Correlative light and electron microscopy using immunolabeled sections.

In correlative microscopy, light microscopy provides the overview and orientation of the complex cells and tissue, while electron microscopy offers the detailed localization and correlation of subcellular structures. In this chapter we offer detailed high-quality electron microscopical preparation methods for optimum preservation of the cellular ultrastructure. From such preparations serial thin sections are collected and used for comparative histochemical, immunofluorescence, and immunogold staining.In light microscopy histological stains identify the orientation of the sample and immunofluorescence labeling facilitates to find the region of interest, namely, the labeled cells expressing the macromolecule under investigation. Sections, labeled with immunogold are analyzed by electron microscopy in order to identify the label within the cellular architecture at high resolution.

Transmission disequilibrium of small CNVs in simplex autism.

We searched for disruptive, genic rare copy-number variants (CNVs) among 411 families affected by sporadic autism spectrum disorder (ASD) from the Simons Simplex Collection by using available exome sequence data and CoNIFER (Copy Number Inference from Exome Reads). Compared to high-density SNP microarrays, our approach yielded ∼2× more smaller genic rare CNVs. We found that affected probands inherited more CNVs than did their siblings (453 versus 394, p = 0.004; odds ratio [OR] = 1.19) and that the probands' CNVs affected more genes (921 versus 726, p = 0.02; OR = 1.30). These smaller CNVs (median size 18 kb) were transmitted preferentially from the mother (136 maternal versus 100 paternal, p = 0.02), although this bias occurred irrespective of affected status. The excess burden of inherited CNVs among probands was driven primarily by sibling pairs with discordant social-behavior phenotypes (p < 0.0002, measured by Social Responsiveness Scale [SRS] score), which contrasts with families where the phenotypes were more closely matched or less extreme (p > 0.5). Finally, we found enrichment of brain-expressed genes unique to probands, especially in the SRS-discordant group (p = 0.0035). In a combined model, our inherited CNVs, de novo CNVs, and de novo single-nucleotide variants all independently contributed to the risk of autism (p < 0.05). Taken together, these results suggest that small transmitted rare CNVs play a role in the etiology of simplex autism. Importantly, the small size of these variants aids in the identification of specific genes as additional risk factors associated with ASD.

Apocrypha Hiberniae. I. Evangelia infantiae

In 1927 M. R. James published Latin Infancy Gospels, identified by him in two related but not identical manuscripts (one the British Library Arundel 404; the other from Hereford), together with a parallel text from the Irish manuscript known as the Leabhar Breac. Later researches brought to light more manuscripts of this Latin work, and also of the Irish text. James recognized that his apocryphal Latin Infancy text was compiled from a combination of the Protevangelium of James and a hitherto unknown text which he named "The Source". Recent research has identified a full Latin translation of the Protevangelium of James. A hitherto unrecognized Irish Infancy Narrative has also been identified in the Dublin manuscript known as the Liber Flavus Fergusiorum. A deep study of this related tradition was called for. This has been carried out over the past ten years by an Irish team in conjunction with Professor Daniel Kaestli and AELAC. The fruits of this labour are published in these two volumes. Volume 13 has a general introduction with a historical sketch of New Testament apocrypha in Ireland and a history of research on the subject. This is followed by a comparison of the Infancy Narratives in the Leabhar Breac and the Liber Flavus Fergusiorum. There are special introductions to these Infancy texts, followed by critical editions of the Irish texts, accompanied by English translations and rich annotation. Next there is similar treatment of the Irish versified Narrative (from ca. 700) of the Childhood Deeds of Jesus (commonly known as the Infancy Narrative (or Gospel) of Thomas. There is then (in volume 14, but with continuous pagination) the edition and translation of an Irish thirteenth-century poem with elements from Infancy Narratives, and both Latin and Irish texts on the wonders at Christ's birth, accompanied by translations and notes. The edition of the Irish material is followed by a critical edition of the full Arundel and Hereford forms of the Infancy Narrative (here referred to as the "J Compilation"), together with a detailed study of all the questions relating to this work. The volume concludes with a critical edition (by Rita Beyers) of the Latin text of the Protevangelium of James, accompanied by a detailed study of the work.. The work contains a detailed study of the Latin translations of the Protevangelium of James and the transmission of this work in the West. The "J Compilation" (a combination of the Protevangelium and texts of Pseudo-Matthew) can be traced back in manuscript transmission to ca. 800,and must have originated some time earlier. Behind it stands an earlier "I ("I" for Irish) Compilation" without influence from Pseudo-Matthew, the form found in the Irish witnesses. It is argued that M. R. James's "Source" may be of Judaeo-Christian origin and may really be the Gospel of the Nazoreans. Among the indexes there is a list of all the Irish words found in the texts. This edition of the Irish and related Latin texts is a major contribution to the study of the apocryphal Infancy Narratives. It should also be of particular interest to Celtic scholars, to students of Irish ecclesiastical learning, and in general to all medievalists.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.

