Computational adaptive optics for live three-dimensional biological imaging

Light microscopy of thick biological samples, such as tissues, is often limited by aberrations caused by refractive index variations within the sample itself. This problem is particularly severe for live imaging, a field of great current excitement due to the development of inherently fluorescent proteins. We describe a method of removing such aberrations computationally by mapping the refractive index of the sample using differential interference contrast microscopy, modeling the aberrations by ray tracing through this index map, and using space-variant deconvolution to remove aberrations. This approach will open possibilities to study weakly labeled molecules in difficult-to-image live specimens.

Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells

Antipyretic analgesics, taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring. Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), phenacetin, and caffeine. The mechanism of toxicity is unclear. We tested the effects of ASA, acetaminophen (APAF, the active metabolite of phenacetin), caffeine, and other related drugs individually and in combination on mouse inner medullary collecting duct cells (mIMCD3). The number of rapidly proliferating cells was reduced by ≈50% by 0.5 mM ASA, salicylic acid, or APAF. The drugs had less effect on confluent cells, which proliferate slowly. Thus, the slow in vivo turnover of IMCD cells could explain why clinical toxicity requires very high doses of these drugs over a very long period. Caffeine greatly potentiated the effect of acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase (COX) inhibitors, indomethacin and NS-398, did not reduce cell number except at concentrations greatly in excess of those that inhibit COX. Therefore, COX inhibition alone is not toxic. APAF arrests most cells in late G1 and S and produces a mixed form of cell death with both oncosis (swollen cells and nuclei) and apoptosis. APAF is known to inhibit the synthesis of DNA and cause chromosomal aberrations due to inhibition of ribonucleotide reductase. Such effects of APAF might account for renal medullary cell death in vivo and development of uroepithelial tumors from surviving cells that have chromosomal aberrations.

UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Molecular cytogenetic delineation of a novel critical genomic region in chromosome bands 11q22.3-923.1 in lymphoproliferative disorders.

Aberrations of the long arm of chromosome 11 are among the most common chromosome abnormalities in lymphoproliferative disorders (LPD). Translocations involving BCL1 at 11q13 are strongly associated with mantle cell lymphoma. other nonrandom aberrations, especially deletions and, less frequently, translocations, involving bands 11q21-923 have been identified by chromosome banding analysis. To date, the critical genomic segment and candidate genes involved in these deletions have not been identified. In the present study, we have analyzed tumors from 43 patients with LPD (B-cell chronic lymphocytic leukemia, n = 40; mantle cell lymphoma, n = 3) showing aberrations of bands 11q21-923 by fluorescence in situ hybridization. As probes we used Alu-PCR products from 17 yeast artificial chromosome clones spanning chromosome bands 11q14.3-923.3, including a panel of yeast artificial chromosome clones recognizing a contiguous genomic DNA fragment of approximately 9-10 Mb in bands 11q22.3-923.3. In the 41 tumors exhibiting deletions, we identified a commonly deleted segment in band 11q22.3-923.1; this region is approximately 2-3 Mb in size and contains the genes coding for ATM (ataxia telangiectasia mutated), RDX (radixin), and FDX1 (ferredoxin 1). Furthermore, two translocation break-points were localized to a 1.8-Mb genomic fragment contained within the commonly deleted segment. Thus, we have identified a single critical region of 2-3 Mb in size in which 11q14-923 aberrations in LPD cluster. This provides the basis for the identification of the gene(s) at 11q22.3-923.1 that are involved in the pathogenesis of LPD.

Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.

