Scanning Slit Confocal Microscopy Research Articles

In vivo Slit Scanning Confocal Microscopic Observation in a Patient with Keratoconus Wearing Miniscleral Contact Lens for One Year: A Case Report

The purpose of the present case report was to evaluate corneal microstructure in a patient with keratoconus wearing miniscleral contact lens (MS CL) for 1 year. A 38 years old patient diagnosed with stage III keratoconus in right eye and corneal graft (penetrating keratoplasty) in left eye was referred to optometry department of a public hospital in Malaysia. Based on the MS CLs fitting performance on the patient’s eye, MS CLs were prescribed in both eyes. However, patient opted for MS CL only in the right eye. In vivo slit scanning confocal microscopy was performed at baseline and after 1 year of contact lens wear in right eye. Qualitative analysis was done using a grading scale designed by Hollingsworth et al. (2005a), while quantitative analysis was performed using NAVIS software in a fixed frame of 0.06 mm². After 1 year of MS CL wear, qualitative observation of right cornea showed no change in stromal haze and architecture of nerve fibers. However, morphological alterations like dark bands were observed both in anterior and posterior stroma along with tendency of clustering keratocyte nuclei in posterior stroma. Quantitative analysis showed that anterior and posterior stroma keratocyte density and endothelium cell density were relatively low after 1 year of MS CL wear (817.3, 667.2, and 2577 cells/mm², respectively) caompared to baseline observation (850.7, 705.0, and 2666 cells/mm², respectively). Mean cell area of anterior stroma, posterior stroma and endothelium were also different in post MS CL wear. Polymegathism and pleomorphism calculated by NAVIS software were varied after 1 year (49.6% and 37.5%, respectively) compared to baseline (67.1% and 29.6%, respectively). In the discussed case, noticeable changes were observed in corneal microstructure after 1 year of MS CL wear. Thereby, in conclusion we recommend future studies with more number of keratoconic eyes wearing MS CLs to emphasize the findings on microstructural changes in keratoconic cornea wearing MS CLs.

Effects of contact lens wearing on keratoconus: a confocal microscopy observation.

To evaluate the corneal cell morphology of new keratoconus patients wearing two different types of rigid gas-permeable (RGP) contact lenses for 1y. Thirty nine eyes of 39 new keratoconus patients were selected and randomly fitted with two types of RGP contact lenses. Group 1 had 21 eyes with regular rigid gas-permeable (RRGP) contact lens and rest 18 eyes were in group 2 with specially designed rigid gas-permeable (SRGP) contact lens. Corneal cell morphology was evaluated using a slit scanning confocal microscope at no-lens wear and after 1y of contact lens wearing. After 1y of contact lens wearing in group 1, the mean anterior and posterior stromal keratocyte density were significantly less (P=0.006 and P=0.001, respectively) compared to no-lens wear. The mean cell area of anterior and posterior stromal keratocyte were also significantly different (P=0.005 and P=0.001) from no-lens wear. The anterior and posterior stromal haze increased by 18.74% and 23.81%, respectively after 1y of contact lens wearing. Whereas in group 2, statistically significant changes were observed only in cell density & area of anterior stroma (P=0.001 and P=0.001, respectively) after 1y. While, level of anterior and posterior stromal haze increased by 16.67% and 11.11% after 1y of contact lens wearing. Polymegathism and pleomorphism also increased after 1y of contact lens wearing in both the contact lens groups. Confocal microscopy observation shows the significant alterations in corneal cell morphology of keratoconic corneas wearing contact lenses especially in group 1. The type of contact lens must be carefully selected to minimize changes in corneal cell morphology.

Corneal profile in primary congenital glaucoma.