The degradation of HFR1, a putative bHLH class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1.

All developmental transitions throughout the life cycle of a plant are influenced by light. In Arabidopsis, multiple photoreceptors including the UV-A/blue-sensing cryptochromes (cry1-2) and the red/far-red responsive phytochromes (phyA-E) monitor the ambient light conditions. Light-regulated protein stability is a major control point of photomorphogenesis. The ubiquitin E3 ligase COP1 (constitutively photomorphogenic 1) regulates the stability of several light-signaling components. HFR1 (long hypocotyl in far-red light) is a putative transcription factor with a bHLH domain acting downstream of both phyA and the cryptochromes. HFR1 is closely related to PIF1, PIF3, and PIF4 (phytochrome interacting factor 1, 3 and 4), but in contrast to the latter three, there is no evidence for a direct interaction between HFR1 and the phytochromes. Here, we show that the protein abundance of HFR1 is tightly controlled by light. HFR1 is an unstable phosphoprotein, particularly in the dark. The proteasome and COP1 are required in vivo to degrade phosphorylated HFR1. In addition, HFR1 can interact with COP1, consistent with the idea of COP1 directly mediating HFR1 degradation. We identify a domain, conserved among several bHLH class proteins involved in light signaling , as a determinant of HFR1 stability. Our physiological experiments indicate that the control of HFR1 protein abundance is important for a normal de-etiolation response.

The role of neutralizing antibodies for mouse mammary tumor virus transmission and mammary cancer development.

Mouse mammary tumor virus (MMTV) infection establishes chronic germinal centers and a lifelong neutralizing Ab response. We show that removal of the draining lymph node after establishment of the germinal center reaction led to complete loss of neutralizing Abs despite comparable infection levels in peripheral lymphocytes. Importantly, in the absence of neutralization, only the exocrine organs mammary gland, salivary gland, pancreas, and skin showed strikingly increased infection, resulting in accelerated mammary tumor development. Induction of stronger neutralization did not influence chronic infection levels of peripheral lymphoid organs but strongly inhibited mammary gland infection and virus transmission to the next generation. Taken together, we provide evidence that a tight equilibrium in virus neutralization allows limited infection of exocrine organs and controls cancer development in susceptible mouse strains. These experiments show that a strong neutralizing Ab response induced after infection is not able to control lymphoid MMTV infection. Strong neutralization, however, is capable of blocking amplification of mammary gland infection, tumor development, and virus transmission to the next generation. The results also indicate a role of neutralization in natural resistance to MMTV infection.

Characterization of pupil responses to blue and red light stimuli in autosomal dominant retinitis pigmentosa due to NR2E3 mutation.

PURPOSE: We characterized the pupil responses that reflect rod, cone, and melanopsin function in a genetically homogeneous cohort of patients with autosomal dominant retinitis pigmentosa (adRP). METHODS: Nine patients with Gly56Arg mutation of the NR2E3 gene and 12 control subjects were studied. Pupil and subjective visual responses to red and blue light flashes over a 7 log-unit range of intensities were recorded under dark and light adaptation. The pupil responses were plotted against stimulus intensity to obtain red-light and blue-light response curves. RESULTS: In the dark-adapted blue-light stimulus condition, patients showed significantly higher threshold intensities for visual perception and for a pupil response compared to controls (P = 0.02 and P = 0.006, respectively). The rod-dependent, blue-light pupil responses decreased with disease progression. In contrast, the cone-dependent pupil responses (light-adapted red-light stimulus condition) did not differ between patients and controls. The difference in the retinal sensitivity to blue and red stimuli was the most sensitive parameter to detect photoreceptor dysfunction. Unexpectedly, the melanopsin-mediated pupil response was decreased in patients (P = 0.02). CONCLUSIONS: Pupil responses of patients with NR2E3-associated adRP demonstrated reduced retinal sensitivity to dim blue light under dark adaptation, presumably reflecting decreased rod function. Rod-dependent pupil responses were quantifiable in all patients, including those with non-recordable scotopic electroretinogram, and correlated with the extent of clinical disease. Thus, the chromatic pupil light reflex can be used to monitor photoreceptor degeneration over a larger range of disease progression compared to standard electrophysiology.

