2.1.

Visualization of Neurons in Hippocampus and Whole Brain

Using ultramicroscopy we have been able to visualize fine details of neurons and neuronal networks of the mouse brain. For this purpose, we used transgenic mice with GFP-labeled neurons, which display a high contrast ratio.^18–20 We were able to visualize neuronal cell bodies in the hippocampus, including some of their dendrites, in the whole mouse brain. To obtain even higher resolution, we dissected hippocampi out of the whole brain and simultaneously visualized the detailed morphology of many neurons, which is not possible with standard fluorescence microscopy (Fig. 2). The complex dendritic network of pyramidal neurons became apparent in the optical sections. Using these sections, 3-D reconstructions of the dendritic networks were obtained including dendritic spines. Dendrites of granule cells could be also visualized through the overlying cortex.¹⁶ Neuronal cell bodies and the initial part of the apical dendrite can also be visualized without GFP staining. The fixation process causes some autofluorescence of the interstitial tissue, exempting neurons. Thus, by recording with a sensitive EM-CCD camera and inverting the contrast, neurons become visible to some extent.²¹

Fig. 2

Green fluorescent protein (GFP)-labeled neurons in the hippocampus.

2.2.

Visualization of Neurons in Spinal Cord

The above-mentioned clearing method needs strong GFP expression and cannot be easily applied to strongly myelinated structures as the adult spinal cord. Therefore, we developed the method further to be able to investigate processes of neuronal regeneration in the injured spinal cord. Initially, we substituted alcohol for the dehydration process by tetrahydrofuran (THF), which proved to affect GFP fluorescence much less.²² We were able to trace single axons in the spinal cord and identify regenerative axons. In addition, we could label macrophages and microglia with different colors, YFP, and CFP and image all three colors in the same specimen.

2.3.

Visualization of Mouse Embryos

Mouse embryos are ideal objects for visualization as they can be rendered completely transparent by our clearing procedure due to their low content of absorbing or refracting material. In whole mouse embryos, we were able to visualize peripheral nerves by immunostaining (Fig. 3).²³ The signal of the neurofilament antibody (anti-NF-160) was clearly distinguishable from the unspecific autofluorescence signal originating from the rest of the tissue. This allowed us to visualize the 3-D arrangement of nerves in peripheral limbs of the mouse embryo in collaboration with Edgar Kramer from the ZMNH in Hamburg.

Fig. 3

Three-dimensional (3-D) reconstruction of a mouse embryo. (a) The surface rendering shows the external view and (b) nervous structures inside the embryo made visible with fluorescent antibody staining.

2.4.

Visualization of Flies

Ultramicroscopy can also be applied to the study of insects. Despite their strong pigmentation, flies can be made transparent by our clearing procedure and small anatomical details can be visualized. In Drosophila melanogaster, the entire body, including muscles and the optic lobes in the nervous system, becomes transparent. Muscle development in Drosophila is the subject of large studies. It was possible to reconstruct whole flies by imaging their autofluorescence (Fig. 4). We used this approach in cooperation with Frank Schnorrer of the MPI of Biochemistry in Munich to screen a large number of different strains of Drosophila melanogaster. In this way, we were able to identify the gene responsible for the development of the indirect flight muscles of Drosophila.^24,25

Fig. 4

3-D reconstruction of Drosophila melanogaster: (a) surface and (b) internal features clearly depicting flight muscles.

3.1.

Correction of Objectives

A severe problem for the imaging of cleared specimens is the lack of suitable objectives designed for high refractive index clearing solutions. The 3DISCO clearing has a refractive index of 1.56, out of the range of any commercially available objective. The resulting refractive index mismatch strongly degrades image quality.²⁶ To solve this problem, we have adopted a new optical approach (WO 2015/010783 A1). We use air objectives with a magnification of up to $20\times$ and a NA of up to 0.45. We then equip them with a corrected dipping cap on the front side and with a complement optical unit on the back-side placed between the objective and the microscope. This enables us to obtain high resolution images with long working distance through clearing solution as shown in Fig. 5.

Fig. 5

(a) Images obtained with $4\times$ objective (NA, 0.28) in air and DBE without and with correction, (b) optical arrangement for objective (without and with correction optics), (c) $4\times$ objective with correction optics, and (d) three corrected air objectives, $2\times$ (NA, 0.14), $4\times$ (NA, 0.28) and $20\times$ (NA, 0.45).

