A perspective on robotic assistance for knee arthroplasty.

Knee arthroplasty is used to treat patients with degenerative joint disease of the knee to reduce pain and restore the function of the joint. Although patient outcomes are generally quite good, there are still a number of patients that are dissatisfied with their procedures. Aside from implant design which has largely become standard, surgical technique is one of the main factors that determine clinical results. Therefore, a lot of effort has gone into improving surgical technique including the use of computer-aided surgery. The latest generation of orthopedic surgical tools involves the use of robotics to enhance the surgeons' abilities to install implants more precisely and consistently. This review presents an evolution of robot-assisted surgical systems for knee replacement with an emphasis on the clinical results available in the literature. Ever since various robotic-assistance systems were developed and used clinically worldwide, studies have demonstrated that these systems are as safe as and more accurate than conventional methods of manual implantation. Robotic surgical assistance will likely result in improved surgical technique and improved clinical results.

Biosensors for characterizing the dynamics of rho family GTPases in living cells.

The biosensors developed in the authors' laboratory have been based on different designs, each imparting specific strengths and weaknesses. Here we describe detailed protocols for the application of three biosensors exemplifying different designs-first, a design in which an environmentally sensitive dye is used to report the activation of endogenous Cdc42, followed by two biosensors based on FRET, one using intramolecular and the other intermolecular FRET. The design differences lead to the need for different approaches in imaging and image analysis.

Three-dimensional sonography with needle tracking: role in diagnosis and treatment of prostate cancer.

OBJECTIVE: Image-guided prostate biopsy has become routine in medical diagnosis. Although it improves biopsy outcome, it mostly operates in 2 dimensions, therefore lacking presentation of information in the complete 3-dimensional (3D) space. Because prostatic carcinomas are nonuniformly distributed within the prostate gland, it is crucial to accurately guide the needles toward clinically important locations within the 3D volume for both diagnosis and treatment. METHODS: We reviewed the uses of 3D image-guided needle procedures in prostate cancer diagnosis and cancer therapy as well as their advantages, work flow, and future directions. RESULTS: Guided procedures for the prostate rely on accurate 3D target identification and needle navigation. This 3D approach has potential for better disease diagnosis and therapy. Additionally, when fusing together different imaging modalities and cancer probability maps obtained from a population of interest, physicians can potentially place biopsy needles and other interventional devices more accurately and efficiently by better targeting regions that are likely to host cancerous tissue. CONCLUSIONS: With the information from anatomic, metabolic, functional, biochemical, and biomechanical statuses of different regions of the entire gland, prostate cancers will be better diagnosed and treated with improved work flow.

Digital differential interference contrast autofocus for high-resolution oil-immersion microscopy.

Continued advances in cellular fluorescent biosensors enable studying intracellular protein dynamics in individual, living cells. Autofocus is valuable in such studies to compensate for temperature drift, uneven substrate over multiple fields of view, and cell growth during long-term high-resolution time-lapse studies of hours to days. Observing cellular dynamics with the highest possible resolution and sensitivity motivates the use of high numerical aperture (NA) oil-immersion objectives, and control of fluorescence exposure to minimize phototoxicity. To limit phototoxicity, to maximize light throughput of the objective for biosensor studies, and because phase contrast is distorted by the meniscus in microtiter plates, we studied autofocus in differential interference contrast (DIC) microscopy with a 60x 1.45 NA oil objective after removing the analyzer from the fluorescent light path. Based on a study of the experimental DIC modulation transfer function, we designed a new bandpass digital filter for measuring image sharpness. Repeated tests of DIC autofocus with this digital filter on 225 fields-of-view resulted in a precision of 8.6 nm (standard deviation). Autofocus trials on specimens with thicknesses from 9.47 to 33.20 mum, controlled by cell plating density, showed that autofocus precision was independent of specimen thickness. The results demonstrated that the selected spatial frequencies enabled very high-precision autofocus for high NA DIC automated microscopy, thereby potentially removing the problems of meniscus distortion in phase contrast imaging of microtiter plates and rendering the toxicity of additional fluorescence exposure unnecessary.

Rapid motion compensation for prostate biopsy using GPU.

Image-guided procedures have become routine in medicine. Due to the nature of three-dimensional (3-D) structure of the target organs, two-dimensional (2-D) image acquisition is gradually being replaced by 3-D imaging. Specifically in the diagnosis of prostate cancer, biopsy can be performed using 3-D transrectal ultrasound (TRUS) image guidance. Because prostatic cancers are multifocal, it is crucial to accurately guide biopsy needles towards planned targets. Further the gland tends to move due to external physical disturbances, discomfort introduced by the procedure or intrinsic peristalsis. As a result the exact position of the gland must be rapidly updated so as to correspond with the originally acquired 3-D TRUS volume prior to biopsy planning. A graphics processing unit (GPU) is used in this study to compute rapid updates performing 3-D motion compensation via registration of the live 2-D image and the acquired 3-D TRUS volume. The parallel computational framework on the GPU is exploited resulting in mean compute times of 0.46 seconds for updating the position of a live 2-D buffer image containing 91,000 pixels. A 2x sub-sampling resulted in a further improvement to 0.19 seconds. With the increase in GPU multiprocessors and sub-sampling, we observe that real time motion compensation can be achieved.

