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INTRODUCTION: The purpose of this study was to evaluate whether vital blue dyes could enhance the contrast of intraoperative optical coherence tomography (OCT) during macular surgery. METHODS: Consecutive patients undergoing elective pars plana vitrectomy for vitreomacular interface disorders were enrolled. Intraoperative OCT was performed with the Artevo 800 microscope (Carl Zeiss Meditec AG, Jena, Germany) before and after injection of 0.2 mL of Trypan Blue and Brilliant Blue G Ophthalmic Solution. The OCT contrast ratio was measured with ImageJ, while the overall scan quality was subjectively classified using a 4-point scale. RESULTS: Ten eyes of 10 patients were enrolled in the study. The OCT contrast ratio was 9.39 ± 5.35 without blue dye and significantly improved to 14.31 ± 10.50 after blue dye injection (p = 0.027). The percentage of patients with a grade 4 scan quality also significantly improved (from 40% without blue dye to 90% with blue dye injection; p = 0.012). CONCLUSION: The use of blue dyes during intraoperative OCT is an effective strategy for improving contrast and scan quality without affecting the surgical time and workflow.

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INTRODUCTION: Recent technologies and new devices continue to be developed in vitreoretinal surgeries, and they provide more details, enhance safety, improve surgeons' comfort, and better visual and anatomical outcomes. Some devices have been used for better visualization during surgery, and some help the operation performance. They are divided into the following titles: Intraoperative OCT (including hand-held, probe-integrated, and microscope-integrated OCT), three-dimensional visualization system, virtual reality system, endoscopic vitrectomy (fiber optics and non-fiber optics), wide-angle viewing systems (contact and non-contact lenses), endo-illumination, light filters, chromovitrectomy, the retinal prosthesis (including epiretinal, subretinal, and suprachoroidal devices), robot-assisted vitreoretinal surgery, newer Vitreoretinal instruments, gene and cell therapy. AREAS COVERED: In this narrative review, we focused on PubMed articles between 2010-2023 with these keywords: 'Optical Coherence Tomography,' 'Three-Dimensional,' 'Virtual System,' 'intraoperative,' 'endoscopic,' 'vitrectomy,' 'lens,' 'illumination,' 'filters,' 'chromovitrectomy,' 'prosthesis,' 'robotic surgery,' 'instrument,' 'gene,' 'cell.' EXPERT OPINION: The main aim of this review is to update the reader on the latest progression in intraoperative imaging and surgical vision technologies and to provide an understanding of how each has helped improve operation and surgical outcomes. The surgeons should know recent updates to do their best and achieve the most excellent results.

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PURPOSE: To compare two commercially available staining solutions (MembraneBlue Dual® by D.O.R.C., Netherlands, and TWIN by AL.CHI.MI.A. S.R.L., Italy), in terms of intraoperative handling, staining efficacy and safety, in eyes undergoing surgery for idiopathic epiretinal membrane (ERM). METHODS: In this observational cross-sectional study, the performance of the dyes used during the procedure (cohesion, ERM and internal limiting membrane [ILM] staining efficacy) was scored by the surgeon using a customized questionnaire after 10 procedures with each of the two dyes. Best-corrected visual acuity (BCVA), central foveal thickness (CFT), blue-light fundus autofluorescence (BAF), and microperimetry-determined retinal sensitivity were reviewed preoperatively and then at 1 and 3 months after surgery. RESULTS: ILM staining efficacy with TWIN was scored 2.89 ± 0.33 by the surgeons, which turned out to be higher than with MembraneBlue Dual® (1.90 ± 0.31, P = 0.0002). The cohesion score was 2.70 ± 0.48 for TWIN and resulted significantly higher than with MembraneBlue Dual® (1.60 ± 0.51, P = 0.0010). BCVA, CFT and retinal sensitivity were similar in the two groups, 1 and 3 months postoperatively (P nonsignificant for all). CONCLUSIONS: Both TWIN and MembraneBlue Dual® dyes showed suitable staining properties and equivalent safety and efficacy profiles, both intra- and postoperatively. The TWIN dye might offer a solution for surgeons who prefer a more cohesive and stable dye.

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PURPOSE: To evaluate epiretinal membrane (ERM) removal utilizing internal limiting membrane (ILM) forceps and visualization with triamcinolone acetonide (TA). METHODS: Retrospective interventional case series of eyes undergoing ERM removal with TA visualization with follow-up of up to five years. Best-corrected visual acuity (BCVA), central macular thickness (CMT), and incidence of complications were reviewed. RESULTS: A total of 132 eyes were included with 54 and 16 eyes completing 3- and 5-year follow-up, respectively. Mean BCVA and CMT improved significantly at all postoperative evaluations compared to baseline (p<0.001). Pre-operative presence of PVD did not affect outcome measures. No intraoperative complications were reported. Immediate post-operative complications included one case of sterile endophthalmitis and one case of vitreous and perimacular hemorrhage. At one year, complications included progression of cataract in phakic eyes (65.4%), steroid-induced glaucoma (2.2%), retinal tear (0.8%), recurrent ERM (4.5%), and recurrent macular edema (11.3%). No further complications were reported at three- and five-year follow-up. CONCLUSION: Combination ILM forceps and TA visualization offers an affordable and safe option for ERM removal with comparable or better outcomes than traditional methods of ERM surgery.

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To assess the cellular stress evoked by exposure of Brilliant Blue-G (BBG), adult retinal pigment epithelial (ARPE-19) cells were treated with various dilutions of BBG in balanced salt solution plus (BSS-PLUS) with and without endoillumination (Alcon Constellation Vision System). The treatments lasted for acute periods of 2 and 5 min. MTT and presto blue assays were performed to assess the changes in cell viability; reactive oxygen species (ROS) production was quantified by DCFDA (dichlorofluorescin diacetate) assay, and the expression of inflammatory stress and endoplasmic reticulum (ER) genes were quantified by qPCR. We observed no reduction in cell viability at 2 min of dye treatment with and without endoillumination while at 5 min exposure, a reduction in cell viability at all concentrations of the dye was observed compared to control. Though there was an increase in ROS with endoillumination, it was insignificant. There was no change in the mRNA expression of TNF-α while that of GRP78, and inflammatory genes viz. IL-8, IL-1ß showed a significant increase at 0.5 mg/ml dye with endoillumination. BBG reduced cell viability with increasing concentration and time. The undiluted concentration of the dye results in inflammatory stress compared to the diluted formulations. Interestingly, increased GRP78 at undiluted concentration indicates a protective response in cells exposed to light. However, further studies are needed to evaluate the effect of cellular stress on the visual outcome. We infer that the commercially available formulation of BBG is safe for the RPE, at the recommended dose for a short duration however its toxicity to other cell types need to be addressed.

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PURPOSES: To compare surgeons' opinions regarding idiopathic full-thickness macular hole (MH) surgery by using traditional microscopy and three-dimensional (3-D) visualization system. To analyze the required time for pars plana vitrectomy (PPV) and for internal limiting membrane (ILM) rhexis by using both visualization methods. To evaluate anatomical surgical results. METHODS: Four surgeons (surgeon 1, fellows 1, 2, 3) performed the total of 40 surgeries for treating MHs. Each one performed 10 surgeries (5 with traditional microscopy and 5 with 3-D visualization). The completion time for PPV and ILM rhexis was determined by using both methods. Ergonomics, educational value, image sharpness, depth perception, field of view and technical skills were analyzed through answering a questionnaire. RESULTS: Forty patients were included in the study. The MH size for surgeon 1, fellows 1, 2 and 3 groups, individually, ranged from 237 to 602 µm; 228 to 590 µm, 271 to 611 µm and 289 to 600 µm, respectively. In the 3-D and in the traditional microscopy subgroups (which includes all 4 physicians on the use of one or the other method), the MH size ranged from 228 to 602 µm and 237 to 611 µm, respectively. Comparisons between the average time for full PPV and ILM rhexis by using the two methods were non-significant, neither in each individual case of 3-D surgery for each surgeon. Surgeon 1 had always been faster than his fellows. Depth perception was rated as similar for both methods. Field of view and educational values were rated as superior when using the 3-D system. Image resolution and ergonomics were rated as superior when using traditional microscopy. Technical skills strongly tended toward 'superiority' when using traditional microscopy. Thirty-six (90%) full-thickness MHs were successfully closed with one surgery. CONCLUSION: The 3-D system for MH surgery had a short learning curve and was a refined educational tool, when used with reduced illumination and precise focus. Concerning MH surgery, heads-up method was similar to traditional microscopy regarding length of time and anatomical surgical results. Heads-up surgery may become a new pattern for ophthalmic surgery as ongoing improvements are applied.

