SignificanceAccurate cell segmentation and classification in three-dimensional (3D) images are vital for studying live cell behavior and drug responses in 3D tissue culture. Evaluating diverse cell populations in 3D cell culture over time necessitates non-toxic staining methods, as specific fluorescent tags may not be suitable, and immunofluorescence staining can be cytotoxic for prolonged live cell cultures.AimWe aim to perform machine learning-based cell classification within a live heterogeneous cell culture population grown in a 3D tissue culture relying only on reflectance, transmittance, and nuclei counterstained images obtained by confocal microscopy.ApproachIn this study, we employed a supervised convolutional neural network (CNN) to classify tumor cells and fibroblasts within 3D-grown spheroids. These cells are first segmented using the marker-controlled watershed image processing method. Training data included nuclei counterstaining, reflectance, and transmitted light images, with stained fibroblast and tumor cells as ground-truth labels.ResultsOur results demonstrate the successful marker-controlled watershed segmentation of 84% of spheroid cells into single cells. We achieved a median accuracy of 67% (95% confidence interval of the median is 65-71%) in identifying cell types. We also recapitulate the original 3D images using the CNN-classified cells to visualize the original 3D-stained image’s cell distribution.ConclusionThis study introduces a non-invasive toxicity-free approach to 3D cell culture evaluation, combining machine learning with confocal microscopy, opening avenues for advanced cell studies.
Circulating tumor cells form metastases by reaching a distant microcirculation, undergoing transendothelial migration, entering into the remote tissue and proliferating. Microfluidic assays have recently been developed that enable the visualization and quantification of this process within vascular networks that recapitulate many aspects of the in vivo microcirculation. The assays are created by seeding endothelial cells in co-culture with fibroblasts or pericytes within a fibrin gel. In 1 day, the networks form, and in 4 to 7 days they are perfusable with medium. At that point, tumor cells, with or without accompanying immune cells, are streamed into the network, some fraction of which will arrest and extravasate into the surrounding matrix. Models of this type have been used to study several aspects of this process. These studies have provided detailed data on the ability of different tumor cell types to extravasate, the adhesion molecules they use to pull themselves through the endothelial monolayer, and the effects of various other cell types in the intravascular space (neutrophils and platelets), and the extracellular matrix (fibroblasts, pericytes, myoblasts, and osteoblasts). Some studies have been carried out for over one week in order to observe the initial stages of growth of the metastatic tumor. Other vascular network models have also been developed that can be used for longer-term studies, with more realistic network morphologies and remodeled matrix composition.
Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.
We present a multicolor fluorescence imaging modality to visualize in real-time tissue structures emitting multispectral fluorescent light from different focal depths. Each designated spectrum of fluorescent emission from a specific depth within a volumetric tissue is probed by a depth-spectrum selective holographic grating. The grating for each fluorescent color are multiplexed within a volume hologram, which enables simultaneously obtaining multicolored fluorescent information at different depths within a biological tissue sample. We demonstrate the imaging modality's ability to obtain laser-induced multicolored fluorescence images of a biological sample from different depths without scanning. We also experimentally demonstrate that the imaging modality can be simultaneously operated at both fluorescent and bright field modes to provide complementary information of volumetric tissue structures at different depths in real-time.
A real-time three dimensional (3D) fluorescence imaging system incorporating wavelength-coded and multiplexed
holographic gratings is presented. Holographic gratings formed in thick Phenanthrenquinone- (PQ-) Doped Poly (methyl
methacrylate) (PMMA) have narrowband spectral-spatial transmittance filtering properties to generate wavelengthspectrum
selective multi-focal planes within a biological object. We demonstrate the imaging modality to obtain laserinduced
fluorescent tissue structures from different depths at the excitation wavelength of 355nm.
The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses non- invasively. This paper describes the development of a micro manipulation workstation integrating two-photon, 3-D imaging with a high-force, uniform-gradient, magnetic manipulator. The uniform-gradient magnetic field applies nearly equal forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5 micrometers diameter paramagnetic particles and over 800 pN on 5.0 micrometers ferromagnetic particles. These forces vary less than 10% over an area 200 x 200 micrometers 2. The compatibility with the use of high numerical aperture (approximately equals 1.0) objectives is an integral part of the workstation design allowing sub- micron resolution 3-D two-photon imaging. Three dimensional maps of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses is not always a local deformation.
We present the design of a magnetic tweezers microscope for cellular manipulation. Our design allows versatile and significant 3D stress application over a large sample region. For linear force application, forces up to 250 pN per 4.5 micrometers magnetic bead can be applied. Finite element analysis shows that variance in force level is around 10 percent within an area of 300 X 300 micrometers 2. Our eight-pole design potentially allows 3D liner force application and exertion of torsional stress. Furthermore, our design allows high resolution imaging using high numerical aperture objective. Both finite element analysis of magnetic field distribution and force calibration of our design are presented. As a feasibility study, we incubated fibronectin coated 4.5 micrometers polystyrene beads with Swiss 3T3 mouse fibroblast cells. Under application around 250 pN of force per magnetic particle, we observed relative movement between attached magnetic and polystyrene beads to be on the order of 1 micrometers . Elastic, viscoelastic, and creeping responses of cell surfaces were observed. Our results are consistent with previous observations using similar magnetic techniques.
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