Nirmalya Bag

Bio: Nirmalya Bag is an academic researcher from Cornell University. The author has contributed to research in topics: Fluorescence correlation spectroscopy & Membrane. The author has an hindex of 12, co-authored 28 publications receiving 620 citations. Previous affiliations of Nirmalya Bag include National University of Singapore & Indian Institute of Technology Bombay.

Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms

Abstract: Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS. Here we provide guidelines for imaging FCS/FCCS measurements on commercial and custom-built microscopes (including sample preparation, setup calibration, data acquisition and evaluation), as well as anticipated results for a variety of in vitro and in vivo samples. For a skilled user of an available SPIM or TIRF setup, sample preparation, microscope alignment, data acquisition and data fitting, as described in this protocol, will take ∼1 d, depending on the sample and the mode of imaging.

Calibration and Limits of Camera-Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

Abstract: Camera-based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR-FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements. We demonstrate that the PSF can be accurately determined by ITIR-FCS and that the laser power and time resolution can be varied over a wide range with limited influence on the measurement of the diffusion coefficient whereas the concentration measurements are sensitive to changes in the measurement parameters. One advantage of ITIR-FCS is that the measurement of the PSF has to be performed only once for a given optical setup, in contrast to confocal FCS in which calibrations have to be performed at least once per measurement day. Using optimized experimental conditions we provide diffusion coefficients for over ten different lipid membranes consisting of one, two and three constituents, measured in over 200000 individual correlation functions. Using software binning and thus the inherent advantage of ITIR-FCS of providing multiple observation areas in a single measurement we test the FCS diffusion law and show how they can be complemented by the local information provided by the difference in cross-correlation functions (ΔCCF). With the determination of the PSF by ITIR-FCS and the optimization of measurement conditions ITIR-FCS becomes a calibration-free method. This allows us to provide measurements of absolute diffusion coefficients for bilayers with different compositions, which were stable over many different bilayer preparations over a time of at least one year, using a single PSF calibration.

Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements.

Abstract: Imaging fluorescence correlation spectroscopy (FCS) performed using array detectors has been successfully used to quantify the number, mobility, and organization of biomolecules in cells and organisms. However, there have not been any systematic studies on the errors in these estimates that are introduced due to instrumental and experimental factors. State-of-the-art array detectors are still restricted in the number of frames that can be recorded per unit time, sensitivity and noise characteristics, and the total number of frames that can be realistically recorded. These limitations place constraints on the time resolution, the signal-to-noise ratio, and the total measurement time, respectively. This work addresses these problems by using a combination of simulations and experiments on lipid bilayers to provide characteristic performance parameters and guidelines that govern accuracy and precision of diffusion coefficient and concentration measurements in camera-based FCS. We then proceed to demonstrate the effects of these parameters on the capability of camera-based FCS to determine membrane heterogeneity via the FCS diffusion laws, showing that there is a lower length scale limit beyond which membrane organization cannot be detected and which can be overcome by choosing suitable experimental parameters. On the basis of these results, we provide guidelines for an efficient experimental design for camera-based FCS to extract information on mobility, concentration, and heterogeneity.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Abstract: Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Single-photon avalanche diode imagers in biophotonics: review and outlook

Abstract: Single-photon avalanche diode (SPAD) arrays are solid-state detectors that offer imaging capabilities at the level of individual photons, with unparalleled photon counting and time-resolved performance. This fascinating technology has progressed at a very fast pace in the past 15 years, since its inception in standard CMOS technology in 2003. A host of architectures have been investigated, ranging from simpler implementations, based solely on off-chip data processing, to progressively "smarter" sensors including on-chip, or even pixel level, time-stamping and processing capabilities. As the technology has matured, a range of biophotonics applications have been explored, including (endoscopic) FLIM, (multibeam multiphoton) FLIM-FRET, SPIM-FCS, super-resolution microscopy, time-resolved Raman spectroscopy, NIROT and PET. We will review some representative sensors and their corresponding applications, including the most relevant challenges faced by chip designers and end-users. Finally, we will provide an outlook on the future of this fascinating technology.

Multiscale Simulations of Biological Membranes: The Challenge To Understand Biological Phenomena in a Living Substance.

Abstract: Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytosk...

The Centenary of the Stern-Volmer Equation of Fluorescence Quenching: From the single line plot to the SV quenching map

Abstract: In the year of 2019, we celebrate the centenary of the publication of the remarkable paper by Otto Stern and Max Volmer concerning the kinetics analysis of fluorescence quenching. Their achievement changed enormously many fields of science with great impact in the studies of electronic spectroscopy and kinetics of organic, inorganic and biological compounds in condensed phase. The development of molecular photochemistry was in great part based on the use of the Stern-Volmer (SV) approach to investigate bimolecular interaction in the electronic excited-state. This paper reviews and summarizes the assumptions behind the Stern-Volmer equation and the extensions of the theory to other important situations. Discussions about the use of the SV approach to investigate probe and quencher distribution, association, diffusion and reaction at the molecular level obtained from advanced fluorescence microscopy methods are also included.