Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy

The mucosa of the gastrointestinal tract is a dynamic tissue composed of numerous cell types with complex cellular functions. Study of the vital intestinal mucosa has been hampered by lack of suitable model systems. We here present a novel animal model that enables highly resolved three-dimensional imaging of the vital murine intestine in anaesthetized mice. Using intravital autofluorescence 2-photon (A2P) microscopy we studied the choreographed interactions of enterocytes, goblet cells, enteroendocrine cells and brush cells with other cellular constituents of the small intestinal mucosa over several hours at a subcellular resolution and in three dimensions. Vigorously moving lymphoid cells and their interaction with constituent parts of the lamina propria were examined and quantitatively analyzed. Nuclear and lectin staining permitted simultaneous characterization of autofluorescence and admitted dyes and yielded additional spectral information that is crucial to the interpretation of the complex intestinal mucosa. This novel intravital approach provides detailed insights into the physiology of the small intestine and especially opens a new window for investigating cellular dynamics under nearly physiological conditions

Noncontact holographic detection for photoacoustic tomography

A holographic method for high-speed, noncontact photoacoustic tomography is introduced and evaluated. Relative changes of the object's topography, induced by the impact of thermoelastic pressure waves, were determined at nanometer sensitivity without physical contact. The object's surface was illuminated with nanosecond laser pulses and imaged with a high-speed CMOS camera. From two interferograms measured before and after excitation of the acoustic wave, surface displacement was calculated and then used as the basis for a tomographic reconstruction of the initial pressure caused by optical absorption. The holographic detection scheme enables variable sampling rates of the photoacoustic signal of up to 50 MHz. The total acquisition times for complete volumes with 230 MVoxel is far below 1 s. Measurements of silicone and porcine skin tissue phantoms with embedded artificial absorbers, which served as a model for human subcutaneous vascular networks, were possible. Three-dimensional reconstructions of the absorbing structures show details with a diameter of 310 aem up to a depth of 2.5 mm. Theoretical limitations and the experimental sensitivity, as well as the potential for in vivo imaging depending on the detection repetition rate, are analyzed and discussed. (C) 2017 Society of Photo-Optical Instrumentation Engineers (SPIE)

Non-Invasive Multi-Dimensional Two-Photon Microscopy enables optical fingerprinting (TPOF) of immune cells

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non-invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two-photon microscopy to non-invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence-based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients

Advantages of microscope-integrated intraoperative online optical coherence tomography: usage in Boston keratoprosthesis type I surgery (vol 21, 016005, 2016)

Boston keratoprosthesis (KPro) type I is a technique to treat patients with corneal diseases that are not amenable to conventional keratoplasty. Correct assembly and central implantation of the prosthesis are crucial for postoperative visual recovery. This study investigates the potential benefit of intraoperative optical coherence tomography (OCT) to monitor KPro surgery. Retrospective case series are presented for two patients who underwent Boston KPro type I implantation. The surgery in both patients was monitored intraoperatively using a commercially available intraoperative OCT (iOCT) device mounted on a surgical microscope. Microscope-integrated intraoperative OCT was able to evaluate the correct assembly and implantation of the KPro. All parts of the prosthesis were visible, and interfaces between the corneal graft and titanium backplate or anterior optics were clearly depictable. Moreover, iOCT visualized a gap between the backplate and graft in one case, and in the other case, a gap between the anterior optic and graft. Neither gap was visible with a conventional surgical microscope. The gap between the anterior optic and the graft could easily be corrected. Microscope-integrated iOCT delivers enhanced information, adding to the normal surgical microscope view during KPro surgery. Correct assembly can be controlled as well as the correct placement of the Boston KPro into the anterior chamber. (C) 2016 Society of Photo-Optical Instrumentation Engineers (SPIE

Advantages of microscope-integrated intraoperative online optical coherence tomography: usage in Boston keratoprosthesis type I surgery

Boston keratoprosthesis (KPro) type I is a technique to treat patients with corneal diseases that are not amenable to conventional keratoplasty. Correct assembly and central implantation of the prosthesis are crucial for postoperative visual recovery. This study investigates the potential benefit of intraoperative optical coherence tomography (OCT) to monitor KPro surgery. Retrospective case series are presented for two patients who underwent Boston KPro type I implantation. The surgery in both patients was monitored intraoperatively using a commercially available intraoperative OCT (iOCT) device mounted on a surgical microscope. Microscope-integrated intraoperative OCT was able to evaluate the correct assembly and implantation of the KPro. All parts of the prosthesis were visible, and interfaces between the corneal graft and titanium backplate or anterior optics were clearly depictable. Moreover, iOCT visualized a gap between the backplate and graft in one case, and in the other case, a gap between the anterior optic and graft. Neither gap was visible with a conventional surgical microscope. The gap between the anterior optic and the graft could easily be corrected. Microscope-integrated iOCT delivers enhanced information, adding to the normal surgical microscope view during KPro surgery. Correct assembly can be controlled as well as the correct placement of the Boston KPro into the anterior chamber. (C) 2016 Society of Photo-Optical Instrumentation Engineers (SPIE

Future Prospects: Assessment of Intraoperative Optical Coherence Tomography in Ab Interno Glaucoma Surgery

Purpose: Ab interno glaucoma surgery, such as trabecular aspiration or ab interno trabeculotomy, has been introduced as an alternative, non-filtering procedure to lower intraocular pressure in glaucoma patients. The purpose of the present study was to assess the feasibility of intraoperative spectral domain optical coherence tomography (iOCT) for improving ab interno glaucoma surgery. Materials and Methods: Intraoperative optical coherence tomography was performed to visualize the anterior chamber angle, the aspiration canula during trabecular aspiration, and the trabectome device during ab interno trabeculotomy. A commercially available 840-nm iOCT was used in a clinical setting, a 1310-nm spectral-domain OCT in an experimental setting. Results: In the clinical setting using the 840-nm iOCT, visualization of the aspiration canula and the trabectome device was possible, however, with rather limited visibility of the chamber angle and the trabecular meshwork. In the experimental setting, the 1310-nm OCTenabled excellent visualization of the chamber angle and detailed evaluation of the instrument-trabecular meshwork relation. Conclusions: Intraoperative use of optical coherence tomography may be helpful for ab interno glaucoma surgery. Adjustments of the commercially available iOCT, including longer wavelengths and oblique scanning options, are necessary for the clinical use

Development, Alteration and Real Time Dynamics of Conjunctiva-Associated Lymphoid Tissue

Purpose: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model. Methods: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy. Results: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 mu m/min. Conclusions: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration

Optimising deep anterior lamellar keratoplasty (DALK) using intraoperative online optical coherence tomography (iOCT)

Background/aims To describe the use of intraoperative online optical coherence tomography (iOCT) for improving deep anterior lamellar keratoplasty (DALK) surgery. Methods Retrospective case series of 6 eyes of 6 male patients with keratokonus, corneal dystrophy or herpetic stromal scars undergoing DALK were investigated using intraoperative optical coherence tomography and postsurgical image/video analysis. Main outcome measures were: visibility of surgical steps, especially, assessment of placement depth of injection needle, preparation of bare Descemet's membrane and drainage of interface fluid. Results iOCT enables real-time visualisation of all surgical steps of DALK procedure in all patients. Placement of air injection needle above Descemet's membrane was reliably monitored as was presence of bare Descemet's membrane and potential interface fluid. Conclusions iOCT assists with visualisation of injection needle placement and with assessment of bare Descemet's membrane as well as interface fluid during the DALK procedure. Overall iOCT may be a helpful device that supports surgeons in all steps of DALK procedure