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The successful detection of retinal breaks is a critical step in rhegmatogenous retinal detachment surgery in order to prevent persistent/recurrent retinal detachments. Not all retinal breaks causing retinal detachments are obvious. Retinal breaks may be obscured by opacities that are either anterior segment related, lens related, or posterior segment related. Rules to identify breaks based on subretinal fluid configuration are more difficult to apply in pseudophakic, aphakic, and scleral buckle encircled eyes-and in eyes with repeat detachments and those with proliferative vitreoretinopathy. Exudative detachments exhibit characteristic features and must be ruled out. A thorough clinical examination preoperatively is important even if a vitrectomy is planned. We review the incidence and causes of undetected breaks, along with preoperative/clinical issues that may hinder break detection. We review the literature with respect to investigative approaches and techniques that are available to the vitreoretinal surgeon when primary breaks remain clinically undetected during the preoperative examination. We broadly divide the surgical approaches into ones where the surgeon utilizes techniques to pursue actively a search for breaks versus adopting a purely speculative approach. Advantages and disadvantages of various techniques are appraised. Intuitively one might argue that an encircling scleral buckle combined with vitrectomy would give higher single operation success than pars plana vitrectomy alone because "undetected" retinal breaks would be addressed by a 360° plombage. We could not confirm this concept. Newer techniques, such as pars plana vitrectomy augmented with dye extrusion or endoscopic-assisted pars plana vitrectomy, show encouraging results. Technological advances such as intraoperative optical coherence tomography will also help to broaden the vitreoretinal surgeon's armamentarium. At this time, there is no gold standard in terms of the recommended approach, and this is reflected in the many options that are available for management. The surgeon must consider the benefits versus the risk of their preferred approach.

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PURPOSE: The vitreous cortex, epiretinal membrane (ERM), and inner limiting membrane (ILM) are transparent tissues and are thus difficult to visualize. Staining these structures can increase the efficiency of a nontraumatic removal. METHODS: The surgeon performs a partial core vitrectomy and induces a posterior vitreous detachment. The vital dye is then injected into the retrohyaloid space in balanced salt solution (BSS). The dyes used are TWIN (Alchimia srl, Padova, Italy), MembraneBlue-Dual (DORC International, Zuidland, the Netherlands), and Doubledyne (Alfa Intes, Casoria, Italy). The surgeon can complete the vitrectomy and gradually aspirate the dye with the probe. Once the vitrectomy is complete, the surgeon can perform the peeling of the ERM without the need to reinject the vital dye over the macula. RESULTS: The presence of the dye over the macula facilitates visualization of the vitreous cortex by blocking the red reflex and increasing the contrast power of the coaxial light probe during the vitrectomy. This allows a negative coloration of the vitreous because the dye acts by increasing the visibility of the surrounding BSS and not the vitreous itself. CONCLUSIONS: We describe a new chromovitrectomy technique using the same dye to increase the visualization of the vitreous, posterior hyaloid, ERM, and ILM.

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PURPOSE: To describe a surgical technique for combined peeling of epiretinal and internal limiting membranes. METHODS: The authors present their procedure of choice for epiretinal membrane surgery: negative staining effect using Brilliant Blue G and single block removal of the epiretinal and internal limiting membranes in a single step. RESULTS: A total of 26 eyes were operated with the described technique. In all cases, the peeling was performed successfully and with no complications. Minimum postoperative follow-up was 12 months. There were no recurrences of epiretinal membranes. CONCLUSIONS: The ideal surgical approach for epiretinal membranes should attempt to reduce mechanical trauma, light exposure, and dye toxicity.

