Aims
Microscopic evaluation of prostate specimens for both clinical and research purposes is generally performed on 5‐μm‐thick tissue sections. Because cross‐sections give a two‐dimensional (2D) ...representation, little is known about the actual underlying three‐dimensional (3D) architectural features of benign prostate tissue and prostate cancer (PCa). The aim of this study was to show that a combination of tissue‐clearing protocols and confocal microscopy can successfully be applied to investigate the 3D architecture of human prostate tissue.
Methods and results
Optical clearing of intact fresh and formalin‐fixed paraffin‐embedded (FFPE) clinical prostate specimens allowed us to visualize tissue structures up to a depth of 800 μm, whereas, in uncleared tissue, detection of fluorescence was only possible up to 70 μm. Fluorescent labelling with a general nuclear dye and antibodies against cytokeratin (CK) 5 and CK8‐18 resulted in comprehensive 3D imaging of benign peripheral and transition prostate zones, as well as individual PCa growth patterns. After staining, clearing, and imaging, samples could still be processed for 2D (immuno)histochemical staining and DNA analysis, enabling additional molecular and diagnostic characterization of small tissue specimens.
Conclusions
In conclusion, the applicability of 3D imaging to archival FFPE and fresh clinical specimens offers unlimited opportunities to study clinical and biological topics of interest in their actual 3D context.
The International Society for Clinical Electrophysiology of Vision (ISCEV) Standard for full-field electroretinography (ERG) describes a minimum procedure, but encourages more extensive testing. This ...ISCEV extended protocol describes an extension to the ERG Standard, namely the photopic negative response (PhNR) of the light-adapted flash ERG, as a well-established technique that is broadly accepted by experts in the field. The PhNR is a slow negative-going wave after the
b
-wave that provides information about the function of retinal ganglion cells and their axons. The PhNR can be reduced in disorders that affect the innermost retina, including glaucoma and other forms of optic neuropathy. This document, based on existing literature, provides a protocol for recording and analyzing the PhNR in response to a brief flash. The protocol includes full-field stimulation, a frequency bandwidth of the recording in which the lower limit does not exceed 0.3 Hz, and a spectrally narrowband stimulus, specifically, a red flash on a rod saturating blue background. Suggested flash strengths cover a range up to and including the minimum required to elicit a maximum amplitude PhNR. This extended protocol for recording the PhNR provides a simple test of generalized retinal ganglion cell function that could be added to standard ERG testing.
Purpose
Electroretinograms elicited by photopigment isolating white noise stimuli (wnERGs) in mice were measured. The dependency of rod- and cone-opsin-driven wnERGs on mean luminance was studied.
...Methods
Temporal white noise stimuli (containing all frequencies up to 20 Hz, equal amplitudes, random phases) that modulated either rhodopsin, S-opsin or L*-opsin, using the double silent substitution technique, were used to record wnERGs in mice expressing a human L*-opsin instead of the native murine M-opsin. Responses were recorded at 4 mean luminances (MLs).
Impulse response functions (IRFs) were obtained by cross-correlating the wnERG recordings with the corresponding modulation of the photopigment excitation elicited by the stimulus. So-called modulation transfer functions (MTFs) were obtained by performing a Fourier transform on the IRFs.
Potentials of two repeated wnERG recordings at corresponding time points were plotted against each other. The correlation coefficient (r
2
repr
) of the linear regression through these data was used to quantify reproducibility. Another correlation coefficient (r
2
ML
) was used to quantify the correlations of the wnERGs obtained at different MLs with those at the highest (for cone isolating stimuli) or lowest (for rod isolating stimuli) ML.
Results
IRFs showed an initial negative (a-wave like) trough N1 and a subsequent positive (b-wave like) peak P1. No oscillatory potential-like components were observed. At 0.4 and 1.0 log cd/m
2
ML robust L*- and S-opsin-driven IRFs were obtained that displayed similar latencies and dependencies on ML. L*-opsin-driven IRFs were 2.5–3 times larger than S-opsin-driven IRFs. Rhodopsin-driven IRFs were observed at −0.8 and − 0.2 log cd/m
2
and decreased in amplitude with increasing ML. They displayed an additional pronounced late negativity (N2), which may be a correlate of retinal ganglion cell activity.
R
2
repr
and r
2
ML
values increased for cones with increasing ML whereas they decreased for rods. For rhodopsin-driven MTFs at low MLs and L*-opsin-driven MTFs at high MLs amplitudes decreased with increasing frequency, with much faster decreasing amplitudes for rhodopsin. A delay was calculated from MTF phases showing larger delays for rhodopsin- vs. low delays for L*-opsin-driven responses.
Conclusion
Opsin-isolating wnERGs in mice show characteristics of different retinal cell types and their connected pathways.
