Dataset Information

A Practical Solution for 77 K Fluorescence Measurements Based on LED Excitation and CCD Array Detector.

ABSTRACT: The fluorescence emission spectrum of photosynthetic microorganisms at liquid nitrogen temperature (77 K) provides important insights into the organization of the photosynthetic machinery of bacteria and eukaryotes, which cannot be observed at room temperature. Conventionally, to obtain such spectra, a large and costly table-top fluorometer is required. Recently portable, reliable, and largely maintenance-free instruments have become available that can be utilized to accomplish a wide variety of spectroscopy-based measurements in photosynthesis research. In this report, we show how to build such an instrument in order to record 77K fluorescence spectra. This instrument consists of a low power monochromatic light-emitting diode (LED), and a portable CCD array based spectrometer. The optical components are coupled together using a fiber optic cable, and a custom made housing that also supports a dewar flask. We demonstrate that this instrument facilitates the reliable determination of chlorophyll fluorescence emission spectra for the cyanobacterium Synechocystis sp. PCC 6803, and the green alga Chlamydomonas reinhardtii.

SUBMITTER: Lamb J

PROVIDER: S-EPMC4503352 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A Practical Solution for 77 K Fluorescence Measurements Based on LED Excitation and CCD Array Detector.

Lamb Jacob J Forfang Kristin K Hohmann-Marriott Martin M

PloS one 20150715 7

The fluorescence emission spectrum of photosynthetic microorganisms at liquid nitrogen temperature (77 K) provides important insights into the organization of the photosynthetic machinery of bacteria and eukaryotes, which cannot be observed at room temperature. Conventionally, to obtain such spectra, a large and costly table-top fluorometer is required. Recently portable, reliable, and largely maintenance-free instruments have become available that can be utilized to accomplish a wide variety of ...[more]

PMID: 26177548

Similar Datasets

Project description:Background:Ca2+ plays an important role in many physiological processes and an accurate study of these signals is important. In modern fluorescence microscopy, a charge-coupled device (CCD) camera is widely deployed for calcium imaging. The ratiometric method is used for the fluorescence dye Fura-2 and Grynkiewitz's formula (Grynkiewicz et al., 1985) is commonly used to convert fluorescence to free Ca2+ concentration ([Ca2+]). But the need to subtract the background signal can lead to a big error in ratiometric calcium measurements. When the error due to background subtraction occurs, the fluorescence ratio of 340 nm divided by 380 nm lights may be twice as large as the actual value. Under conditions when the excitation intensity is not adjusted to ensure the same throughput of the objective lens for ultraviolet dye illumination, the indicator does not gradually bleach out for channels with a wavelength of 340 nm and 380 nm light, which lead to an additional error in determining the concentration of Ca2+. New method:Here we present a new approach for calculating [Ca2+] from the ratiometric fluorescence of Fura-2 dye imaged by a CCD camera. It is designed to optimize [Ca2+] measurements with photobleaching correction without background subtraction error. A mathematical method is also provided for removing the existing underestimated value of fluorescence at an excitation wavelength of 340 nm and compensating for the bleaching rate for both channels with wavelengths of 340 nm and 380 nm using a power function. Results:In cultured neurons, the calculations of the free Ca2+ concentration during Ca2+ transients estimated by the old and new methods, determine it to the same extent. This comparison was made under conditions without errors through background subtraction. If there is this error, the old method calculates [Ca2+] with a much higher, rather than the actual value. Conclusions:We present a modified Grynkiewitz's formula for calculation [Ca2+] for ratiometric dye, such as Fura-2 imaged by a CCD camera, with photobleaching correction without background subtraction error.

Dataset Information

A Practical Solution for 77 K Fluorescence Measurements Based on LED Excitation and CCD Array Detector.

Publications

A Practical Solution for 77 K Fluorescence Measurements Based on LED Excitation and CCD Array Detector.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets