 1 and 0.2 cm thickness of the adductor and the mantle (Figure 2B, inset), and 10–2 cm–1 optical absorption coefficient of the tissues at the Alexa 555 excitation wavelength (545 nm), we have determined the scattering coefficients μs for the mantle and adductor before (5.3 × 103 cm–1 and 2 × 103 cm–1) and after (41 and 12 cm–1) the optical clearing. Intriguing is that we have revealed a 2 order decrease of the μs, yielding the sufficient increase of the transmittance (Figure 2B) and Dapi/Alexa 555 FL signal from the whole body of an adult animal. Indeed, Figure 3A,B demonstrates an increase of FL signal from the tissues (mantle, adductor, gills, and ganglia) after the optical clearing, while Figure 3C summarizes these results as a 1- to 2-order increase of the FL signal from the tissues, statistically confirmed for different areas of the body.

Figure 3

Figure 3. FL signal enhanced by the optical clearing of bivalve oyster Crassostrea gigas. (A,B) Dapi and Alexa 555 FL spectra in log scale before and after the optical clearing of tissues under the same conditions. (C) The enhancement of the FL signal from organs and tissues compared to the FL signal from the uncleared ones. In the case of uncleared dense tissues such as adductor and ganglia, the FL signal was zero; therefore, the 102 estimated enhancement is a low limit.

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Bivalves

In this section and further, we look at different examples (for invertebrate animals of different taxonomic groups) of using the developed protocol for optical clearing and imaging their neurostructures. As a first example, we consider bivalve mollusks, which are completely covered with a calcium-based shell consisting of two flaps fixed on hinges. These mollusks are among the most difficult to pass immunohisto- and immunocytochemistry due to the large amount of calcium (46) and the presence of lectin molecules possessing immunoglobulin-binding domains that give nonspecific binding to antibodies when cells and tissues are stained. (47) Therefore, it is important to have access to the first and second antibodies during the immunohistochemistry procedure. Moreover, all the internal organs of mollusks Mytillus trossulus are opaque (Figure 4A) and, therefore, most morphological studies of the structure of organs and tissues are carried out by a complex routine of histocutting methods to assemble a general picture of the structure of the animal/organs. In our case, to test the staining and the optical clearing protocols, we have considered an adult bivalve, since it contained a large number of pigments and colored tissues (gonads, adductor). As one can see in Figure 4A–A3, immunostainings of the whole animal followed by its optical clearing allows us to identify individual main ganglia (cerebral) without gilding and isolation of nerve structures (Figure 4B–C1). In addition to the general morphology of the ganglion, the internal structure of the ganglion is also clearly distinguishable in bivalves Mytillus trossulus (size 40 mm in length) and Dreissena polymorpha (size 50 mm in length) (Figure 4B–C1), while the neuropile is visible in the center and on the periphery in the zone of the outer cell layer of main ganglia (Figure 4B1,C1). Next, the ganglion neurites are visible, the path of which can be traced at different levels of the stacks. Based on the overall picture of the staining, a 3D model of the entire nervous system of the mollusks can be made, as we performed for an oyster in Figure 1E. Protobranchia mollusks Acila insignis and Nucula sp. were also immunostained by antibodies (FMRFamide) and cleared by BABB (Figure S3A,C). Individual ganglia were visible in deep Z-stacks (Supplementary Figure S3B, D).

Figure 4

Figure 4. General view and nervous system morphology of adult mussel Mytillus trossulus before and after clearance. Immunodetection of peptide FMRF amide by whole mount immunostainings with FMRFamide antibodies. (A,A2) Before clearing, (A1,A3) after clearing, (B–C1) immunostaninsg with 5-HT antibody (red color) mussels Mytillus trossulus (B–B1) and FMRFamide (green color) antibody Dreissena polymorpha (C–C1). Scale bars, 1 cm (B,C), and 500 μm (B1,C1). Abbreviations: CG–cerebral ganglia, VG–visceral ganglia, PG–pedal ganglia, p.ad–posterior adductor.