To investigate biomechanical and ultrastructural corneal parameters and ocular biometrics in the affected eyes of patients with unilateral primary congenital glaucoma (PCG) as compared to unaffected fellow eyes and age-matched healthy controls. A total of 12 patients with treated unilateral PCG and 10 normal subjects were evaluated. LENSTAR was performed to determine biometric parameters; the ocular response analyser was employed to determine biomechanical properties and slit-scanning confocal microscopy was used for evaluation of corneal ultrastucture. Axial length was significantly higher and mean keratometry in affected eyes was significantly flatter in affected eyes as compared to fellow eyes and normal controls (p<0.05), and a negative correlation was present between axial length and mean keratometry (p<0.05). Mean aqueous depth and anterior chamber depth were increased in affected eyes as compared to fellow eyes and normal controls (p<0.05). There was no significant difference in central corneal thickness (CCT) among affected eyes, fellow eyes and normal controls. Corneal hysteresis (CH) was significantly reduced in affected eyes (p<0.05) and corneal resistance factor (CRF) was also reduced in the affected eyes as compared to fellow eyes and normal controls, although not statistically significant. Mean endothelial cell density was reduced in the affected eyes compared to fellow eyes and normal controls (p<0.05). Corneal biometrics, biomechanical parameters and ultrastructural features are altered in eyes affected with PCG despite clinically normal and clear corneas. These findings should be considered in the preoperative assessment of intraocular or corneal surgery in these patients.

Corneal Cell Morphology in Keratoconus: A Confocal Microscopic Observation.

To evaluate corneal cell morphology in patients with keratoconus using an in vivo slit scanning confocal microscope. A cross-sectional study was conducted to evaluate the corneal cell morphology of 47 keratoconus patients and 32 healthy eyes without any ocular disease. New keratoconus patients with different disease severities and without any other ocular co-morbidity were recruited from the ophthalmology department of a public hospital in Malaysia from June 2013 to May 2014. Corneal cell morphology was evaluated using an in vivo slit-scanning confocal microscope. Qualitative and quantitative data were analysed using a grading scale and the Nidek Advanced Visual Information System software, respectively. The corneal cell morphology of patients with keratoconus was significantly different from that of healthy eyes except in endothelial cell density (P = 0.072). In the keratoconus group, increased level of stromal haze, alterations such as the elongation of keratocyte nuclei and clustering of cells at the anterior stroma, and dark bands in the posterior stroma were observed with increased severity of the disease. The mean anterior and posterior stromal keratocyte densities and cell areas among the different stages of keratoconus were significantly different (P < 0.001 and P = 0.044, respectively). However, the changes observed in the endothelium were not significantly different (P > 0.05) among the three stages of keratoconus. Confocal microscopy observation showed significant changes in corneal cell morphology in keratoconic cornea from normal healthy cornea. Analysis also showed significant changes in different severities of keratoconus. Understanding the corneal cell morphology changes in keratoconus may help in the long-term monitoring and management of keratoconus.

Post-photorefractive Keratectomy Pain and Corneal Sub-basal Nerve Density.

Purpose:The perceived and reported pain of patients receiving photorefractive keratectomy (PRK) widely varies. We assessed the potential role of the subbasal nerve plexus density as a predictor of postoperative pain level. Consecutive patients scheduled to undergo PRK at the Refractive Surgery Clinic of Farabi Eye Hospital, Tehran, were approached.Methods:Forty-nine myopic left eyes from 49 patients who consented to undergo scanning slit confocal microscopy assessments preoperatively were included. ImageJ (1.48v) was used to measure the captured subbasal nerve length. Postoperative pain intensity was assessed by the Visual Analog Scale (VAS) (score range: 0 for no pain to 10 for the maximum possible) on the next day of surgery.Results:The mean age of the patients was 27.55 (range: 19–40) years. The median reported pain level was 5. Approximately 32.7% of the subjects reported a pain score of 6 or higher. Mean nerve density was 19.54 (range: 14.34–24.73) mm/mm2. Nerve density was not correlated with the reported intensity of pain (P = 0.172). However, pain was correlated with the reported ocular discomfort, i.e., a pooled index of foreign body sensation, photophobia, burning sensation, and tearing (P < 0.001), and also with the pooled index of ocular inflammatory signs (conjunctival injection and eyelid edema) (P = 0.027).Conclusion:Crude density of corneal nerves may not be a good predictor of post-PRK pain while wearing bandage contact lenses. The predominant pain mechanism appears to be of an inflammatory nature (not nociceptive or neuropathic).

Evaluation of the Corneal Layers in Meibomian-Gland-Dysfunction-Related Dry Eye by In Vivo Slit-Scanning Confocal Microscopy

ABSTRACTIn the current study it was aimed to evaluate the findings of cornea in Dry Eye related Meibomian Gland Dysfunction through in vivo confocal microscopy. 30 patients of Dry Eye related Meibomian Gland Dysfunction (DEMGD) and 30 healty individuals were included. 46 eyes of 30 DEMGD patients (group 1) and 46 eyes of 30 healthy individuals (group 2) were formed as control group and images were captured from the centre of the cornea. 26 of the patients (%86,6) in Group 1 were male, and four of them (%13,4) were female and 25 of healthy individuals (% 83,3) were male and 5 of them(%16,7) were female. The ages of the patients in Group 1 were between 23-67 (51,58±13,4 on average). The ages of the healthy individuals in Group 2 were between 23-67 (51,45±10,4 on average). Tear film break-up time and Schirmer 1 values were significantly lower in the MGDDE group than the control group (p<0.001). There were statistically significant intergroup differences in basal epithelial cell density and area and stromal nerve thickness (p<0.05). Surface epithelium changes, anterior stromal hyperreflectivity and subepithelial nerve morphology changes were not observed in the control group. As a result, some of the effects on cornea tissue caused by Dry Eye related Meibomian Gland Dysfunction were able to be visualized with confocal microscopy at micro level.

Evaluation of Subbasal Nerve Morphology and Corneal Sensation after Accelerated Corneal Collagen Cross-linking Treatment on Keratoconus

Purpose: The aim of this study was to report on the evaluation of corneal nerve fiber density and corneal sensation after accelerated corneal collagen cross-linking on keratoconus patients.Methods: The study was performed on 30 keratoconus eyes (30 participants: 16 M, 14 F; 17–32 years old) treated with accelerated collagen cross-linking for disease stabilization. Mean outcome measures were corneal sensation evaluation by Cochet–Bonnet esthesiometry and subbasal nerve fiber density assessment by corneal in vivo confocal microscopy. All corneal measurements were performed using scanning slit confocal microscopy (ConfoScan 4, Nidek Technologies, Padova, Italy).Results: The accelerated corneal collagen cross-linking procedure was performed on 30 eyes of 30 patients (19 right, 63.3%; 11 left, 27.7%). The mean age was 23.93 ± 4. The preoperative mean keratometry, apex keratometry and pachymetry values were 47.19 ± 2.82 D, 56.79 ± 5.39 and 426.1 ± 25.6 μm, respectively. Preoperative mean corneal sensation was 56.3 ± 5.4 mm (with a range from 40 to 60 mm), it was significantly decreased at 1st and 3rd month visit and increased to preoperative values after 6th month visit. Preoperative mean of subbasal nerve fiber density measurements was 22.8 ± 9.7 nerve fiber/mm2 (with a range of 5–45 mm), it was not still at the preoperative values at 6th month (p = 0.0001), however reached to the preoperative values at 12th month (p = 0.914).Conclusions: Subbasal nerve fibers could reach the preoperative values at the 12th month after accelerated corneal collagen cross-linking treatment although the corneal sensation was improved at 6th month. These findings imply that the subjective healing process is faster than the objective evaluation of the keratoconus patients’ cornea treated with accelerated corneal collagen cross-linking.