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins.

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique that offers precise spatiotemporal control of protein inactivation, enabling better understanding of the protein's role in cell function. EGFP has been used effectively as a CALI chromophore, and its cotranslational attachment to the target protein avoids having to use exogenously added labeling reagents. A potential drawback to EGFP-CALI is that the CALI phenotype can be obscured by the endogenous, unlabeled protein that is not susceptible to light inactivation. Performing EGFP-CALI experiments in deficient cells rescued with functional EGFP-fusion proteins permits more complete loss of function to be achieved. Here, we present a modified lentiviral system for rapid and efficient generation of knockdown cell lines complemented with physiological levels of EGFP-fusion proteins. We demonstrate that CALI of EGFP-CapZbeta increases uncapped actin filaments, resulting in enhanced filament growth and the formation of numerous protrusive structures. We show that these effects are completely dependent upon knocking down the endogenous protein. We also demonstrate that CALI of EGFP-Mena in Mena/VASP-deficient cells stabilizes lamellipodial protrusions.

Digital autofocus methods for automated microscopy.

Automatic focusing of microscope images is an essential part of modern high-throughput microscopy. This chapter describes implementation of a robust autofocus system appropriate for using either air or oil immersion objectives in robotic imaging. Both hardware and software algorithms are described, and caveats of using viscous immersion media with multifield scanning are detailed.

Toward complete laser ablation of melanoma contaminant cells in a co-culture outgrowth model via image cytometry.

BACKGROUND: Contaminant cancer cells in autologous transplant tissue can cause relapse and the rates are unknown. A method capable of removing all contaminant cells with a high probability detected by cytomic analyses would be useful. Neither 100% cell purging nor techniques for measuring the probability of success have been developed. Here, we report a method for removing 100% of the cells under ideal staining conditions and quantify the probability of success. METHODS: Laser ablation was combined with previously reported automated microscopy to purge contaminant cells and evaluate 100% ablation in a co-culture model of prestained mouse melanoma cells mixed with mouse NIH-3T3 cells. Melanoma passage efficiency was measured by: (1) micropipetting single cells into microtiter wells and (2) ablating all but one melanoma cell in co-cultures. RESULTS: (74 +/- 5)% of single melanoma cells pipetted into microtiter plate wells divided at least once. With ablation of all but one contaminant cell in co-cultures, melanoma dominated in (62 +/- 8)% cultures in 21 days. With 100% ablation in six additional experiments, no melanoma outgrowth was observed, giving a >99.1% probability that all contaminant melanoma cells were purged. CONCLUSIONS: We successfully demonstrated a model for complete ablation within a defined probability using automated high-content image cytometry with ideal staining conditions. The results show that the instrumentation is capable of delivering 100% ablation at a defined probability and establishes the basis for further studies with clinical models wherein pretherapeutic cytomic analyses of unique cellular expression and/or morphological characteristics will be key for contaminant cancer cell identification.

Functional proteometrics for cell migration.

BACKGROUND: Advances in living cellular fluorescence biosensors and computerized microscopy enable a vision of fully automated high-resolution measurements of the detailed intracellular molecular dynamics directly linked to cellular behaviors. Given the heterogeneity of cell populations, a statistically relevant study of molecular-cellular dynamics is a key motivation for improved automation. METHODS: We explored automating computerized, microscope-based data extraction and analyses that monitor cell locomotion, rates of mitoses, and spatiotemporal activities of intracellular proteins via ratiometric fluorescent biosensors in mouse fibroblasts. Novel image processing methods included K-means clustering segmentation preprocessing followed by modified discrete, normalized cross-correlational alignment of two-color images; ratiometric processing for fluorescence resonance energy transfer (FRET) measurements; and intracellular spatial distribution measurements of RhoA GTPase activity. RESULTS: The interdivision time was 19.4 h (mean) +/- 6.0 h (SD) (n = 7) for the GFP-histone cells in the two-by-two field that was scanned for 72 h. After registration and ratioing of the cells with the RhoA biosensor, increases in both cell protrusion and retraction were coincident with to increases in RhoA activity. CONCLUSIONS: These advances lay the foundation for extracting and correlating measurements characterizing the functional relationships of spatial localization and protein activation with features of cell migration such as velocity, polarization, protrusion, retraction, and mitosis.

Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation.

This chapter details quantitative imaging of the Mero-CBD biosensor, which reports activation of endogenous Cdc42 in living cells. The procedures described are appropriate for imaging any biosensor that uses two different fluorophores on a single molecule, including FRET biosensors. Of particular interest is an algorithm to correct for fluorophore photobleaching, useful when quantitating activity changes over time. Specific topics include procedures and caveats in production of the Mero-CBD sensor, image acquisition, motion artifacts, shading correction, background subtraction, registration, and ratio imaging.