RNA-based gene duplication sheds new light on mammalian sex chromosome evolution

ABSTRACT : Gene duplication is a fundamental source of raw material for the origin of genetic novelty. It has been assumed for a long time that DNA-based gene duplication was the only source of new genes. Recently however, RNA-based gene duplication (retroposition) was shown in multiple organisms to contribute significantly to their genetic diversity. This mechanism produces intronless gene copies (retrocopies) that are inserted in random genomic position, independent of the position of the parental source genes. In human, mouse and fruit fly, it was demonstrated that the X-linked genes spawned an excess of functional retroposed gene copies (retrogenes). In human and mouse, the X chromosome also recruited an excess of retrogenes. Here we further characterized these interesting biases related to the X chromosome in mammals. Firstly, we have confirmed presence of the aforementioned biases in dog and opossum genome. Then based on the expression profile of retrogenes during various spermatogenetic stages, we have provided solid evidence that meiotic sex chromosome inactivation (MSCI) is responsible for an excess of retrogenes stemming from the X chromosome. Moreover, we showed that the X-linked genes started to export an excess of retrogenes just after the split of eutherian and marsupial mammalian lineages. This suggests that MSCI has originated around this time as well. More fundamentally, as MSCI reflects the spread of recombination barrier between the X and Y chromosomes during their evolution, our observation allowed us to re-estimate the age of mammalian sex chromosomes. Previous estimates suggested that they emerged in the common ancestor of all mammals (before the split of monotreme lineage); whereas, here we showed that they originated around the split of marsupial and eutherian lineages, after the divergence of monotremes. Thus, the therian (marsupial and eutherian) sex chromosomes are younger than previously thought. Thereafter, we have characterized the bias related to the recruitment of genes to the X chromosome. Sexually antagonistic forces are most likely driving this pattern. Using our limited retrogenes expression data, it is difficult to determine the exact nature of these forces but some conclusions have been made. Lastly, we looked at the history of this biased recruitment: it commenced around the split of marsupial and eutherian lineages (akin to the biased export of genes out of the X). In fact, the sexually antagonistic forces are predicted to appear just around that time as well. Thereby, the history of the recruitment of genes to the X, provides an indirect evidence that these forces are responsible for this bias.

FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor.

The phytochrome (phy) family of photoreceptors is of crucial importance throughout the life cycle of higher plants. Light-induced nuclear import is required for most phytochrome responses. Nuclear accumulation of phyA is dependent on two related proteins called FHY1 (Far-red elongated HYpocotyl 1) and FHL (FHY1 Like), with FHY1 playing the predominant function. The transcription of FHY1 and FHL are controlled by FHY3 (Far-red elongated HYpocotyl 3) and FAR1 (FAr-red impaired Response 1), a related pair of transcription factors, which thus indirectly control phyA nuclear accumulation. FHY1 and FHL preferentially interact with the light-activated form of phyA, but the mechanism by which they enable photoreceptor accumulation in the nucleus remains unsolved. Sequence comparison of numerous FHY1-related proteins indicates that only the NLS located at the N-terminus and the phyA-interaction domain located at the C-terminus are conserved. We demonstrate that these two parts of FHY1 are sufficient for FHY1 function. phyA nuclear accumulation is inhibited in the presence of high levels of FHY1 variants unable to enter the nucleus. Furthermore, nuclear accumulation of phyA becomes light- and FHY1-independent when an NLS sequence is fused to phyA, strongly suggesting that FHY1 mediates nuclear import of light-activated phyA. In accordance with this idea, FHY1 and FHY3 become functionally dispensable in seedlings expressing a constitutively nuclear version of phyA. Our data suggest that the mechanism uncovered in Arabidopsis is conserved in higher plants. Moreover, this mechanism allows us to propose a model explaining why phyA needs a specific nuclear import pathway.

Chromatic pupil responses: preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex.