To record and analyse electroretinograms (ERGs) to luminance stimuli with white noise temporal profiles in mice. White noise stimuli are expected to keep the retina in a physiologically more natural ...state than, e.g., flashes. The influence of mean luminance (ML) was studied.
Electroretinograms to luminance temporal white noise (TWN) modulation (wnERGs) were measured. The white noise stimuli contained all frequencies up to 20 Hz with equal amplitudes and random phases. Responses were recorded at 7 MLs between -0.7 and 1.2 log cd/m
. Impulse response functions (IRFs) were calculated by cross correlating the averaged white noise electroretinogram (wnERG) responses with the stimulus. Amplitudes and latencies of the initial trough and subsequent peak in the IRFs were measured at each ML. Fourier transforms of the IRFs resulted in modulation transfer functions (MTFs). wnERGs were averaged across different animals. They were measured twice and the responses at identical instances in the 1st and 2nd recordings were plotted against each other. The correlation coefficient (
) of the linear regression quantified the reproducibility. The results of the first and second measurement were further averaged. To study the underlying ERG mechanisms, the ERG potentials at the different MLs were plotted against those at the lowest and highest ML. The correlation coefficients (
) were used to quantify their similarities.
The amplitudes of the initial (a-wave-like) trough of the IRFs increased with increasing ML. The following positive (b-wave-like) peak showed a minimum at -0.4 log cd/m
above which there was a positive correlation between amplitude and ML. Their latencies decreased monotonously with increasing ML. In none of the IRFs, oscillatory potential (OP)-like components were observed.
values were minimal at a ML of -0.1 log cd/m
, where the MTFs changed from low-pass to band-pass.
values increased and decreased with increasing ML when correlated with responses obtained at the highest or the lowest ML, respectively.
White noise electroretinograms can be reliably recorded in mice with luminance stimuli. IRFs resemble flash ERGs superficially, but they offer a novel procedure to study retinal physiology. New components can be described in the IRFs. The wnERGs are either rod- or cone-driven with little overlap.
Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer ...from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.
Liver transplantation is the only effective treatment for end-stage liver disease, but absolute donor shortage remains a limiting factor. Recent advances in tissue engineering focus on generation of ...native extracellular matrix (ECM) by decellularized complete livers in animal models. Although proof of concept has been reported for human livers, this study aims to perform whole liver decellularization in a clinically relevant series using controlled machine perfusion. In this study, we describe a mild nondestructive decellularization protocol, effective in 11 discarded human whole liver grafts to generate constructs that reliably maintain hepatic architecture and ECM components using machine perfusion, while completely removing cellular DNA and RNA. The decellularization process preserved the ultrastructural ECM components confirmed by histology, electron microscopy, and proteomic analysis. Anatomical characteristics of the native microvascular network and biliary drainage of the liver were confirmed by contrast computed tomography scanning. Decellularized vascular matrix remained suitable for normal suturing and no major histocompatibility complex molecules were detected, suggesting absence of allo-reactivity when used for transplantation. After extensive washing, decellularized scaffolds were nontoxic for cells after reseeding human mesenchymal stromal or umbilical vein endothelial endothelium cells. Indeed, evidence of effective recellularization of the vascular lining was obtained. In conclusion, we established an effective method to generate clinically applicable liver scaffolds from human discarded whole liver grafts and show proof of concept that reseeding of normal human cells in the scaffold is feasible. This supports new opportunities for bioengineering of transplantable grafts in the future.
Aims
Many glandular lesions can mimic prostate cancer microscopically, including atrophic glands, adenosis and prostatic intraepithelial neoplasia. While the characteristic histopathological and ...immunohistochemical features of these lesions have been well established, little is known about their three‐dimensional architecture. Our objective was to evaluate the three‐dimensional organisation of common prostate epithelial lesions.
Methods and results
500 μm‐thick punches (n = 42) were taken from radical prostatectomy specimens, and stained with antibodies targeting keratin 8–18 and keratin 5 for identification of luminal and basal cells, respectively. Tissue samples were optically cleared in benzyl alcohol:benzyl benzoate and imaged using a confocal laser scanning microscope. The three‐dimensional architecture of peripheral and transition zone glands was acinar, composed of interconnecting and blind‐ending saccular tubules. In simple atrophy, partial atrophy and post‐atrophic hyperplasia, the acinar structure was attenuated with branching blind‐ending tubules from parental tubular structures. Three‐dimensional imaging revealed a novel variant of prostate atrophy characterised by large Golgi‐like atrophic spaces parallel to the prostate surface, which were represented by thin, elongated tubular structures on haematoxylin and eosin (H&E) slides. Conversely, adenosis lacked acinar organisation, so that it closely mimicked low‐grade prostate cancer. High‐grade prostatic intraepithelial neoplasia displayed prominent papillary intraluminal protrusions but retained an acinar organisation, whereas intraductal carcinoma predominantly consisted of cribriform proliferations with either spheroid, ellipsoid or complex interconnecting lumens.