BCL-2-deficient T cells demonstrate accelerated cell cycle progression and increased apoptosis following activation. Increasing the levels of BCL-2 retarded the G0-->S transition, sustained the levels of cyclin-dependent kinase inhibitor p27Kip1, and repressed postactivation death. Proximal signal transduction events and immediate early gene transcription were unaffected. However, the transcription and synthesis of interleukin 2 and other delayed early cytokines were markedly attenuated by BCL-2. In contrast, a cysteine protease inhibitor that also blocks apoptosis had no substantial affect upon cytokine production. InterleUkin 2 expression requires several transcription factors of which nuclear translocation of NFAT (nuclear factor of activated T cells) and NFAT-mediated transactivation were impaired by BCL-2. Thus, select genetic aberrations in the apoptotic pathway reveal a cell autonomous coregulation of activation.

The genetic basis of epidermolysis bullosa simplex with mottled pigmentation.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical stress-induced degeneration of basal epidermal cells. It is now well-established that the three major subtypes of EBS are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here we show that a rare subtype, referred to as EBS with mottled pigmentation (MP), is also a disorder of these keratins. Affected members of two seemingly unrelated families with EBS-MP had a C to T point mutation in the second base position of codon 24 of one of two K5 alleles, leading to a Pro: Leu mutation. This mutation was not present in unaffected members nor in 100 alleles from normal individuals. Linkage analyses mapped the defect to this type II keratin gene (peak logarithm of odds score at phi = 0 of 3.9), which is located on chromosome 12q11-q13. This provides strong evidence that this mutation is responsible for the EBS-MP phenotype. Only conserved between K5 and K6, and not among any of the other type II keratins, Pro-24 is in the nonhelical head domain of K5, and only mildly perturbs the length of 10-nm keratin filaments assembled in vitro. However, this part of the K5 head domain is likely to protrude on the filament surface, perhaps leading to additional aberrations in intermediate filament architecture and/or in melanosome distribution that are seen ultrastructurally in patients with the mutation.

Gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix.

We have chosen tumors of the uterine cervix as a model system to identify chromosomal aberrations that occur during carcinogenesis. A phenotype/genotype correlation was established in defined regions of archived, formalin-fixed, and hematoxylin/eosin-stained tissue sections that were dissected from normal cervical epithelium (n = 3), from mild (n = 4), moderate (n = 6), and severe dysplasias/carcinomas in situ (CIS) (n = 13), and from invasive carcinomas (n = 10) and investigated by comparative genomic hybridization. The same tissues were analyzed for DNA ploidy, proliferative activity, and the presence of human papillomavirus (HPV) sequences. The results show that an increase in proliferative activity and tetraploidization had occurred already in mildly dysplastic lesions. No recurrent chromosomal aberrations were observed in DNA extracted from normal epithelium or from mild and moderate dysplasias, indicating that the tetraploidization precedes the loss or gain of specific chromosomes. A gain of chromosome 3q became visible in one of the severe dysplasias/CIS. Notably, chromosome 3q was overrepresented in 90% of the carcinomas and was also found to have undergone a high-level copy-number increase (amplification). We therefore conclude that the gain of chromosome 3q that occurs in HPV16-infected, aneuploid cells represents a pivotal genetic aberration at the transition from severe dysplasia/CIS to invasive cervical carcinoma.

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends.

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

Left-handed metamaterial coatings for subwavelength-resolution imaging

We report on a procedure to improve the resolution of far-field imaging by using a neighboring high-index medium that is coated with a left-handed metamaterial. The resulting plot can also exhibit an enhanced transmission by considering proper conditions to retract backscattering. Based on negative refraction, geometrical aberrations are considered in detail since they may cause a great impact in this sort of diffraction-unlimited imaging by reducing its resolution power. We employ a standard aberration analysis to refine the asymmetric configuration of metamaterial superlenses. We demonstrate that low-order centrosymmetric aberrations can be fully corrected for a given object plane. For subwavelength-resolution imaging, however, high-order aberrations become of relevance, which may be balanced with defocus. Not only the point spread function but also numerical simulations based on the finite-element method support our theoretical analysis, and subwavelength resolution is verified in the image plane.