OBJECTIVE: To weight the rod-, cone-, and melanopsin-mediated activation of the retinal ganglion cells, which drive the pupil light reflex by varying the light stimulus wavelength, intensity, and duration. DESIGN: Experimental study. PARTICIPANTS: Forty-three subjects with normal eyes and 3 patients with neuroretinal visual loss. METHODS: A novel stimulus paradigm was developed using either a long wavelength (red) or short wavelength (blue) light given as a continuous Ganzfeld stimulus with stepwise increases over a 2 log-unit range. The pupillary movement before, during, and after the light stimulus was recorded in real time with an infrared illuminated video camera. MAIN OUTCOME MEASURES: The percent pupil contraction of the transient and sustained pupil response to a low- (1 cd/m(2)), medium- (10 cd/m(2)), and high-intensity (100 cd/m(2)) red- and blue-light stimulus was calculated for 1 eye of each subject. From the 43 normal eyes, median and 25th, 75th, 5th, and 95th percentile values were obtained for each stimulus condition. RESULTS: In normal eyes at lower intensities, blue light evoked much greater pupil responses compared with red light when matched for photopic luminance. The transient pupil contraction was generally greater than the sustained contraction, and this disparity was greatest at the lowest light intensity and least apparent with bright (100 cd/m(2)) blue light. A patient with primarily rod dysfunction (nonrecordable scotopic electroretinogram) showed significantly reduced pupil responses to blue light at lower intensities. A patient with achromatopsia and an almost normal visual field showed selective reduction of the pupil response to red-light stimulation. A patient with ganglion cell dysfunction owing to anterior ischemic optic neuropathy demonstrated global loss of pupil responses to red and blue light in the affected eye. CONCLUSIONS: Pupil responses that differ as a function of light intensity and wavelength support the hypothesis that selected stimulus conditions can produce pupil responses that reflect phototransduction primarily mediated by rods, cones, or melanopsin. Use of chromatic pupil responses may be a novel way to diagnose and monitor diseases affecting either the outer or inner retina.

The Irish Infancy Narratives and their Relationship with Latin Sources

(Résumé de l'ouvrage) In 1927 M. R. James published Latin Infancy Gospels, identified by him in two related but not identical manuscripts (one the British Library Arundel 404; the other from Hereford), together with a parallel text from the Irish manuscript known as the Leabhar Breac. Later researches brought to light more manuscripts of this Latin work, and also of the Irish text. James recognized that his apocryphal Latin Infancy text was compiled from a combination of the Protevangelium of James and a hitherto unknown text which he named "The Source". Recent research has identified a full Latin translation of the Protevangelium of James. A hitherto unrecognized Irish Infancy Narrative has also been identified in the Dublin manuscript known as the Liber Flavus Fergusiorum. A deep study of this related tradition was called for. This has been carried out over the past ten years by an Irish team in conjunction with Professor Daniel Kaestli and AELAC. The fruits of this labour are published in these two volumes. Volume 13 has a general introduction with a historical sketch of New Testament apocrypha in Ireland and a history of research on the subject. This is followed by a comparison of the Infancy Narratives in the Leabhar Breac and the Liber Flavus Fergusiorum. There are special introductions to these Infancy texts, followed by critical editions of the Irish texts, accompanied by English translations and rich annotation. Next there is similar treatment of the Irish versified Narrative (from ca. 700) of the Childhood Deeds of Jesus (commonly known as the Infancy Narrative (or Gospel) of Thomas. There is then (in volume 14, but with continuous pagination) the edition and translation of an Irish thirteenth-century poem with elements from Infancy Narratives, and both Latin and Irish texts on the wonders at Christ's birth, accompanied by translations and notes. The edition of the Irish material is followed by a critical edition of the full Arundel and Hereford forms of the Infancy Narrative (here referred to as the "J Compilation"), together with a detailed study of all the questions relating to this work. The volume concludes with a critical edition (by Rita Beyers) of the Latin text of the Protevangelium of James, accompanied by a detailed study of the work.. The work contains a detailed study of the Latin translations of the Protevangelium of James and the transmission of this work in the West. The "J Compilation" (a combination of the Protevangelium and texts of Pseudo-Matthew) can be traced back in manuscript transmission to ca. 800,and must have originated some time earlier. Behind it stands an earlier "I ("I" for Irish) Compilation" without influence from Pseudo-Matthew, the form found in the Irish witnesses. It is argued that M. R. James's "Source" may be of Judaeo-Christian origin and may really be the Gospel of the Nazoreans. Among the indexes there is a list of all the Irish words found in the texts. This edition of the Irish and related Latin texts is a major contribution to the study of the apocryphal Infancy Narratives. It should also be of particular interest to Celtic scholars, to students of Irish ecclesiastical learning, and in general to all medievalists.

