Correlated optical and isotopic nanoscopy

Abstract

The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures.

High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS

“…One of our goals was to define (and quantify) the relationship between cell morphology and PFO* and ALO-D4 binding. NanoSIMS is ideal for this purpose because it provides high-resolution images-higher than those obtained by confocal microscopy (12). Also, NanoSIMS offers the unique capability to correlate chemical information (e.g., specific secondary ions) to high-resolution morphology.…”

Review of combined isotopic and optical nanoscopy

“…35 In such studies, a Cs þ primary ion beam of 16-keV energy is usually applied, and stable isotopes, such as 2 H, 13 Therefore, the preparation of biological specimens requires attention due to the possible migration or complete loss of soluble compounds during fixation and embedding phases. 30 NanoSIMS has proven to be capable of imaging proteins by pulsing isotopically labeled amino acids 36 or nucleotides 35 into the cell and imaging cellular turnover. However, understanding metabolism at the subcellular level, gene, and protein expression, the study of protein complexes and single organelles are still limited as this technique cannot provide chemical maps of specific proteins.…”

“…However, understanding metabolism at the subcellular level, gene, and protein expression, the study of protein complexes and single organelles are still limited as this technique cannot provide chemical maps of specific proteins. 34,36 Attempts to improve the specificity of the protein imaging have been done by Angelo et al, 37 where NanoSIMS was used for imaging cells labeled with metal conjugated antibodies. Primary antibodies were coupled with up to 100 isotopically pure lanthanides, and positive secondary ions of metals were sputtered away with an oxygen primary ion beam.…”

“…The former depends on the number of ions that can be determined from the particular structure. For example, a gold particle of ∼50 to 100 nm in diameter will provide excellent contrast, 36 but small particles, such as the 5-nm ones used commonly in electron microscopy, are extremely difficult to detect. At the same time, the image contrast depends on the difference in the abundances of the isotopes measured between the structure of interest and the surrounding material.…”

“…In biological samples, this has been determined to be in the order of 10 to 20 nm. 36 The main limitation in this type of experiment is the sample drift, which should be minimized as it otherwise distorts both the lateral and the axial measurements.…”