Challenges in quantitative single molecule localization microscopy

Single molecule localization microscopy (SMLM), which can provide up to an order of magnitude improvement in spatial resolution over conventional fluorescence microscopy, has the potential to be a highly useful tool for quantitative biological experiments. It has already been used for this purpose in varied fields in biology, ranging from molecular biology to neuroscience. In this review article, we briefly review the applications of SMLM in quantitative biology, and also the challenges involved and some of the solutions that have been proposed. Due to its advantages in labeling specificity and the relatively low overcounting caused by photoblinking when photo-activable fluorescent proteins (PA-FPs) are used as labels, we focus specifically on Photo-Activated Localization Microscopy (PALM), even though the ideas presented might be applicable to SMLM in general. Also, we focus on the following three quantitative measurements: single molecule counting, analysis of protein spatial distribution heterogeneity and co-localization analysis. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

Progress in quantitative single-molecule localization microscopy

With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM)

Progress in quantitative single-molecule localization microscopy

With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy(PALM)

Infrared light emission from semiconductor devices

We present results using near-infrared (NIR) cameras to study emission of common defect classes for integrated circuits. The cameras are based on a liquid nitrogen cooled HgCdTe imaging array with high quantum efficiency and very low read noise. The array was developed for infrared astronomy and has high quantum efficiency in the wavelength range from 0.8 to 2.5 {mu}m. For comparison, the same set of samples used to characterize the performance of the NIR camera were studied using a non-intensified, liquid-nitrogen-cooled, slow scan CCD camera (with a spectral range 400-1100 nm). Results show that the NIR camera images all of the defect classes studied here with much shorter integration times than the cooled CCD, suggesting that photon emission beyond 1 {mu}m is significantly stronger than at shorter wavelengths

Effect of head pillow and shoulder roll on diameter of the right internal jugular vein

Background: Right internal jugular vein (RIJV) is the most commonly used site for central venous cannulation. Ultrasound guidance has increased success rate and reduced complications of central venous cannulation. The main aim before cannulation is to increase the size of the vein by optimal positioning. We used ultrasound to assess changes in right internal jugular diameter with use of a head pillow and shoulder roll and their effect on the degree of overlap between RIJV and carotid. Methodology: 106 patients were recruited in the study. After intubation the patients were placed in the following 3 positions (i) P1 -without head pillow or shoulder roll, (ii) P2 - with head pillow and (iii) P3- with shoulder roll. All measurements were made in 15 degree Trendelenberg tilt and head turned to the left by 30 degree. The following measurements were recorded in all 3 positions. (a) Transverse and Antero-Posterior diameter of the RIJV. (b) Transverse diameter of the right carotid. (c) Overlap between RIJV and carotid. Results: In our study the mean transverse diameter of RIJV was 1.87cm in P1 and P2 and 1.72 cm in P3respectively. The mean antero-posterior diameter was 1.39cm, 1.37cm and 1.13 cm in P1, P2 and P3 respectively. The increase in diameter in P1 was statistically significant (P < 0.001). The mean overlap percentage between right carotid and RIJV was 49.41%, 50.97% and 35.7% in P1, P2 and P3 respectively. This difference between P3 and other two position was also statistically significant (P < 0.00). Conclusion: We conclude that placing the patient supine in 15 degree Trendelenberg tilt and a30 degree head rotation to the opposite side with or without use of a head pillow would lead to greater chance of first pass success during R IJV cannulation as the diameter was found to be maximum in this position. We do not recommend use of a shoulder roll as there was significant reduction in diameter though the overlap between RIJV and carotid was found to be minimal. Use of ultrasound and proper positioning of the patient will reduce the possible catastrophic complications associated with RIJV cannulation