nanoFLeye

The Way to a Deeper Insight into the Cell

nanoFLeye (nanoFluorescenceEye) is the innovative reply on demands and needs in the field of superresolving optical imaging based on the localization microscopy technique SPDM (spectral precision distance microscopy).

nanoFLeye excels with

3D Multicolour Imaging based on SPDM

High Stability

High Flexibility

Choose up to Four Different Excitation Wavelengths Suitable to your Desired Dyes

Choose your Favoured Microscope Objective

Clear and User-Friendly Interface

Possibility of Remote Control

Opportunity of High Level Automatization such as Autofocus, Easily Programmable Measurement Sequences and Automated Data Analysis

SPECIAL FEATURE

nanoFLeye is equipped with the pioneering ReconFlex camera developed by Surface Concept GmbH making localization microscopy substantially easier and faster.

 

Localization microscopy in general is time and disk space consuming by recording an image stack comprising tens of thousands of images for post-processing of the data to determine the position of each molecule (reconstruction).

 

ReconFlex supports a much faster data analysis for localization microscopy.

 

 

It offers ultra-high flexibilty in terms of its different modes:

Normal Camera Mode: For  Live Imaging, adjusting the Microscope and recording Imaging Stacks

Reconstruction Support Mode:  A Fast Detection Mode of Localization Areas by the nanoFLeye System

Both modes can also be applied simultaneously.

Advantages

Enormous Reduction in Data Transfer, Time and Disk Space

Select Fiducial Markers prior to the Measurement to correct Possible Drifts in Real-Time

The User retains Full Control over the Algorithms used in the Reconstruction Mode

Provided with ReconFlex, nanoFLeye opens up a new door to real-time, easy and user-friendly superresolving localization microscopy.

APPLICATION EXAMPLES

nanoFLeye allows to reveal structures well below the Abbe limit being of interest for biomedical as well as material science applications.

The inverted setup of nanoFLeye and its flexible sample holder allows for the investigation of a huge variety of sample configurations.

 

Protein-Polymers in a Cell Culture Specimen

The images show a segment of fluorescence-labeled microtubules in a cell culture (HeLa-cells)

nanoFLeye uncovers sub-structures within the polymer configuration

The corresponding linescans show the intensity distribution along the implied line in the images

Conventional epifluorescent microscopy image of Alexa647-labeled microtubuli of HeLa-cells.

Superresolved image of the identical sample position recorded by nanoFleye. The scale bar (horizontal line bottom left) corresponds to 1µm.

Linescan (vertical line in the upper image) of the epifluorescent image.

Linescan (vertical line in the upper image) of the superresolved image.

Alexa680-labeled human platelets (HuPLTs, PF4, A680, native)

sample preparation courtesy of: Dr. M. Schmitt, LMU München

left: conventional epifluorescent microscopy image

right: superresolved image recorded by nanoFLeye

Human Thrombocytes

The images show the pf4 distribution inside the platlets

nanoFLeye uncovers the number of the labeled cytokines as well as their formation in the platelets

Conventional epifluorescent microscopy image of Alexa680-labeled HuPLTs. The image size is 5µm x 5µm.

Superresolved image of the identical sample position recorded by nanoFLeye.

Protein-Polymers in a Tissue Sample

Epifluorescence (30µm x 30µm),

scale bar 5µm

Immunofluorescence micrographs

of a 30µm thick rat brain tissue

ICV 10 Hippocampus (DG)

Staining: microtubules (Alexa647)

The images show a segment of fluorescence-labeled microtubules in 30µm thick rat brain tissue

The histogram shows the distribution of the localization accuracy of the detected events.

A mean localzation accuracy of 18nm could be achieved

Mean localization accuracy: 18nm

Sample preparation courtesy of:

Dr. Sebastian Bauer

Leiter AG Translationale Epileptologie

Epilepsiezentrum Frankfurt Rhein-Main

Klinik für Neurologie

Goethe Universität Frankfurt

Epifluorecence, scale bar 1µm

SPDM of the identical section,

scale bar 1µm

METHOD

Convential fluorescence microscopy is a versatile tool to perform functional cell biology analysis. Fluorophores are being coupled to antibodies which bind to their corresponding proteins in the cell. By analyzing the fluorescence signals in the microscopy image, one can get insight to  the distribution of the chosen proteins inside the cell.

 

However, in terms of nano-science and detailed insight into biological processes on a molecular level, convential fluorescence microscopy is stretched to its limit.

 

In an epifluorescence microscope the lateral resolution is determined by the diffraction limit, i.e. you cannot distinguish two molecules having a distance less than ~200 nm from each other. In a confocal setup it is possible to improve the resolution slightly, but not sufficient to detect single molecules.

Localization Microscopy

Spectral features are used to achieve optical isolation

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In Conventional Fluorescence Microscopy the FWHM of the Point-Spread-Function (PSF) >200 nm. Signals of adjacent dyes overlap, therefore single molecules cannot be resolved

 

Using SPDM, randomly activated dyes are „optically isolated“, i.e. no overlap of the signal of adjacent molecules can occur

 

The locations of the optically isolated fluorophores are determined by the localization algorithm with a precision down to 20 nm

 

All localizations found in a stack of usually ten thousands of images are displayed in a single reconstructed super-resolved image

 

CONTACT

Surface Concept GmbH

 

Am Sägewerk 23a

55124 Mainz

Germany

 

phone: +49 6131 62716 0

fax:        +49 6131 62716 29

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3D Multicolour Imaging based on SPDM

High Stability

High Flexibility

Clear and User-Friendly Interface

Possibility of Remote Control

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