We have assembled such corrected objectives also for CLARITY clearing.²⁷ Figure 6 shows in detail the typical neuroanatomy of the hippocampus imaged inside the whole mouse brain.

Fig. 6

(a) Anatomy of the hippocampus (20 optical sections) imaged in the whole mouse brain with a corrected $4\times$ objective (NA, 0.28). (b) Neurons in the neocortex imaged with corrected $4\times$ objective (300 optical sections).

Although specialized long working distance objectives for the CLARITY clearing are becoming commercially available (Olympus); they are extremely expensive (in the 30,000 Euro range). We have thus begun to apply our postcorrection approach also to standard high-NA water-immersion objectives (e.g., $40\times$, NA 0.8). They provide 3-mm working distance, which is sufficient for many biological experiments and come at a tenth of the cost of the specialized clarity objectives (Fig. 7). As these objectives are often already in the lab for patch clamping purposes, the addition of a postcorrection device may be financially a very attractive way to generate a high-NA-clarity objective. Of course, whole field and color correction may not be perfect, but this is not the case even for the expensive specialized objectives. We intend to make the described corrected air and water immersion objectives commercially available.

Fig. 7

(a) $40\times$ (NA, 0.8) and $10\times$ (NA, 0.3) water immersion objectives corrected for $n=1.45$ (CLARITY). (b) Already with the corrected $10\times$ objective, spines become visible in a CLARITY-cleared whole mouse brain.

3.2.

Generation of a Super-Thin Light Sheet

The lateral resolution that is obtained in light sheet microscopy mainly depends on the NA of the microscope objectives that can be used. However, the axial resolution depends on the light sheet thickness, as the light sheet can be made thinner than the axial extension of the point-spread function of the objectives. A light sheet generated with just an ordinary cylinder lens attains a thickness considerably larger than $100\mu \mathrm{m}$ at its ends if it is long enough to section the whole mouse brain. We have, therefore, developed a much more elaborate optical system (DE 10 2010 046 133) that uses a Powell lens and several aspheric lenses to generate a very thin, long light sheet Fig. 8.²⁸

Fig. 8

(a) Light sheet-generating optics, (b) profile of light sheets obtained with two different optical systems: red Gaussian, green generated with one Powell lens, and (c) comparison of the focus of the two light sheets.

3.3.

Further Improvement of Tissue Clearing and Preservation of Green Fluorescent Protein Fluorescence

Apart from optics, the clearing procedure is of paramount importance. A crucial problem related to our first clearing agent, benzyl alcohol and benzyl benzoate (BABB), is that it is not fully compatible with GFP.²³ It is known that benzyl benzoate, as well as benzyl alcohol, significantly diminishes the fluorescence of GFP and, under prolonged incubation, can completely eliminate it. We, therefore, used mice with strong neuronal GFP expression and always kept the clearing times to a minimum.

Because lipophilic organic clearing agents are not mixable with water, their application requires a careful dehydration of the tissue. Generally, this is done by using a graded series of ethanol. Ethanol can, however, destroy the fluorescence of GFP if the incubation times are not kept short. An optimal dehydration medium for ultramicroscopy should be mixable with water in any proportion and should not interact with GFP. We found THF to be the most promising. As a clearing agent, we found dibenzyl ether (DBE) to work best (3DISCO).^29,30 BABB, DBE, and other clearing and dehydration agents tend to build organic peroxides under prolonged storage, especially in the presence of oxygen or when exposed to light. We found concentrations of peroxides of about $5-50\mathrm{mg}/\mathrm{l}$ in commercial THF and DBE. These peroxide concentrations clearly diminish the success of chemical clearing since they quench the fluorescence of GFP.³¹ Also, benzyl alcohol and benzyl benzoate used for the preparation of BABB are already often contaminated with peroxides when delivered by the manufacturer. Thus, it is necessary to eliminate peroxides by means of solvent extraction or column chromatography and to then store the chemicals under argon to prevent them from reacting with oxygen. The content of peroxides can easily be determined by test strips, e.g., Quantofix 25 peroxide test, Sigma-Aldrich, Germany.