Clinical and quality of life data correlation with a single-optic accommodating intraocular lens

Purpose: To examine a single-optic accommodating intraocular lens (IOL) visual performance by correlating IOL implanted eyes’ defocus curve with the intraocular aberrometric profile and the impact on the quality of life (QOL). Methods: Prospective consecutive case series study including a total of 25 eyes of 14 patients with ages ranging between 52 and 79 years old. All cases underwent cataract surgery with implantation of the single-optic accommodating IOL Crystalens HD (Bausch & Lomb). Distance and near visual acuity outcomes, intraocular aberrations, the defocus curve and QOL (NEI VFQ-25) were evaluated 3 months after surgery. Results: A significant improvement in distance visual acuity was found postoperatively (p = 0.02). Mean postoperative LogMAR uncorrected near visual acuity was 0.44 ± 0.23 (20/30). 60% of eyes had a postoperative addition between 0 and 1.5 diopters (D). The defocus curve showed an area of maximum visual acuity for the levels of defocus corresponding to distance and intermediate vision (−1 to +0.5 D). Postoperative intermediate visual acuity correlated significantly some QOL indices (r ≥ 0.51, p ≤ 0.03; difficulty in going down steps or seeing how people react to things that patient says) as well as with J0 component of manifest cylinder. Postoperative distance-corrected near visual acuity correlated significantly with age (r = 0.65, p < 0.01). Conclusions: This accommodating IOL seems to be able to restore the distance visual function as well as to provide an improvement in intermediate and near vision with a significant impact on patient's QOL, although limited by age and astigmatism. Future studies with larger sample sizes should confirm all these trends.

Visual and optical performance and quality of life after implantation of posterior chamber phakic intraocular lens

Purpose To evaluate visual, optical, and quality of life (QoL) outcomes and intercorrelations after bilateral implantation of posterior chamber phakic intraocular lenses. Methods Twenty eyes with high to moderate myopia of 10 patients that underwent PRL implantation (Phakic Refractive Lens, Carl Zeiss Meditec AG) were examined. Refraction, visual acuity, photopic and low mesopic contrast sensitivity (CS) with and without glare, ocular aberrations, as well as QoL outcomes (National Eye Institute Refractive Error Quality of Life Instrument-42, NEI RQL-42) were evaluated at 12 months postoperatively. Results Significant improvement in uncorrected (UDVA) and best-corrected distance (CDVA) visual acuities were found postoperatively (p < 0.01), with significant reduction in spherical equivalent (p < 0.01). Low mesopic CS without glare was significantly better than measurements with glare for 1.5, 3, and 6 cycles/degree (p < 0.01). No significant correlations between higher order root mean square (RMS) with CDVA (r = −0.26, p = 0.27) and CS (r ≤ 0.45, p ≥ 0.05) were found. Postoperative binocular photopic CS for 12 cycles/degree and 18 cycles/degree correlated significantly with several RQL-42 scales. Glare index correlated significantly with CS measures and scotopic pupil size (r = −0.551, p = 0.04), but not with higher order RMS (r = −0.02, p = 0.94). Postoperative higher order RMS, postoperative primary coma and postoperative spherical aberration was significant higher for 5-mm pupil diameter (p < 0.01) compared with controls. Conclusions Correction of moderate to high myopia by means of PRL implantation had a positive impact on CS and QoL. The aberrometric increase induced by the surgery does not seem to limit CS and QoL. However, perception of glare is still a relevant disturbance in some cases possibly related to the limitation of the optical zone of the PRL.

