Confocal sample-scanning microscope for single-molecule spectroscopy and microscopy with fast sample exchange at cryogenic temperatures

Abstract

The construction of a microscope with fast sample transfer system for single-molecule spectroscopy and microscopy at low temperatures using 2D/3D sample-scanning is reported. The presented construction enables the insertion of a sample from the outside (room temperature) into the cooled (4.2 K) cryostat within seconds. We describe the mechanical and optical design and present data from individual Photosystem I complexes. With the described setup numerous samples can be investigated within one cooling cycle. It opens the possibility to investigate biological samples (i) without artifacts introduced by prolonged cooling procedures and (ii) samples that require preparation steps like plunge-freezing or specific illumination procedures prior to the insertion into the cryostat.

Stable Single‐Molecule Lines of Terrylene in Polycrystalline para‐Dichlorobenzene at 1.5 K

“…[32] Capillaries used in confocal fluorescence spectroscopy have been shown to increase the sensitivity to sub-picomolar concentrations. Here, we tested up to six samples in the same experiment by using multiple glass capillaries (or later hollow fibers) to test either different samples and/or different concentrations at the same time.…”

“…Here, we tested up to six samples in the same experiment by using multiple glass capillaries (or later hollow fibers) to test either different samples and/or different concentrations at the same time. [32] Capillaries used in confocal fluorescence spectroscopy have been shown to increase the sensitivity to sub-picomolar concentrations. [33] Starting from a mixture with a well-known concentration, 10 À4 m of Tr/p-DCB, we obtained two other samples with concentrations of approximately 10 À6 and 10 À8 m after dilution.…”

Effects of Irregular Bimetallic Nanostructures on the Optical Properties of Photosystem I from Thermosynechococcus elongatus

“…To ensure the observation of individual coupled pairs in the entire width of the excitation beam's focus, a very low concentration of PSI trimers (∼ 3 pM) in the solution was used. The samples were prepared outside of the cryostat and subsequently were inserted quickly onto the scanning desk in the cooled cryostat (4.2 K) by using our fast sample transfer mechanism introduced in [34]. The samples were scanned in the xy direction over a specific range.…”

“…To form a hybrid, less than 1 µL of this highly diluted solution was sandwiched between metallic nanostructures and bare glass coverslips and transferred to a pre-cooled (4.2 K) cryostat. SMS is performed by using a home-built confocal microscope setup operating at cryogenic temperature ∼1.6 K [34]. A cw diode laser at a wavelength of 665 nm was used as an excitation source for fluorescence measurements.…”

Single Nanoparticle Photoluminescence Studies of Visible Light-Sensitive TiO2and ZnO Nanostructures

“…Therefore, in order to solve these problems, it is necessary to probe individual nanostructure behaviors hidden within the uncorrelated ensemble systems by using single nanoparticle spectroscopic techniques similar to single molecule spectroscopy. 21,22 Since Bawendi et al reported the first observation of the unique PL intermittency from a single ZnS-overcoated CdSe QDs, 23 the single nanoparticle spectroscopy has been applied to study the surface state behaviors including exciton dynamics of different single semiconductor QDs such as ZnS-overcoated CdS, and CdTe, [24][25][26][27] InGaAs/GaAs 28 and Y2O2S:Eu…”