Corneal Changes in Neurosurgically Induced Neurotrophic Keratitis

Neurotrophic keratitis (NK) represents a sight-threatening complication after trigeminal impairment. To our knowledge, the duration for which trigeminal injury may affect corneal structures and function has not been investigated previously. To describe the long-term clinical, morphological, and functional outcomes of NK after neurosurgical trigeminal damage. Observational case series performed at a corneal and ocular surface diseases referral center in 2010. Eight consecutive patients with monolateral NK from 1 to 19 years after neurosurgery and 20 age- and sex-matched healthy participants were included. Complete eye examination, tear film function tests, corneal staining, and Cochet-Bonnet esthesiometry were performed. The number and density of corneal nerves, number of hyperreflective keratocytes, and corneal epithelial, endothelial, and keratocyte cell densities were evaluated by in vivo slit scanning confocal microscopy. Clinical and morphological data were compared with the contralateral unaffected eyes and with the eyes of healthy control participants. All patients showed superficial punctate keratitis and dry eye in the NK eye and a healthy contralateral eye. Decreased corneal sensitivity was observed in all affected eyes (mean [SD], 2.0 [1.9] mm in the affected eyes vs 5.8 [0.3] mm in the contralateral unaffected eyes; P = .01) and was related to decreased subbasal nerve length (P = .04; R = 0.895). Corneal epithelial and endothelial cell densities were significantly decreased and the number of hyperreflective keratocytes was significantly increased in NK eyes compared with contralateral unaffected eyes and with the eyes of healthy participants. A longer duration of NK was associated with lower endothelial cell density (P = .046; R = -0.715). Corneal morphology and function were impaired even years after neurosurgical trigeminal damage, suggesting that assessment of tear film and corneal sensitivity as well as in vivo confocal microscopy examination should be performed in all patients with trigeminal impairment.

The corneal endothelium after keratoplasty for keratoconus

BackgroundThe aim was to assess the corneal endothelium, post‐operative visual outcome and complications following keratoplasty for keratoconus.MethodsSeventy‐six corneal grafts (57 per cent penetrating keratoplasties and 43 per cent deep anterior lamellar keratoplasties) were assessed as part of routine follow‐up at the Ophthalmology Department at Gartnavel General Hospital in Glasgow. Routine ophthalmic assessment included visual acuity, slitlamp biomicroscopy and scanning slit confocal microscopy of the central cornea. Case records were reviewed and demographic, surgical and post‐operative data retrieved. Linear and logistic regression models were used to determine associations between endothelial cell density, visual acuity and post‐operative complications. Kaplan–Meier analysis was used to estimate the probability for grafts to retain more than 700 endothelial cells per mm2 and to have 0.3 logMAR or better vision. Odds ratios were calculated to assess the risk for low cell counts and poor visual outcome.ResultsThe mean and standard deviation of the endothelial cell count was predictably higher in lamellar (2033 ± 643 cells per mm2) than penetrating keratoplasties (898 ± 380 cells per mm2) and the mean post‐operative cell count for grafts without post‐operative complications was higher (1585 ± 775) than those with complications (1312 ± 747 cells per mm2). Mean visual acuity was 0.25 ± 0.20 and 0.29 ± 0.41 for lamellar and penetrating keratoplasties, respectively. A higher number of post‐operative events was associated with an increased risk for graft rejection (odds ratio 2.40, p = 0.008, multiple logistic regression) and with poorer visual outcome (odds ratio 1.38, p = 0.044). After keratoplasty, the visual outcome and endothelial cell density were not associated, either in penetrating or deep anterior lamellar grafts (p > 0.05).ConclusionsIn keratoplasties performed for keratoconus, visual outcome cannot be predicted reliably by post‐operative endothelial cell density but post‐operative events can lead to poorer visual outcomes in both types of grafts.