Pilot study of hyperopic LASIK using the solid-state laser technology

Background To evaluate and report the visual, refractive, and aberrometric outcomes of LASIK for the correction of low to moderate hyperopia in a pilot group using a commercially available solid-state laser. Methods Prospective pilot study including 11 consecutive eyes with low to moderate hyperopia of six patients undergoing LASIK surgery using the Pulzar Z1 solid-state laser (CustomVis Laser Pty Ltd., currently CV Laser). Visual, refractive, and aberrometric changes were evaluated. Potential complications were evaluated as well. Mean follow-up time was 6.6 months (range, 3 to 11 months). Results A significant improvement in LogMAR uncorrected distance visual acuity (UDVA) was observed postoperatively (p = 0.01). No significant change was detected in LogMAR corrected distance visual acuity (CDVA) (p = 0.21). Postoperative LogMAR UDVA was 0.1 (about 20/25) or better in ten eyes (90.9 %). Mean overall efficacy and safety indices were 1.03 and 1.12. Postoperatively, no losses of lines of CDVA were observed. Postoperative spherical equivalent was within ±1.00 D in ten eyes (90.9 %). With regard to aberrations, no statistically significant changes were found in higher order and primary coma RMS postoperatively (p ≥ 0.21), and only minimal but statistically significant negativization of primary spherical aberration (p = 0.02) was observed. No severe complications were observed. Conclusion LASIK surgery using the solid-state laser technology seems to be a useful procedure for the correction of low to moderate hyperopia, with minimal induction of higher order aberrations.

The posterior chamber phakic refractive lens (PRL): a review

Implantation of phakic intraocular lenses (pIOLs) is a reversible refractive procedure, preserving the patient’s accommodative function with minimal induction of higher order aberrations compared with corneal photoablative procedures. Despite this, as an intraocular procedure, it has potential risks such as cataracts, chronic uveitis, pupil ovalization, corneal endothelial cell loss, pigmentary dispersion syndrome, pupillary block glaucoma, astigmatism, or endophthalmitis. Currently, only two models of posterior chamber pIOLs are commercially available, the implantable collammer lens (STAAR Surgical Co.) and the phakic refractive lens (PRL; Zeiss Meditec). The number of published reports on the latter is very low, and some concerns still remain about its long-term safety. The present article reviews the published literature on the outcomes after PRL implantation in order to provide a general overview and evaluate its real potential as a surgical refractive option.

Deep Anterior Lamellar Keratoplasty versus Penetrating Keratoplasty for Macular Corneal Dystrophy: A Randomized Trial

Purpose: To compare outcomes of big-bubble deep anterior lamellar keratoplasty (DALK) and penetrating keratoplasty (PK) for macular corneal dystrophy. Design: Prospective, randomized, interventional case series. Methods: Setting: Single hospital. Patients: Eighty-two eyes of 54 patients requiring keratoplasty for the treatment of macular corneal dystrophy without endothelial involvement were included. Main outcome measures: Operative complications, uncorrected visual acuity, best-corrected visual acuity, contrast sensitivity function, higher-order aberrations, and endothelial cell density were evaluated. Results: The DALK and PK group consisted of 35 and 41 eyes, respectively. Best-corrected visual acuity after surgery was 20/40 or better 68.5% and 70.7% of the eyes in the DALK and PK groups, respectively (P > .05). No statistically significant differences between groups were found in contrast sensitivity function with and without glare for any spatial frequency (P > .05). Significantly higher levels of higher-order aberrations were found in the DALK group (P < .01). In both groups, a progressive and statistically significant reduction in endothelial cell density was found (P < .01). At the last follow-up, the mean endothelial cell loss was 18.1% and 26.9% in DALK and PK groups, respectively (P = .03). Graft rejection episodes were seen in 5 eyes (12.1%) in the PK group, and regrafting was necessary in 3 eyes (7.3%). Recurrence of the disease was documented in 5.7% and 4.8% of the eyes in the DALK and PK groups, respectively. Conclusions: Deep anterior lamellar keratoplasty with the big-bubble technique provided comparable visual and optical results as PK and resulted in less endothelial damage, as well as eliminating endothelial rejection in macular corneal dystrophy. Deep anterior lamellar keratoplasty surgery is a viable option for macular corneal dystrophy without endothelial involvement.

Advanced techniques for the human eye study: ocular aberrometry

Lectures about the course module "Advanced techniques for the human eye study: ocular aberrometry".