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Nemertines

Next, we considered Nemertines, which are a taxon of invertebrates, also known as proboscis worms. These animals are often brightly colored and have a dense muscular body wall, leading to a complicated immunohistochemistry associated with difficult penetration of antibodies. In our case, we have utilized again the developed protocol for optical clearing to identify the neurostructure and to compare the results: immunocytochemistry without DMSO (Figure 5A), without DMSO and with optical clearing (Figure 5A1), and with DMSO and optical clearing (Figure 5A2). The latter appeared to be more successful for simple, fast, and efficient identification of tiny neurostructures in nemertea species Quasitetrastemma stimpsoni, Lineus alborostratus, Micruna Kulikova, and Hubrechtella sp. Important is that the combination of immunostaining with DMSO and BABB clearing provided a positive staining of the “brain” in nemertines (Figure 5A2): 5-HT-immunopositive processes have been identified in the epidermis layer of all adult nemertines. Finally, the structure and neurogenesis of Quasitetrastemma stimpsoni larvae have been tracked using this protocol.

Figure 5

Figure 5. (A–A1) Optical images of adult worm nemertines Quasitetrastemma stimpsoni before and after optical clearing. Scale bar, 3 cm. Testing immunofluorescence and the optical clearing protocol on adult worm nemertines Quasitetrastemma stimpsoni: (B) immunofluorescence with 5-HT antibody (red) without DMSO and without clearing, (B1) immunofluorescence with 5-HT (red) antibody without DMSO and with clearing, (B2) immunofluorescence with 5-HT (red) antibody with DMSO and with clearing, and (C–F) testing several worm species using immunofluorescence with 5-HT antibody with DMSO and with clearing. Colors: red–5-HT-lir, green–cilia (acetylated tubulin immunoreactivity), blue–nuclei (Dapi). Scale bars, 5 mm.

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Annelids

Like the nemertine worms, annelids have a rigid cuticle consisting of collagen fibers secreted by the epidermis. Some species of annelids have glands that secrete mucus in the epidermis, protecting their skin. (48) Under the epidermis is the dermis, which consists of connective tissue. Below are two layers of muscles: circular and longitudinal muscles. Some annelids also have oblique internal muscles that connect the underside of the body to each side. As a result, all this multilayered structure of the annelids’ integuments makes them complex animals for the whole-body innumeration and reconstruction of internal organs and tissues. In our case, we have used annelid Fabricia sp with a length of 5 mm and 0.5 mm width, annelid Flabelligeridae sp with a length of 10 mm and 1.5 mm width, and annelid Owenia borealis with a length of 10 mm and 3 mm width (Figure 6). Despite such a huge size of species for confocal microscopy, all three annelids were successfully immunostained and optically cleared by the protocol described above (Table 2). Intriguingly, the neurostructures of the central nervous system and a nerve ring have been easily identified for Flabelligeridae (Figure 6B), Owenia sp (Figure 6C–C1), as well as a nerve trunk for Fabricia (Figure 6A–A2).

Figure 6

Figure 6. Testing immunofluorescence and the optical clearing protocol on adult annelids. (A–A2) Fabricia sp, (B) Flabelligerida, (C–C1) Owenia borealis. (A–A2) Organization of the full nervous system of Fabricia using 5-HT (green), acetylated alpha-tubulin (red) with Dapi (blue). (A1,A2) The same thing with other Z-projections. (B) Immunofluorescence and the optical clearing protocol applied for an adult annelid from Flabelligerida taxon. Color: 5-HT (green), acetylated alpha-tubulin (red) with Dapi (blue). (C–C1) Organization of the nervous system of the head of Owenia borealis. (C) General anatomy of the nervous system viewed from the ventral side: Z-projection after double immunostaining against acetylated alpha-tubulin (red) and serotonin (green) and staining with Dapi (blue). (C1) View from above. Scale bars, 100 μm (A–A2), 200 μm (B), 500 μm (C–C1).

