Biology subjects

Djayakarsana, D.

Publications and source records attributed to Djayakarsana, D..

3 recordsLinked to original sources

bioRxiv2021

Deuterium Magnetic Resonance Spectroscopy of Early Treatment-Induced Changes in Tumour Lactate in vitro

Elevated production of lactate is a key characteristic of aberrant tumour cell metabolism and can be non-invasively measured as an early marker of tumour response using deuterium (2H) magnetic resonance spectroscopy (MRS). Following treatment, changes in the 2H-labeled lactate signal could identify tumour cell death or impaired metabolic function, which precede morphological changes conventionally used to assess tumour response. In this work, the association between apoptotic cell death, extracellular lactate concentration, and early treatment-induced changes in the 2H-labeled lactate signal was established in an in vitro tumour model. Experiments were conducted at 7 T on acute myeloid leukemia cells which had been treated with 10 {micro}g/mL of the chemotherapeutic agent cisplatin. At 24 and 48 hours after cisplatin treatment, the cells were injected with 20 mM of [6,6-2H2]glucose and scanned over two hours using a two-dimensional 2H MR spectroscopic imaging sequence. The resulting signals from 2H-labeled glucose, lactate, and water were quantified using a spectral fitting algorithm implemented on the OXford Spectroscopy Analysis (OXSA) MATLAB toolbox. After scanning, the cells were processed for histological stains (TUNEL [terminal deoxynucleotidyl transferase UTP nick end labeling] and H&E [hematoxylin and eosin]) to assess apoptotic area fraction and cell morphology respectively, while a colorimetric assay was used to measure extracellular lactate concentrations in the supernatant. Significantly lower levels of 2H-labeled lactate were observed in the 48-hour treated cells compared to the untreated and 24-hour treated cells, and these changes were significantly correlated with an increase in apoptotic fraction and a decrease in extracellular lactate. By establishing the biological processes associated with treatment-induced changes in the 2H-labeled lactate signal, these findings suggest that 2H MRS of lactate may be valuable in evaluating early tumour response.

biophysics↗

bioRxiv2019

FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) yields an image resolution 1-2 orders of magnitude below that of conventional light microscopy, resolving fine details on intracellular structure and macromolecular organization. The massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Density based clustering algorithms, such as DBSCAN, can provide spatial statistics on protein/nucleic acid aggregation or dispersion while explicitly identifying macromolecular clusters. The performance of DBSCAN, however, is typically dependent upon an arbitrary, or at least highly subjective, parametric tuning of the algorithm. Moreover, DBSCAN can be computationally expensive, which makes it arduous to evaluate on large image stacks. This is all the more important in 3-dimensions where there exist limited alternatives for quantifying clustering in SMLM data, and where a 2-dimensional analysis of true 3-dimensional data may give rise to image artefacts. We have developed an open-source software package in Python for both identifying and quantifying spatial clustering in 3-dimensional SMLM datasets. FOCAL3D is an extension of our previously developed, 2-dimensional, grid based clustering algorithm FOCAL. FOCAL3D provides a highly efficient way to spatially cluster SMLM datasets, scaling linearly with the number of localizations, and the algorithmic parameters may be systematically optimized so that the resulting analysis is insensitive to variation over a range of parameter choices. We initially validate the performance and parametric insensitivity of FOCAL3D on simulated datasets, then apply the algorithm to 3-dimensional, astigmatic dSTORM images of the nuclear pore complex in human osteosarcoma cells.\n\nThe data and software package are available at: http://www.utm.utoronto.ca/milsteinlab/software/

bioinformatics↗

bioRxiv2019

Nanoscopic Stoichiometry and Single-Molecule Counting

Single-molecule localization microscopy (SMLM) has the potential to revolutionize proteomic and genomic analyses by providing information on the number and stoichiometry of proteins or nucleic acids aggregating at spatial scales below the diffraction limit of light. Here we present a method for molecular counting with SMLM built upon the exponentially distributed blinking statistics of photoswitchable fluorophores, with a focus on organic dyes. We provide a practical guide to molecular counting, highlighting many of the challenges and pitfalls, by benchmarking the method on fluorescently labeled, surface mounted DNA origami grids. The accuracy of the results illustrates SMLMs utility for optical -omics analysis.

biophysics↗