Small fibre neuropathy in Fabry disease

Fabry disease is an X-linked inherited lysosomal storage disorder associated with glycosphingolipid accumulation in organs and vascular endothelium [1]. Neuropathic pain is common in Fabry disease and is associated with small fibre neuropathy [2, 3]. The diagnosis of small fibre neuropathy is based on symptoms like paresthesias, dysesthesias and pain and clinical signs with abnormal pin prick sensation, quantitative sensory testing (QST) and skin biopsy [4]. Corneal confocal microscopy examination of patients with diabetes mellitus as well as Fabry disease has demonstrated a reduction in sub-basal nerve density, also reflecting small fibre neuropathy [5–7]. In the present study we have compared these tests in patients with Fabry disease. Fifteen patients (five males with mean age of 16.2 years and 10 females with mean age of 33.8 years) with Fabry disease, all verified by mutation analysis, were recruited from the Bergen Fabry cohort at Haukeland University Hospital, Bergen, Norway (2004–2009). Blood levels of alpha-galactosidase varied from 2.2 to 37.8 lkat/kg protein (N: 17.7–26.4). All patients had episodes of neuropathic pain aggravated by heat. Eleven sexand age-matched healthy individuals served as controls for the assessment of small fibre affection. The disease severity scoring system (DS3) was used to measure the general neurological, cardiovascular and renal symptoms and signs in the Fabry patients [8]. Age was positively correlated with the DS3 total score (r = 0.68, p = 0.005; Pearson’s test), while it was negatively associated with the DS3 peripheral nervous system (PNS) score (r = -0.52, p = 0.045; Pearson’s test). Males had higher DS3 PNS score than females (p = 0.043; t test), but there was no sex association with DS3 total score (p = 0.73; t test). The alpha-galactosidase levels were not associated with the DS3 PNS score (r = 0.40, p = 0.135; Pearson’s test). Routine clinical and neurological examination, nerve conduction studies, electromyography, and QST (vibrametry and thermotests) were performed. A slit scanning confocal microscope (Confoscan 3, Nidek Technologies, Vigonza, Italy) was used to measure the sub-basal cornea nerve fibre length (CNFL) (mm/mm) in the central cornea of one eye of each subject. Skin-punch biopsy from the A. K. Bertelsen J. Aarseth C. A. Vedeler (&) Department of Neurology, Haukeland University Hospital, Bergen, Norway e-mail: christian.vedeler@helse-bergen.no

Standardization of Corneal Haze Measurement in Confocal Microscopy

Corneal stromal haze cannot be compared in longitudinal studies or across laboratories without standardization. In this study, a method was devised of standardizing image brightness in confocal microscopy of the cornea. Thirty-six normal corneas of 18 untreated volunteers and 35 corneas of 18 patients 3 years after LASIK were examined by slit scanning confocal microscopy. The mean image intensity of each frame was adjusted for daily variations in sensitivity of the microscope by using scans through a solution of Amco Clear (GSF Chemicals, Columbus, OH). Adjusted image intensities were expressed in scatter units (SU), representing the concentration of Amco Clear that produced the same intensity as the image. The intensity from the stroma in corneas after LASIK was compared to that in untreated corneas by using generalized estimating equation models. In the untreated corneas, image brightness was 1079 ± 242 and 758 ± 142 SU in the anterior and mid stroma, respectively. Three years after LASIK, image intensity in the flap was 740 ± 186 SU, approximately 30% lower than in corresponding stroma of the untreated corneas (P < 0.001). At mid stroma, brightness was 715 ± 117 SU after LASIK, and was not significantly different from brightness in untreated corneas (P = 0.26). Clinical confocal microscopy provides a high-resolution measurement of corneal haze, and Amco Clear provides a means of standardizing these measurements. This method can detect subtle decreases in haze in the corneal flap 3 years after LASIK and could be used to examine changes in haze after lamellar keratoplasty.

Real-Time Imaging and Quantification of Amyloid-β Peptide Aggregates by Novel Quantum-Dot Nanoprobes

BackgroundProtein aggregation plays a major role in the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease. However, direct real-time imaging of protein aggregation, including oligomerization and fibrillization, has never been achieved. Here we demonstrate the preparation of fluorescent semiconductor nanocrystal (quantum dot; QD)-labeled amyloid-β peptide (QDAβ) and its advanced applications.Methodology/Principal FindingsThe QDAβ construct retained Aβ oligomer-forming ability, and the sizes of these oligomers could be estimated from the relative fluorescence intensities of the imaged spots. Both QDAβ coaggregation with intact Aβ42 and insertion into fibrils were detected by fluorescence microscopy. The coaggregation process was observed by real-time 3D imaging using slit-scanning confocal microscopy, which showed a typical sigmoid curve with 1.5 h in the lag-time and 12 h until saturation. Inhibition of coaggregation using an anti-Aβ antibody can be observed as 3D images on a microscopic scale. Microglia ingested monomeric QDAβ more significantly than oligomeric QDAβ, and the ingested QDAβ was mainly accumulated in the lysosome.Conclusions/SignificanceThese data demonstrate that QDAβ is a novel nanoprobe for studying Aβ oligomerization and fibrillization in multiple modalities and may be applicable for high-throughput drug screening systems.