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Anomura

Finally, as one of the most difficult animals for optical clearing, we chose the king crab Paralithodes camtschaticus (anomura). As in the case of bivalves, nemertines, and annelids, the conventional optical clearing protocols developed for vertebrates provided conflicting results for crabs, which are difficult to analyze. Therefore, we have changed some protocol settings and introduced additional steps for immuno-coloring and the optical clearing of the earlier (zoea I) and late-stage (zoea IV) king crab larvae Paralithodes camtschaticus equipped with maxims and mandibles (Table 2). (49) An additional step of decalcification with EDTA and bleaching with H2O2 significantly improved the immunostainings of the neurostructure and increased the transparency of the whole-body of larvae. As a result, neurostructure of the larvae has been easily observed using confocal microscopy after immunostaining with synapsin and nuclei (Dapi) (Figure 7). We have shown that larval P. camtschaticus is composed of a brain and a ventral nerve cord (VNC) including fused sub esophageal ganglia (SEG), five thoracic ganglia associated with the pereiopods (walking legs), and six pleonic (abdominal) ganglia (AG) (Figure 7A–D). Thereby, the developed protocol expands the list of animals to those containing the calcified shell in order to investigate their morphological and neuroanatomy.

Figure 7

Figure 7. (A–B) Optical images of king crab P. camtschaticus before (A) and after (B) optical clearing. Scale bar, 100 μm. Brain and ventral nerve cord (VNC) in king crab P. camtschaticus at different larval stages: confocal laser-scanning microscopy (CLSM) 3D images showing the brain and VNC (sagittal plane) in zoea I (C) and zoea IV (D). General diagram of CNS (F). Abbreviations: Ey–compound eye; mBr–median brain; SEG–subesophageal ganglia; TG–thoracic ganglia; CG–commissural ganglion; AG(1–6)–abdominal ganglia; T(1–5)–thoracic neuropils; mPC–median protocerebrum; DC–deutocerebrum; TC–tritocerebrum; OC–esophageal connective; LP–lateral protocerebrum. Scale bars, 100 μm (C–F).

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Discussion

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Listing the current problems of modern optical clearing for confocal microscopy (low speed of clearing, different tissues and organs with pigments and complex morphology, any ages of the animals, and decreased FL signal), we can speculate that the developed protocol made it possible to carry out an efficient optical clearing and bioimaging of a new class of organisms─marine animals (Table 2), whose body structure, pigmentation, shells, and neurostructure are much more complex and diverse in comparison with vertebrates.
In particular, we have managed to reduce the optical clearing time to seconds and tens of minutes for the whole body, which, with a number of exceptions, (9) is 2 orders of magnitude faster than existing protocols for vertebrates provide. Moreover, a detailed analysis of the optical clearing time made it possible to establish both the dynamics of such a process and the evolution of the optical parameters. Thus, we have proved that the optical clearing reduces the light scattering coefficients by 2 orders of magnitude, making the whole body transparent up to 90% and allowing one to increase the FL signal by 2 orders of magnitude.
Then, regardless of the complex chemistry of marine animals (high concentration of pigments, lectin, calcium, etc.) preventing both staining and optical clearing, the developed protocol allowed us to implement these processes for the uniform staining of both larvae and adult invertebrates. All these effects allow us to identify the fine neurostructures of various marine animals easily, quickly, and in detail.