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PURPOSE: Evaluate toxicity of acai fruit (Euterpe oleracea) dye concentrations in a rabbit model. METHODS: Rabbits were injected intravitreously with 10%, 25%, and 35% acai dye concentrations. Control eyes received balanced salt solution (BSS). Electroretinogram (ERG), fundus imaging, fluorescein angiography (FA), optical coherence tomography (OCT), and light and transmission electron microscopy (LM/TEM) were performed. RESULTS: Fundus imaging showed increased vitreous opacity with increased dye concentrations. FA and OCT showed normality with all concentrations. Comparisons between BSS and dye concentrations were analyzed using Kruskal-Wallis and Mood's median test (p < 0.05). At 24 h, ERGs showed reduced amplitudes from baseline in all eyes. Median b-wave amplitudes nonsignificantly decreased and latency increased with 10% and 25%; findings were significant (p < 0.05) for 35%. LM and TEM showed no abnormalities for 10% and 25%. With 35%, TEM showed ganglion cell edema at 24 h that resolved after 7 days. Vacuolization, multilamellar bodies, and nerve bundle damage occurred at 24 h/7 days in the inner nuclear layer. Mitochondrial cristae disruption occurred in the inner photoreceptor segment at 24 h that decreased by 7 days. CONCLUSION: Ten and twenty-five percent concentrations were safe and may improve identification of the posterior hyaloid and internal limiting membrane during chromovitrectomy in humans.

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PURPOSE: This study was performed to investigate the chemical stability of different dyes used in chromovitrectomy and the influence of various product parameters on it. METHODS: Buffered dye solutions were prepared containing 1.5 g/L acid violet 17, 0.25 g/L brilliant blue G, 1.3 g/L bromophenol blue, and 1.5 g/L trypan blue, combined with deuterium oxide, polyethylene glycol 3350, and D-mannitol as additives. For accelerated storage testing, samples were incubated for 400 h at 80°C corresponding to 2 years according to the Van 't Hoff equation. After different incubation times samples were taken for UV/Vis spectroscopy, pH measurement, and osmometry. RESULTS: Depending on dye, additive, and packaging, different solutions exhibit differences in chemical stability and hence shelf life. Packaging in syringes instead of vials increases dye stability. Additives may negatively influence important parameters, e.g. polyethylene glycol 3350 increases osmolality beyond the physiological range. Notably, acid violet 17 is chemically unstable except in D-mannitol-containing buffer, packed in syringes. However, simultaneously, D-mannitol leads to a pH shift below 7.0. CONCLUSION: In summary, dye solutions filled in syringes should be preferred to vials to slow down oxidative degradation. Especially acid violet 17 solutions should be used with caution because the addition of D-mannitol may contribute to pH values beyond the physiological range.

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The term "chromovitrectomy" has been coined to define the use of vital dyes in vitreoretinal surgery. The basic concept for the application of vital dyes during vitreoretinal surgery is to assist in highlighting preretinal membranes and tissues which are very thin and semitransparent and thus difficult to detect. Various dyes are currently being used in routine clinical procedures, however, the ideal staining agent has not yet been found. The vital dyes indocyanine green, infracyanine green, and brilliant blue stain the internal limiting membrane, trypan blue and triamcinolone acetonide help to visualize the epiretinal and vitreous membranes. New dyes with a better safety profile than the synthetic ones are important for optimizing the outcome of modern ophthalmic surgery and natural dyes, such as lutein, offer a potentially safer and more efficient method of identifying intraocular structures such as vitreous and ILM. Any dye, which is intravitreally injected has the potential to become toxic.

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BACKGROUND: The objective of the study was to determine the safety parameters of using brilliant blue green (BBG) for chromovitrectomy by assessing the cytotoxicity of BBG on cultured retinal ganglion cells (RGCs) exposed to illumination. METHODS: RGCs were exposed to two concentrations of BBG (0.25 and 0.5 mg/mL) under metal halide illumination at varying distances (1 and 2.5 cm), intensities (990 and 2,000 Fc), and durations (1, 5 and 15 minutes). Cell viability was assessed using the WST-1 and CellTiter 96(®) AQueous One solution cell proliferation assays. RESULTS: Using the WST-1 assay, with high-intensity illumination, viability of RGCs ranged from 97.5±16.4% of controls with minimum BBG and light exposure (0.25 mg/mL BBG and illuminated for 1 minute at 2.5 cm distance) to 53.1±11.3% of controls with maximum BBG and light exposure (0.50 mg/mL and illuminated for 15 minutes at 1 cm distance; P < 0.01). With medium-intensity illumination, RGCs showed better viability, ranging from 95.1±7.2% of controls with minimum BBG and light exposure to 72.3±12.8% of controls with maximum BBG and light exposure. CellTiter 96(®) AQueous One assay showed similar results. CONCLUSION: RGCs seem to safely tolerate up to 5 minutes of exposure to 0.5 mg/mL BBG under diffuse medium-intensity illumination (990 Fc).