Latin Infancy Gospels: The J Compilation. Introduction and Edition

(Résumé de l'ouvrage) In 1927 M. R. James published Latin Infancy Gospels, identified by him in two related but not identical manuscripts (one the British Library Arundel 404; the other from Hereford), together with a parallel text from the Irish manuscript known as the Leabhar Breac. Later researches brought to light more manuscripts of this Latin work, and also of the Irish text. James recognized that his apocryphal Latin Infancy text was compiled from a combination of the Protevangelium of James and a hitherto unknown text which he named "The Source". Recent research has identified a full Latin translation of the Protevangelium of James. A hitherto unrecognized Irish Infancy Narrative has also been identified in the Dublin manuscript known as the Liber Flavus Fergusiorum. A deep study of this related tradition was called for. This has been carried out over the past ten years by an Irish team in conjunction with Professor Daniel Kaestli and AELAC. The fruits of this labour are published in these two volumes. Volume 13 has a general introduction with a historical sketch of New Testament apocrypha in Ireland and a history of research on the subject. This is followed by a comparison of the Infancy Narratives in the Leabhar Breac and the Liber Flavus Fergusiorum. There are special introductions to these Infancy texts, followed by critical editions of the Irish texts, accompanied by English translations and rich annotation. Next there is similar treatment of the Irish versified Narrative (from ca. 700) of the Childhood Deeds of Jesus (commonly known as the Infancy Narrative (or Gospel) of Thomas. There is then (in volume 14, but with continuous pagination) the edition and translation of an Irish thirteenth-century poem with elements from Infancy Narratives, and both Latin and Irish texts on the wonders at Christ's birth, accompanied by translations and notes. The edition of the Irish material is followed by a critical edition of the full Arundel and Hereford forms of the Infancy Narrative (here referred to as the "J Compilation"), together with a detailed study of all the questions relating to this work. The volume concludes with a critical edition (by Rita Beyers) of the Latin text of the Protevangelium of James, accompanied by a detailed study of the work.. The work contains a detailed study of the Latin translations of the Protevangelium of James and the transmission of this work in the West. The "J Compilation" (a combination of the Protevangelium and texts of Pseudo-Matthew) can be traced back in manuscript transmission to ca. 800,and must have originated some time earlier. Behind it stands an earlier "I ("I" for Irish) Compilation" without influence from Pseudo-Matthew, the form found in the Irish witnesses. It is argued that M. R. James's "Source" may be of Judaeo-Christian origin and may really be the Gospel of the Nazoreans. Among the indexes there is a list of all the Irish words found in the texts. This edition of the Irish and related Latin texts is a major contribution to the study of the apocryphal Infancy Narratives. It should also be of particular interest to Celtic scholars, to students of Irish ecclesiastical learning, and in general to all medievalists.

Congenital Myasthenic Syndromes with COLQ mutations: Long term follow-up

Congenital myasthenic syndromes (CMS) are clinically and genetically heterogeneous inherited disorders characterized by impaired neuromuscular transmission. Mutations in the acetylcholinesterase (AChE) collagenlike tail subunit gene (ColQ) cause recessive forms of synaptic CMS with end plate AChE deficiency. We report the time course of clinical manifestations in 15 COLQ-mutated patients followed from 1987 to 2010. All patients suffered from a muscle weakness with onset at birth or in childhood. Ocular and bulbar signs were found in 60% of the patients and delayed pupillary light response in 20% of our patients. EMG study demonstrated a decrement on repetitive nerve stimulation and repetitive compound muscle action potential in all patients. Clinical symptoms strongly fluctuated daily, weekly, monthly or even yearly. Severe relapses were characterized by a general motor weakness associated with pain which resolved spontaneously after a few months whereas the relapses with these symptoms and bulbar signs could last up to several years. Genetic analyses identified 16 different mutations including 9 novel ones. There was no genotype-phenotype correlation. Our study confirms the predominance of oculobulbar signs and the frequency of respiratory distress in COLQrelated CMS. At the end of the follow up of 23 years, interesting findings were (i) the spontaneous reversibility of severe relapses, some of them lasting for up to 5 years (ii) the good prognosis of COLQ-related CMS, since at the end of the follow-up 80% of patients were ambulant and 87% of patients had no respiratory trouble (iii) the efficacy of Ephedrine and, to a lesser extend, of 3-4 DAP. The triggering factors of relapses were esterase inhibitors, effort, puberty, pregnancy and delivery highlighting the importance of hormonal factors in CMS. In conclusion, patients diagnosed with unknown congenital myopathy should undergo an electrophysiological study of neuromuscular junction to identify ColQ-related CMS.

Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy.

BACKGROUND: Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet's membrane, in the posterior cornea. METHODOLOGY/PRINCIPAL FINDINGS: We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet's membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats. CONCLUSIONS/SIGNIFICANCE: Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.