Conclusions

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We report on a unique protocol providing simultaneously fast (from a few seconds to minutes) and uniform whole-body optical clearing of marine animals such as bivalves (Crassostrea gigas, Mytillus trossulus, Dreissena polymorpha), nemertines (Quasitetrastemma stimpsoni, Lineus alborostratus, Micruna Kulikova, and Hubrechtella sp), annelids (Flabelligeridae sp, Owenia borealis, and Fabricia sp), and anomura (king crab Paralithodes camtschaticus), possessing different structures, integuments, and organization of the body of any age and thickness (up to 2 cm). The protocol developed is based on the joint use of DMSO-based immunostaining of the animals followed by their optical clearing with BABB. Confocal microspectroscopy confirmed that the protocol provides an increase of the FL signal by 2 orders of magnitude and decrease the light scattering coefficient by 2 orders of magnitude, thereby simplifying and accelerating the confocal bioimaging of the whole body. Moreover, by tracking the optical clearing over time with 0.3 s resolution, we revealed that the Gompertz growth function optimally describes the clearing process, allowing us to determine the physical mechanisms of the clearing and its optical parameters such as the rate of clearing, scattering, and absorption coefficients of the tissues. Thereby, we were able to identify in detail and to describe previously unknown neurostructures of the invertebrate animals, paving the way to new discoveries in neuroscience. Moreover, compared to other optical clearing methods, our protocol covers a wide range of uses. Its simplicity and versatility makes it attractive for interdisciplinary studies of diverse nervous systems. For instance, one can take different taxonomic groups of invertebrates, choose the most suitable conditions based on the found clearing effect (Tables 1, 2), and then study the neural networks. We also believe that the developed optical clearing protocol can be successfully used by physicians, biologists, zoologists, and neuroscientists and can contribute to deep understanding of the anatomical structures of different animals.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.3c00087.

  • Figures on confocal imaging of optically cleared mice and fish, as well as comparison of diffusion and Gompertz curves for the clearing dynamics (PDF)

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Author Information

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  • Corresponding Authors
    • Valentin A. Milichko - School of Physics and Engineering, ITMO University, St. Petersburg, 197101, Russia;  Institut Jean Lamour, Universit de Lorraine, UMR CNRS 7198, 54011 Nancy, France;  Orcidhttps://orcid.org/0000-0002-8461-0804;  Email: [email protected]
    • Vyacheslav Dyachuk - A.V. Zhirmunsky National Scientific Center of Marine Biology, Russian Academy of Sciences, Far Eastern Branch, Vladivostok 690041, Russia;  Email: [email protected]
    • Author Contributions

      V.A.M. and V.D., conceptualization and manuscript writing. Separately: V.A.M., optical analysis and modeling; V.D., optical clearing, bioimaging.

    • Notes
      The authors declare no competing financial interest.

    Acknowledgments

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    The authors thank Dr. Elena Temereva for providing annelids; Dr. Timur Magarlamov and Dr. Alexei V. Chernyshev for providing nemertines; Dr. Olga Yurchenko for providing oysters; and Dr. Elena Kotsyuba and Dr. Maslennikov for providing the larvae of king crab. The authors are grateful to the Vostok Biological Station (NSCMB FEB RAS), the Far East Center of Electron Microscopy, Optical Research Group of IDB RAS, and the Biology and Genetic Engineering Center for Collective Use (FCEALTB FEB RAS) for their assistance. This work was supported by the Russian Science Foundation (Grant No. 21-74-30004 (clearance) and 22-14-00245 (immunostainings).

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    • Abstract

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      Figure 1

      Figure 1. Clearing (A–C) and Immunohistochemical whole mounting staining of adult oyster Crassostrea gigas. (A) Common view of oyster with shell. (B) Uncleared soft body of oyster (without shell). (C) Cleared soft body of oyster. (D) Schematic anatomy of inner organ of oyster. (E) 3D reconstruction of nervous systems of adult oyster. (F1–F6) Immunostainings with serotonin (red color) nervous system of oyster abbreviations: lp–labial palp, CG–cerebral ganglia, dg–digestive gland, vnc–ventral nerve cord, add–muscle adductor, VG–visceral ganglia, st–stomach. Scale bars, 50 μm.

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      Figure 2

      Figure 2. Optical transmittance spectra for the bivalve oyster Crassostrea gigas during the optical clearing. (A) Optical transmittance spectra for the mantle, adductor, gills, ganglia of oyster with 1 cm thickness of the samples except the mantle (0.2 cm), and corresponding organic solvents (methanol and BABB). (B) Evolution of the normalized optical transmittance for the mantle and adductor over time. Crosses are experimental points; dotted lines are the Gompeltz growth fit. (C–F) Evolution of the optical transmittance over time for the mantle and adductor with corresponding evolution in log scale of the normalized transmittance at Dapi and Alexa 555 excitation wavelengths (402 and 545 nm, respectively).