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PURPOSE: To study the safety profile of Lutein/Zeaxanthin(L/Z)-based natural dye solutions in in vitro and in vivo models. MATERIAL AND METHODS: In vitro cytotoxicity and cellular growth experiments were carried out on ARPE-19 and human corneal epithelial (HCE) cell lines using different L/Z-based dye solutions, either alone or in association with brilliant blue (BB) or trypan blue (TB). Light and transmission electron microscopy studies were performed seven days after intravitreal injection of dye solutions in rabbits. Electroretinogram (ERG) recordings were taken at baseline and before histopathology. RESULTS: In vitro cytotoxicity assays demonstrated that the different L/Z-based solutions (from 0.3 to 2%), either alone or in association with BB (0.025%) or TB (0.04%), did not significantly alter mitochondrial activity (≤15%) in the cell lines tested. In addition, in vitro cell growth was inhibited by up to 60% depending on the dye solution, and in direct proportion to the concentration assayed. There was no evidence of structural alterations in the neurosensory retina, retinal pigment epithelium (RPE), or choriocapillaris-choroidal complex. b-Wave ERG records showed no significant differences (±15.2%) in comparison with baseline. CONCLUSIONS: L/Z-based dye solutions demonstrated a safe profile in in vitro and in vivo models, and may be a useful tool for staining intraocular structures.

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PURPOSE: To investigate the retinal toxicity by electroretinography (ERG), clinical examination and histology after intravitreal injection of biological stains in two concentrations: Trisodium (0.50 g/L and 1.00 g/L), Orangell (0.25 g/L and 1.00 g/L) and Methyl Violet (0.50 g/L and 1.00 g/L). METHODS: Eighteen New-Zealand albinos rabbits were assigned in six groups (n = 3 in each group). The animals in group 1 received Trisodium in the dose of 0.50 g/L and group 2 received 1.00 g/L; Group 3 received Orangell in the dose of 0.25 g/L and group 4 received 1.00 g/L; Group 5 received Methyl Violet in the dose of 1.00 g/L and group 6 received 0.50 g/L. A volume of 0.05 mL of dye was injected in the right eyes, whereas the left eyes received the same volume of balanced salt solution (BSS) as control. ERG recordings and clinical examination were performed at baseline and seven days after intravitreal injection. The ERG responses at one week after injection were compared with baseline levels. A decrease in the post-injection amplitude of more than 50% was considered remarkable. After the 7-day follow-up, rabbits were euthanized and eye enucleated for light microscopy (LM) histological evaluation. RESULTS: At clinical examination by indirect ophthalmoscopy seven days after dye injection, all eyes were negative for cataract, hemorrhage, retinal detachment, and intraocular opacities. Amplitude analysis of maximum scotopic b-wave showed no significant reduction in either dye injected or control eyes. Neither dye nor BSS caused significant retinal alteration on LM at doses tested. CONCLUSIONS: Trisodium, Orangell and Methyl Violet can be applied in future studies in order to prove the capacity to stain preretinal tissues and vitreous without toxicity. The three dyes did not induce significant ERG amplitude reduction or LM alterations in this preliminary experimental research. Trisodium, Orangell and Methyl Violet may be potentially useful vital dyes for ocular surgery, and deserve further investigation.

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PURPOSE: To investigate the retinal biocompatibility of Brilliant Blue G with deuterated water (BBG-D2O) as a vital dye for chromovitrectomy. METHODS: In this animal study, 0.05 mL of 0.25 g/L Brilliant Blue G (BBG) associated with 0.13 mL/mL of deuterium oxide (D2O) was injected intravitreally in the right eye and the same amount of balanced salt solution (BSS) was injected similarly in the left eye of rabbits. Clinical examination and histology with light microscopy were performed after seven days. Retinal cell layers were evaluated for morphologic alterations. Electroretinographic (ERG) changes were also assessed at baseline and 7 days after the injections. RESULTS: A total of 6 rabbits were included in the study. The gross histopathologic appearance of the retina, choroid, sclera and optic nerve was within normal limits without any sign of severe retinal necrosis or cystic degeneration. Light microscopy showed that BBG-D2O caused no substantial alterations in retinal layers as compared to control eyes. The injection of BBG-D2O did not induce considerable functional ERG alterations. CONCLUSION: Intravitreal injection of BBG-D2O 0.25 g/L seems to induce no retinal toxicity as documented by lack of functional and histological changes.