OCT and In Vivo Confocal Microscopy of a Pigmented Corneal Tumor-like Lesion

A 43-year-old woman presented with a pigmented flat tumor situated at the posterior surface of the cornea nasally in her left eye. Anterior-segment optical coherence tomography revealed that the lesion was similar to the iris leaf, was limited to the cornea, and did not communicate with the iridocorneal angle. In vivo scanning slit confocal microscopy imaged dense hyperreflective tissue behind the endothelium and bright spots dispersed on the adjacent endothelial surface. Multiple hyporeflective formations resembling cell nuclei were visualized within the hyperreflective mass and the cell borders were distinguished. The diagnosis of pigmented nevus or retrocorneal membrane was suspected. The authors conclude that anterior-segment optical coherence tomography and in vivo scanning slit confocal microscopy are useful in assessing the microstructure and penetration of pigmented corneal lesions.

Real-time slit scanning microscopy in the meridional plane

The standard microscope architecture around which confocal microscopes are built imposes fundamental restrictions on the speed with which images (three-dimensional data sets) can be obtained. Commercially available slit scanning confocal microscopes are able to produce optically sectioned images at frame rates well in excess of 100 Hz. However only the focal (x-y) plane can be imaged at this speed. To image a volume specimen it is necessary to physically change the distance between the objective lens and the specimen. This refocusing process is often necessarily slow and represents a bottleneck to the speed of image acquisition. We describe the construction of a slit scanning confocal microscope based on what we know to be a novel microscope architecture, which permits images of other planes and, particular, the meridional (x-z) plane to be acquired in real time.

Distribution and morphological changes of subbasal nerve fiber after different excimer laser operation

目的 观察准分子激光原位角膜磨镶术(LASIK)和准分子激光上皮下角膜磨镶术(LASEK)术后角膜上皮基底膜下神经分布及形态学特征.方法 共焦显微镜观察低、中度近视LASIK及LASEK 27例(54眼),术前、术后1、3、6月的角膜上皮基底膜下神经纤维分布和形态学特征,并测定泪膜破裂时间(BUT)和泪液分泌量(Schirmer I值).结果 LASIK(12例,24眼)与LASEK(15例,30眼)术后角膜上皮基底膜下神经纤维的再生和形态学在各时间点差异均无统计学意义.两组中BUT有统计学意义(P 0.05).结论 对于低、中度近视而言,LASIK与LASEK术后角膜上皮基底膜下神经纤维的再生未显示形态学上的差异。

Improved sectioning in a slit scanning confocal microscope

We describe a simple implementation of a slit scanning confocal microscope to obtain an axial resolution better than that of a point-scanning confocal microscope. Under slit illumination, images of a fluorescent object are captured using an array detector instead of a line detector so that out-of-focus light is recorded and then subtracted from the adjacent images. Axial resolution after background subtraction is 2.2 times better than the slit confocal resolution, and out-of-focus image suppression is calculated to attenuate with defocus faster by 1 order of magnitude than in the point confocal case.

Alterations in corneal sensitivity and nerve morphology in patients with primary Sjögren's syndrome

