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  Introduction

LSM 710 Confocal Laser Scanning Microscope with ConfoCor3 (FCS)
The biomedical sciences are considered some of the most important and future oriented areas of research. Taking advantage of increasingly powerful technologies, they lead to a deeper understanding of the complex mechanisms that are the foundation of living systems at the molecular, cellular and tissue levels. Confocal microscopy systems have laid the groundwork for numerous scientific breakthroughs and have made possible a number of new methods for conducting research.
Confocal microscopy is an optical imaging technique used to increase micrograph contrast and to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane.
In case of new confocal laser scanning microscope, LSM 710, the system adds new potential to all areas of biological research with higher sensitivity, reduced background noise, improved flexibility regarding the use of new fluorochromes, and integrated analytical methods.
Sensitivity is the key feature in a confocal microscope. The LSM 710 achieves a high sensitive image acquisition with low noise level, and provides reduced photo-toxicity for experiments with living cells.
The LSM 710 offers improvements in almost every aspect, whether it involves faster scan speeds at lower zoom factors (i.e., large fields of view with a field number of up to 20 in the intermediate plane) or more constant imaging conditions with, for example, stable laser excitation or control of the focus plane using the definite focus attachment on the Axio Observer microscope stand.
The trend towards more representative experiments with living cells also means analyzing the interactions of structures. Whether it involves cancer research, cell death, the analysis of DNA repair proteins, protein synthesis or the detailed mechanisms of cell division, freely definable ROIs for bleach and photoactivation experiments are essential. The LSM 710 offers the ideal tools for single and multiple ROIs with individual settings and at the fastest speeds possible.
Fast photoactivation experiments used to be very difficult with point scanning confocal microscopes. The faster scan rates and improved signal to noise of the LSM 710 now make it possible to analyze diffusion even of small soluble proteins with such a microscope.

Explaining diffusion processes and molecular interactions and their relation to cellular compartments and structures is essential in the detailed understanding of cell functions.
In many cases it is not the average signal that is of interest but the behavior of single molecules acting in an individual manner.
Fluorescence-based assays at a single molecule level have revolutionized the way we can observe molecules at work in their natural surroundings. Many of these technologies are based on fluorescence correlation spectroscopy (FCS).
ConfoCor 3 with a microscope from LSM 710 is much more than an imaging device. It not only allows you to observe single molecules non-invasively but also to resolve fast dynamic processes with great accuracy

 
  Principles and Benefits of Confocal Microscope
The big advantage of confocal microscopy is the possibility to collect light exclusively from a single plane.
A pinhole sitting conjugated to the focal plane (i.e. confocal) keeps light from the detector that is reflected/emitted from others than the focal plane.
The laser scanning microscope scans the sample sequentially point by point and line by line and assembles the pixel information to one image. That way optical slices of the specimen are imaged with high contrast and high resolution in x, y and z.
By moving the focus plane single images (optical slices) can be put together to build up a three dimensional stack that can be digitally processed afterwards.

Instruments:
Confocal Laser Scanning Microscope with ConfoCor3
Model:
LSM 710 / Carl Zeiss MicroImaging GmbH,
Germany
 
  Key features of LSM 710
Dramatically Increased Sensitivity for Visualizing the faintest signals
Greater Flexibility for Imaging multiple fluorescent markers simultaneously and for every kind of experiment
Precision and Reproducibility
Extended Live Cell Capabilities - analyzing cellular functions, molecular interaction and optical
   manipulation for dynamic live cell studies
Uncompromised Multiphoton Imaging - Gaining deeper insights in thick specimens
Low Maintenance and High Efficiency through self-test and maintenance tools
 
  Specifications
Laser Inserts
(VIS, V)
Pigtail-coupled lasers with polarization preserving single-mode fibers;
Diode 405nm- DAPI, Hoechest, BFP, PA-GFP, KAEDE…)
Multi Ar-laser 458/488/514 nm - CFP, GFP, FITC, Alexa 488, Cy2, YFP...
HeNe-laser 543 nm - Rhodamine, Cy3, Alexa 546, Texas Red, RFP, DsRed…
Objectives Objective EC “Plan-Neofluar” 10x/0.30
EC Plan-NEOFLUAR 20x/0,5
Objective “C-Apochromat” 40x/1,20 W corr
EC Plan-NEOFLUAR 100x/1,3 Oil
Spectral Detection Spectral detector 3 channels with highly sensitive low dark noise PMTs; spectral detection range freely selectable (resolution down to 3 nm); additionally two incident light channels with APDs for imaging and single photon measurements; transmitted light channel with PMT
Scanning speed 14 × 2 speed stages; up to 12.5 frames/sec with 256 × 256 pixels; 5 frames/sec with 512 × 512 pixels (max. 77 frames/sec 512 × 32); min 0.38 ms for a line of 512 pixels; up to 2619 lines per second
Master-pinhole pre-adjusted in size and postion, individually variable for multi-tracking and short wavelengths (e.g. 405 nm)
Incubation system Incubation with control of temperature, CO2 , humidity or O2
 
  Applications
Long-term live cell Imaging using GFP (ex. Cancer cell)
Calcium imaging using calcium indicators
Photomanipulation experiment (ex FRAP, FLIP, FRET…)
Live cell imaging using new fluorescent proteins (PA-GFP, KAEDE)
4D imaging (X,Y,Z-t)
High resolution, hig magnification fluorescent microscopy for all kinds of Biomedical research.
 
  FCS Applications
Investigating molecule distribution in cells
principles of diffusion processes
Studying protein complex formations
Detecting common movement ex. Vesicle transportation
Quantifying receptor ligand interactions - Pharmaceutical drug study
 
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