Finally, we began to embed our specimens in a special resin mixture in order to store them for long time and prevent bleaching. We screened various epoxy, polyester, and acrylic resins as well as different curing agents for their use as an embedding medium for cleared GFP-expressing biological tissues. Finally, we developed a transparent solid–resin formulation, which maintains the specimens’ transparency and provides a constant signal-to-noise ratio even after hours of continuous laser irradiation after an initial small drop. This may be due to preserving the fluorescent structure of GFP in the resin. The refractive index of the cured resin matches the refractive index of DBE ($n=1.56$) but can also easily be adapted to other clearing media. Thus, light can pass from the clearing medium to the resin and the resin-embedded specimen without refraction. The pronounced fluorescence-stabilizing effect of our resin embedding allows high-power illumination or long exposure times with virtually no loss in signal quality. Also, long-term storage of cleared GFP-expressing samples becomes possible for archiving purposes or repetitive investigations.³²

4.1.

Three-Dimensional-Superresolution Imaging with Stimulated Emission Depletion Light Sheets

It has been shown that it is possible to reduce light sheet thickness by applying stimulated emission depletion (STED) to light sheet microscopy (Friedrich et al.³³). The reduction in light sheet thickness described was marginal, but also patent applications describing this approach can be found (e.g., DE 10 2009 008 646). By stepping the STED light sheet in the three planes x, y, and z, a 3-D superresolution reconstruction of the specimen should be obtainable.³⁴ For this approach, it is pivotal that the specimen remains absolutely rigid so that it can be rotated without minute deformations. This is the case for 3DISCO cleared specimens, which become hard as epoxy resin. As the STED technology tends to bleach preparations, the antibleaching effect of the resin described above should be very helpful. The resin would further support the mechanical stability.

At the end, the combination of all these technologies may allow for the reconstruction of the whole mouse brain with single synapse resolution. Data handling would then, of course, become an important isssue.

4.2.

Biology: Towards Visualization of Memory

A very exciting application of ultramicroscopy would be the whole-brain visualization of the neuronal populations, which are involved in development, maintenance, and expression of fear memories in the mouse brain. Although current in vivo imaging techniques (e.g., fMRI) have been helpful in determining the brain areas responsible for particular functions, these methods are technically limited. None of them attains cellular resolution. Cellular resolution can, in principle, be achieved by visualization of immediate early gene expression. For this purpose, transgenic mice could be used, which express the fluorescent proteins eGFP or dtTomato controlled by the promoter of the activity-dependent gene cFos. It has been shown that cFos-eGFP expression could be induced in restricted brain regions after specific activation of neuronal ensembles.^35,36 For this analysis, a combination of two-photon microscopy and histological sectioning was used. This combination proved to be reliable and powerful but also rather costly and limited in its resolution is practice. To allow visualization of activated neurons in the whole brain, high-resolution ultramicroscopy of cleared brains seems to be the method of choice. Using cFos–eGFP, it should be possible to reconstruct the distribution of activated neurons during fear conditioning and the recall of recent and remote fear memories. The establishment of this technique would open completely new avenues for the study of activity changes in complex biological systems.

If once STED light sheet microscopy could be combined with the visualization of functional fluorescent proteins, one might dream of identifying synapses that are active during the storage of certain engrams in memory formation.

4.3.

Clinical Applications of Ultramicroscopy

Ultramicroscopy should be also applicable for diagnosis in pathology regarding cancer staging. It allows a 3-D reconstruction of malignant tissue removed during operation. Of special interest for pathologists is the nature of the tissue at the margins of the operation material. We found that in fixed tumor tissue, the structures become visible just by their autofluorescence. This is probably induced by the formalin fixation (Fig. 9). Exact identification of tumor and normal tissue will need further correlative studies with standard histological staining. However, in Fig. 9, the strong autofluorescent material in milk-duct-like structures is already prominent. As the histological stain of another part of the tumor shows, this bright material seems to be cancerous cells in the milk ducts, typical for this ductal carcinoma (DCIS) in situ. With ultramicroscopy, now the 3-D arrangement of this part of the tumor can be visualized. This first example shows that in the future, ultramicroscopy may become a valuable instrument for tumor staging in clinical medicine.

Fig. 9

Human breast cancer (DCIS) imaged with a $2\times$ objective. Both in the 3-D reconstruction (a) and the standard histological section and (b) the clogged milk-duct-like structures can be seen.