The aim of the study was to assess subjective symptoms and objective clinical signs of dry eye in relation to corneal nerve morphology and sensitivity in primary Sjögren's syndrome. Twenty eyes of 20 primary Sjögren's syndrome patients and ten eyes of 10 healthy age- and sex-matched controls were included in the study. Ocular surface disease index (OSDI) questionnaire and visual analog scales were used to assess subjective symptoms. The mechanical sensitivity of the central cornea was measured using a modified Belmonte non-contact esthesiometer followed by an analysis of corneal nerve morphology using scanning slit confocal microscopy (ConfoScan 3). OSDI symptom scores were high in primary Sjögren's syndrome patients, compared with controls. Accordingly, the mean corneal detection threshold was low in patients implicating corneal mechanical hypersensitivity (54.5 ± 40.1 ml/min vs. 85.0 ± 24.6 ml/min, P = 0.036). However, nerve densities were similar, and no correlation was present between corneal sensitivity and nerve density. In contrast, alterations in nerve morphology were found; stromal nerves appeared thicker, and nerve growth cone-like structures were seen in 20% of patients, often associated with dendritic antigen-presenting cells. Sjögren's syndrome patients presented with corneal mechanical hypersensitivity, although corneal nerve density did not differ from controls. However, alterations in corneal nerve morphology (nerve sprouting and thickened stromal nerves) and an increased amount of antigen-presenting cells, implicating the role of inflammation, were observed. These observations offer an explanation for the corneal mechanical hypersensitivity, or even hyperalgesia often observed in these patients. We hypothesize that patients with primary Sjögren's syndrome dry eye suffer from neuropathic corneal mechanical hypersensitivity induced by ocular surface inflammation.

Multispectral confocal microendoscope for in vivo and in situ imaging

We describe the design and operation of a multispectral confocal microendoscope. This fiber-based fluorescence imaging system consists of a slit-scan confocal microscope coupled to an imaging catheter that is designed to be minimally invasive and allow for cellular level imaging in vivo. The system can operate in two imaging modes. The grayscale mode of operation provides high resolution real-time in vivo images showing the intensity of fluorescent signal from the specimen. The multispectral mode of operation uses a prism as a dispersive element to collect a full multispectral image of the fluorescence emission. The instrument can switch back and forth nearly instantaneously between the two imaging modes (less than half a second). In the current configuration, the multispectral confocal microendoscope achieves 3-microm lateral resolution and 30-microm axial resolution. The system records light from 500 to 750 nm, and the minimum resolvable wavelength difference varies from 2.9 to 8.3 nm over this spectral range. Grayscale and multispectral imaging results from ex-vivo human tissues and small animal tissues are presented.

In Vivo Confocal Microscopy of Corneal Grafts Shortly after Penetrating Keratoplasty

To describe the microstructural status of corneal grafts shortly after penetrating keratoplasty (PK) and to evaluate the efficacy and safety of confocal microscopy in examining corneal grafts at that time. A confocal microscope with a 40 x front lens was used to examine corneal grafts in 32 patients (32 eyes) 4 days after PK. Images were analyzed, and endothelial cell density counts were compared with presurgical, eye bank values determined by specular microscopy. Microstructural alterations of the graft included epithelial and stromal edema, epithelial degeneration in both superficial and basal cell layers, dark stromal striae, activated keratocytes, and needle-like structures in the stroma. Descemet membrane folds were visible in 31 of 32 grafts; in 1 graft, the dense stromal edema did not allow imaging of posterior layers. Stromal nerve fibers were imaged in 28 grafts (88%). Endothelial cell density ranged from 1666 to 2548 cells/mm2 (mean+/-SD, 2125+/-283 cells/mm2); perioperative endothelial cell density loss varied from 0% to 29% (mean, 12%). No adverse reactions or signs of worsening of clinical condition were observed after the examination. White light scanning slit confocal microscopy permits imaging of a graft's microstructure (including epithelium and stromal layers), as well as calculation of endothelium cell density, as soon as 4 days after PK. The most frequently observed morphologic alterations of corneal grafts shortly after PK include epithelial and stromal edema, epithelial degeneration, stromal striae, and Descemet membrane folds. Stromal nerves can still be seen in the graft 4 days after PK.

Virtual slit scanning microscopy

We present a novel slit scanning confocal microscope with a CCD camera image sensor and a virtual slit aperture for descanning that can be adjusted during post-processing. A very efficient data structure and mathematical criteria for aligning the virtual aperture guarantee the ease of use. We further introduce a method to reduce the anisotropic lateral resolution of slit scanning microscopes. System performance is evaluated against a spinning disk confocal microscope on identical specimens. The virtual slit scanning microscope works as the spinning disk type and outperforms on thick specimens.