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Adequate visualization and identification of the posterior hyaloid, epiretinal membranes and the internal limiting membrane are of paramount importance in modern vitreoretinal surgery. "Chromovitrectomy" is a term used for describing the vital dyes use in order to stain these transparent tissues and facilitate their manipulation during vitreous surgery. This article reviews the indications, applications and characteristics of vital dyes in vitreoretinal surgery. Various dyes are currently being used in routine clinical procedures, however the ideal staining agent has not yet been found. Any dye which is injected intravitreally has the potential to become toxic. Triamcinolone acetonide is used to highlight the vitreous and is particularly beneficial in determining the attachment of the posterior hyaloid to the underlying retina. Trypan blue stains epiretinal membranes and facilitates their complete removal. Both indocyanine green and brilliant blue G stain the internal limiting membrane properly, however concerns over indocyanine green toxicity have made surgeons switch to brillliant blue G as a safer alternative.

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Vital dyes have advanced diagnosis and surgical technique in various specialties, including oncology, gastroenterology and ophthalmology. Intra-operative and diagnostic dyes are finding uses in all areas of ophthalmology, including cornea, cataract, retina, glaucoma, orbit and conjunctiva. We provide a summary of current knowledge of the chemical concepts of vital dyes in ophthalmology. We review the properties of dyes, techniques of application, indications and complications in ocular surgery. Vital dyes represent an expanding area of research, and novel dyes deserve further investigation.

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PURPOSE: To assess tight junction integrity in cultured human foetal retinal pigment epithelium (RPE) after exposure to clinically relevant indocyanine green (ICG) concentrations. METHODS: Human foetal RPE was cultured with the Hu & Bok method. The apical compartments of well-differentiated cultures were exposed to 0.125, 0.05 and 0.025 mg/ml ICG with or without 10-min illumination. Vehicle and trypsin/EDTA or EDTA alone served as controls. Three minutes was chosen to mimic surgical exposure time, while 3 h was used for toxicity assays, with subsequent wash out. Cell-cell junctions were studied before and after exposure by phase contrast microscopy and immunofluorescence (ZO-1). Blood-retinal barrier function was measured through transepithelial electrical resistance (TER). RESULTS: At 6-8 weeks postconfluence, RPE had grown into pigmented hexagonal monolayers with stable TER (435-1227 Ω*cm(2) ). After 3 min ICG exposure, cell morphology remained unchanged, with patchy cell-cell dissociation in positive controls. A continuous ZO-1 signal was detected in ICG groups, whereas trypsin controls showed patchy loss of the tight junction stain. TER had dropped at 1.5 h after 3 min exposure to 22.8 ± 3.1%, compared with 10.2 ± 3.9% in positive controls. Surgical light illumination did not affect TER. After 3 h exposure to 0.05 mg/ml ICG, TER decreased to 58.1 ± 8.3%, while vehicle controls maintained similar levels as prior to exposure (92.7 ± 2.4%). TER recovered in all ICG groups to prior levels within 3 days. CONCLUSION: Indocyanine green (ICG) exposure induced a transient decrease in transepithelial electrical resistance, despite unaltered tight junction structure.

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The basic concept for the application of vital dyes during vitreoretinal surgery is to assist in highlighting preretinal membranes and tissues which are very thin and semitransparent and thus difficult to detect. The vital dyes may be classified according to different criteria, where the most commonly applied includes chemical classification. In ophthalmic surgery, vital dyes are widely used in cataract and vitreoretinal surgery. The vital dyes, indocyanine green, infracyanine green, and brilliant blue stain the internal limiting membrane, and trypan blue and triamcinolone acetonide help to visualize epiretinal membranes and vitreous, respectively. This review exhibits the current literature regarding the properties of vital dyes, techniques of application, indications, and toxicities during vitreoretinal surgery and, also suggests that the field of chromovitrectomy represents an expanding area of research.