Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images

Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity.We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field -stack, and by measuring the intensity variations of this stack over the -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. Using the popular CellProfiler freeware cell image analysis software mainly targeted for fluorescence microscopy, we validate our method by automatically segmenting low contrast and rather complex shaped murine macrophage cells.The proposed approach frees up a fluorescence channel, which can be used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright field images match closely to the standard approach where cell areas are localized using fluorescence, and conclude that the high contrast bright field projection image can directly replace one fluorescent channel in whole cell quantification. Matlab code for calculating the projections can be downloaded from the supplementary site: http://sites.google.com/site/brightfieldorstaining

A Third Measure-Metastable State in the Dynamics of Spontaneous Shape Change in Healthy Human's White Cells

Human polymorphonuclear leucocytes, PMN, are highly motile cells with average 12-15 µm diameters and prominent, loboid nuclei. They are produced in the bone marrow, are essential for host defense, and are the most populous of white blood cell types. PMN also participate in acute and chronic inflammatory processes, in the regulation of the immune response, in angiogenesis, and interact with tumors. To accommodate these varied functions, their behavior is adaptive, but still definable in terms of a set of behavioral states. PMN morphodynamics have generally involved a non-equilibrium stationary, spheroid Idling state that transitions to an activated, ellipsoid translocating state in response to chemical signals. These two behavioral shape-states, spheroid and ellipsoid, are generally recognized as making up the vocabulary of a healthy PMN. A third, “random” state has occasionally been reported as associated with disease states. I have observed this third, Treadmilling state, in PMN from healthy subjects, the cells demonstrating metastable dynamical behaviors known to anticipate phase transitions in mathematical, physical, and biological systems. For this study, human PMN were microscopically imaged and analyzed as single living cells. I used a microscope with a novel high aperture, cardioid annular condenser with better than 100 nanometer resolution of simultaneous, mixed dark field and intrinsic fluorescent images to record shape changes in 189 living PMNs. Relative radial roundness, R(t), served as a computable order parameter. Comparison of R(t) series of 10 cells in the Idling and 10 in the Treadmilling state reveals the robustness of the “random” appearing Treadmilling state, and the emergence of behaviors observed in the neighborhood of global state transitions, including increased correlation length and variance (divergence), sudden jumps, mixed phases, bimodality, power spectral scaling and temporal slowing. Wavelet transformation of an R(t) series of an Idling to Treadmilling state change, demonstrated behaviors concomitant with the observed transition

Simplified automatic method for measuring the visual field using the perimeter ZERK 1

Background: Currently available perimeters have limited capabilities of performing measurements of the visual field in children. In addition, they do not allow for fully automatic measurement even in adults. The patient in each case (in any type of perimeter) has at his disposal a button which he uses to indicate that he has seen a light stimulus. Such restrictions have been offset in the presented new perimeter ZERK 1. Methods: The paper describes a new type of automated, computerized perimeter designed to test the visual field in children and adults. The new perimeter and proprietary software enable to carry out tests automatically (without the need to press any button). The presented full version of the perimeter has been tested on a head phantom. The next steps will involve clinical trials and a comparison with measurements obtained using other types of perimeters. Results: The perimeter ZERK 1 enables automatic measurement of the visual field in two axes (with a span of 870 mm and a depth of 525 mm) with an accuracy of not less than 1o (95 LEDs on each arm) at a typical position of the patient's head. The measurement can be carried out in two modes: default/typical (lasting about 1 min), and accurate (lasting about 10 min). Compared with available and known types of perimeters, it has an open canopy, proprietary software and cameras tracking the eye movement, automatic control of fixation points, light stimuli with automatically preset light stimulus intensity in the following ranges: 550-700 mcd (red 620-630 nm), 1100-1400 mcd (green 515-530 nm), 200-400 mcd (blue 465-475 nm). Conclusions: The paper presents a new approach to the construction of perimeters based on automatic tracking of the eye movements in response to stimuli. The unique construction of the perimeter and the software allow for its mobile use in the examination of children and bedridden patients

Targeting cell cycle and hormone receptor pathways in cancer

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response.C E S Comstock, M A Augello, J F Goodwin, R de Leeuw, M J Schiewer, W F Ostrander Jr, R A Burkhart, A K McClendon, P A McCue, E J Trabulsi, C D Lallas, L G Gomella, M M Centenera, J R Brody, L M Butler, W D Tilley and K E Knudse

Detection Of Automatic Digital Image Analysis Problems For The Evaluation Of Immune Markers In Breast Cancer Biopsies

INTRODUCTION / BACKGROUND: Automatic digital image analysis has increased in recent years. There are several applications for image analysis in pathology, among them, immunohistochemistry biomarkers quantification. This is a simple, economic and fast method to quantify stained biomarkers in digitalized biopsies enhancing sensitivity and objectivity. However, automatic procedures do not always work satisfactorily in all digitalized samples. AIMS: To quantify the number of incorrectly analyzed images as a result of errors on the automatic procedure developed for quantifying immune markers in breast cancer biopsies, to evaluate the amount of time spent in this reanalysis and to define which kind of biopsy produces more errors. METHODS: 10,770 cores of breast tumor (intra-tumoral and peri-tumoral areas) and negative and positive axillary lymph nodes areas from ductal invasive breast cancer were included in different tissue microarrays (TMAs). Slides of each TMA were immunohistochemically stained for CD4, CD8, CD57, CD68, S100, LAMP3, CD83, CD1A, CD123 and CD21 immune markers and were digitalized at 40X with the Apperio Scanscope XT scanner. Each core was extracted as an individual digital image in TIFF format. The stained area was automatically quantified using procedures developed with Fiji (Image J) software in a HP Intel Inside Core i.7 computer with 16GB of RAM memory. Firstly, the whole area of each core was evaluated in pixels by using the luminance channel, applying the median filter and gray-scale segmentation. The second step evaluated the positive pixel number stained in brown for obtaining a brown color channel and then, a gray scale and size segmentation for positive objects, including holes inside the segmented area. Finally, this selected brown area was automatically surrounded by an overlay. RESULTS: 2,751 had to be reanalyzed (25.5%). Specifically, intra-tumoral and peri-tumoral cores were those with higher reanalysis levels (35.6% and 34.9%, respectively) while axillary lymph nodes cores present lower levels (Negative Nodes 13.5% and Positive Nodes 16.4%). Regarding the immune biomarker, CD21, LAMP3 and CD123 were those with higher reanalysis levels (38.4%, 35.2% and 34.2%, respectively) in contrast with CD8, CD68 and CD83 that were those with lower levels (16.4%, 17.7%, 18.4%, respectively). The reanalysis levels of the remaining biomarkers were 22% in CD4, 23.9% in S100, 26.% in CD1A and 27.4% in CD57. Each core took a mean of 1.56 minutes to be analyzed so the total time spent in the reanalysis of the images was 71.5 hours. The principal reasons for reanalysis were problems in the TMAs assembly accuracy and presence of adipose tissue, hemosiderin, artifacts, unspecific staining and background noise. Several TMAs presented glue bubbles and different types of dirt, as hairs or dust that were quantified as positive area. Intratumoral and peritumoral cores are those with higher levels of adipose tissue. These adipocytes cause alterations in the automatic quantification due to their different cellular structure. Specifically, in cores stained for S100 marker in which membranes of adipocytes were also stained in brown and, thus, quantified as immune marker. CONCLUSIONS: It could be possible to reduce the time of analysis and to obtain more exact values of immune quantification in digital images improving the accuracy of TMAs assembly, overcoming unspecific staining and background and adapting the parameters of the procedures