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      Figure 3

      Figure 3. FL signal enhanced by the optical clearing of bivalve oyster Crassostrea gigas. (A,B) Dapi and Alexa 555 FL spectra in log scale before and after the optical clearing of tissues under the same conditions. (C) The enhancement of the FL signal from organs and tissues compared to the FL signal from the uncleared ones. In the case of uncleared dense tissues such as adductor and ganglia, the FL signal was zero; therefore, the 102 estimated enhancement is a low limit.

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      Figure 4

      Figure 4. General view and nervous system morphology of adult mussel Mytillus trossulus before and after clearance. Immunodetection of peptide FMRF amide by whole mount immunostainings with FMRFamide antibodies. (A,A2) Before clearing, (A1,A3) after clearing, (B–C1) immunostaninsg with 5-HT antibody (red color) mussels Mytillus trossulus (B–B1) and FMRFamide (green color) antibody Dreissena polymorpha (C–C1). Scale bars, 1 cm (B,C), and 500 μm (B1,C1). Abbreviations: CG–cerebral ganglia, VG–visceral ganglia, PG–pedal ganglia, p.ad–posterior adductor.

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      Figure 5

      Figure 5. (A–A1) Optical images of adult worm nemertines Quasitetrastemma stimpsoni before and after optical clearing. Scale bar, 3 cm. Testing immunofluorescence and the optical clearing protocol on adult worm nemertines Quasitetrastemma stimpsoni: (B) immunofluorescence with 5-HT antibody (red) without DMSO and without clearing, (B1) immunofluorescence with 5-HT (red) antibody without DMSO and with clearing, (B2) immunofluorescence with 5-HT (red) antibody with DMSO and with clearing, and (C–F) testing several worm species using immunofluorescence with 5-HT antibody with DMSO and with clearing. Colors: red–5-HT-lir, green–cilia (acetylated tubulin immunoreactivity), blue–nuclei (Dapi). Scale bars, 5 mm.

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      Figure 6

      Figure 6. Testing immunofluorescence and the optical clearing protocol on adult annelids. (A–A2) Fabricia sp, (B) Flabelligerida, (C–C1) Owenia borealis. (A–A2) Organization of the full nervous system of Fabricia using 5-HT (green), acetylated alpha-tubulin (red) with Dapi (blue). (A1,A2) The same thing with other Z-projections. (B) Immunofluorescence and the optical clearing protocol applied for an adult annelid from Flabelligerida taxon. Color: 5-HT (green), acetylated alpha-tubulin (red) with Dapi (blue). (C–C1) Organization of the nervous system of the head of Owenia borealis. (C) General anatomy of the nervous system viewed from the ventral side: Z-projection after double immunostaining against acetylated alpha-tubulin (red) and serotonin (green) and staining with Dapi (blue). (C1) View from above. Scale bars, 100 μm (A–A2), 200 μm (B), 500 μm (C–C1).

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      Figure 7

      Figure 7. (A–B) Optical images of king crab P. camtschaticus before (A) and after (B) optical clearing. Scale bar, 100 μm. Brain and ventral nerve cord (VNC) in king crab P. camtschaticus at different larval stages: confocal laser-scanning microscopy (CLSM) 3D images showing the brain and VNC (sagittal plane) in zoea I (C) and zoea IV (D). General diagram of CNS (F). Abbreviations: Ey–compound eye; mBr–median brain; SEG–subesophageal ganglia; TG–thoracic ganglia; CG–commissural ganglion; AG(1–6)–abdominal ganglia; T(1–5)–thoracic neuropils; mPC–median protocerebrum; DC–deutocerebrum; TC–tritocerebrum; OC–esophageal connective; LP–lateral protocerebrum. Scale bars, 100 μm (C–F).

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    • Supporting Information

      Supporting Information

      The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.3c00087.

      • Figures on confocal imaging of optically cleared mice and fish, as well as comparison of diffusion and Gompertz curves for the clearing dynamics (PDF)


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