Conclusions
While various prostate epithelial lesions might mimic malignancy on H&E slides, their three‐dimensional architecture is acinar and clearly different from the tubular structure of prostate cancer, with adenosis as an exception.
The Gleason score is one of the most important parameters for therapeutic decision-making in prostate cancer patients. Gleason growth patterns are defined by their histological features on 4- to 5-µm ...cross sections, and little is known about their three-dimensional architecture. Our objective was to characterize the three-dimensional architecture of prostate cancer growth patterns. Intact tissue punches (n = 46) of representative Gleason growth patterns from radical prostatectomy specimens were fluorescently stained with antibodies targeting Keratin 8/18 and Keratin 5 for the detection of luminal and basal epithelial cells, respectively. Punches were optically cleared in benzyl alcohol-benzyl benzoate and imaged using a confocal laser scanning microscope up to a depth of 500 µm. Gleason pattern 3, poorly formed pattern 4, and cords pattern 5 all formed a continuum of interconnecting tubules in which the diameter of the structures and the lumen size decreased with higher grades. In fused pattern 4, the interconnections between the tubules were markedly closer together. In these patterns, all tumor cells were in direct contact with the surrounding stroma. In contrast, cribriform Gleason pattern 4 and solid pattern 5 demonstrated a three-dimensional continuum of contiguous tumor cells, in which the vast majority of cells had no contact with the surrounding stroma. Transitions between cribriform pattern 4 and solid pattern 5 were seen. There was a decrease in the number and size of intercellular lumens from cribriform to solid growth pattern. Glomeruloid pattern 4 formed an intermediate structure consisting of a tubular network with intraluminal epithelial protrusions close to the tubule splitting points. In conclusion, three-dimensional microscopy revealed two major architectural subgroups of prostate cancer growth patterns: (1) a tubular interconnecting network including Gleason pattern 3, poorly formed and fused Gleason pattern 4, and cords Gleason pattern 5, and (2) serpentine contiguous epithelial proliferations including cribriform Gleason pattern 4 and solid Gleason pattern 5.
Microglia are the resident macrophages of the central nervous system and contribute to maintaining brain’s homeostasis. Current 2D “petri-dish”
in vitro
cell culturing platforms employed for ...microglia, are unrepresentative of the softness or topography of native brain tissue. This often contributes to changes in microglial morphology, exhibiting an amoeboid phenotype that considerably differs from the homeostatic ramified phenotype in healthy brain tissue. To overcome this problem, multi-scale engineered polymeric microenvironments are developed and tested for the first time with primary microglia derived from adult rhesus macaques. In particular, biomimetic 2.5D micro- and nano-pillar arrays (diameters = 0.29–1.06 µm), featuring low effective shear moduli (0.25–14.63 MPa), and 3D micro-cages (volume = 24 × 24 × 24 to 49 × 49 × 49 μm
3
) with and without micro- and nano-pillar decorations (pillar diameters = 0.24–1 µm) were fabricated using two-photon polymerization (2PP). Compared to microglia cultured on flat substrates, cells growing on the pillar arrays exhibit an increased expression of the ramified phenotype and a higher number of primary branches per ramified cell. The interaction between the cells and the micro-pillar-decorated cages enables a more homogenous 3D cell colonization compared to the undecorated ones. The results pave the way for the development of improved primary microglia
in vitro
models to study these cells in both healthy and diseased conditions.
Our past anecdotal evidence prompted that a longer response window (RW) in the Trivector test (Cambridge Colour Test) improved mature observers' estimates of chromatic discrimination. Here, we ...systematically explored whether RW variation affects chromatic discrimination thresholds measured by the length of Protan, Deutan and Tritan vectors. We employed the Trivector test with three RWs: 3 s, 5 s, and 8 s. Data of 30 healthy normal trichromats were stratified as age groups: 'young' (20-29 years), 'middle-aged' (31-48 years), and 'mature' (57-64 years). We found that for the 'young' and 'middle-aged', the thresholds were comparable at all tested RWs. However, the RW effect was apparent for the 'mature' observers: their Protan and Tritan thresholds decreased at 8-s RW compared to 3-s RW; moreover, their Tritan threshold decreased at 5-s RW compared to 3-s RW. Elevated discrimination thresholds at shorter RWs imply that for accurate performance, older observers require longer stimulus exposure and are indicative of ageing effects manifested by an increase in critical processing duration. Acknowledging low numbers in our 'middle-aged' and 'mature' samples, we consider our study as pilot. Nonetheless, our findings encourage us to advocate a RW extension in the Trivector protocol for testing mature observers, to ensure veridical measures of their chromatic discrimination by disentangling these from other ageing effects-slowing down of both motor responses and visual processing.
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