SCANNING-LASERCONFOCAL MICROSCOPY

(SLCM)

Hatice Nur Halipçi50030916

Fatih University-Molecular Biology and Genetic

OUTLINE

• Short History• General Definition • Principles of Confocal Microscopy• Types of Confocal Microscopes • Scanning Laser- Confocal Microscopy• Application Areas • Advantages• Disadvantages

HISTORY OF CONFOCAL and SLC MICROSCOPY

The principle of confocal imaging was patented by Marvin Minsky in 1957

In 1978, Thomas and Christoph Cremer designed a laser scanning process

What is the Confocal

Microscopy?

is an optical imaging technique used to increase micrograph contrast and to reconstruct 3-D images by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane

confocal microscope

Why does it called“CONFOCAL?”

•In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information. Also the pinhole is conjugate to the focal point of the lens. – that’s why it’s called “confocal”

•In a conventional fluorescence microscope, the entire specimen is flooded in light from a light source. So, all parts of the specimen throughout the optical path will be excited.

WHAT’S THE PRINCIBLE?

•The principle of confocal microscopy is the elimination of out of focus light, thus producing a high z-resolution image.

• Confocal fluorescence microscopes achieve this, via two principal mechanisms.

First, incident light is focused to a particular point within the specimen by passing it through a very small aperture, the first pinhole. The focusing helps to limit the excitation of fluorophores above and below the plane of focus.

Second, any emission that is above or below the plane of focus is blocked from reaching the detector by passing it through a second pinhole. The specimen is placed in the light-path at a conjugate focal plane such that movement in the vertical (z) direction keeps the focus at a fixed distance from the objective, and effectively scans in layers through the specimen.

WHAT’S THE FEATURE?

•The key feature of confocal microscopy is its ability to produce in-focus images of thick specimens, a process known as optical sectioning..

• Images are taken point-by-point and reconstructed with a computer, rather than projected through an eyepiece.

Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, and fluorophores for highly specific targets.

1. Scanning laser confocal microscopes (CLSM) 2. Spinning disk confocal microscopes (SDCM) 3. Programmable array microscopes (PAM).

Types of Confocal Microscopes

SCANNING -LASER

CONFOCAL MICROSCOPY

SCANNING:

The science and techniques involved in the use of scanners

LASER:

an acronym for Light Amplification by Stimulated Emission of Radiation

CONFOCAL:

having the same focus  

MICROSCOPY:

research with the use of microscopes

Scanning Laser Confocal Microscopy (SLCM)

is a valuable tool for obtaining high resolution images and 3-D reconstructions.

HOW DOES IT WORK?

• A laser is used to provide the excitation light (in order to get very high intensities). The laser light (blue) reflects off a dichroic mirror. From there, the laser hits two mirrors which are mounted on motors; these mirrors scan the laser across the sample.

…..how does it work?

•Dye in the sample fluoresces, and the emitted light (green) gets descanned by the same mirrors that are used to scan the excitation light (blue) from the laser. The emitted light passes through the dichroic and is focused onto the pinhole. The light that passes through the pinhole is measured by a detector.

…..how does it work?

•So, there never is a complete image of the sample -- at any given instant, only one point of the sample is observed. The detector is attached to a computer which builds up the image, one pixel at a time

Some of Microscopy Images

Retina ganglion cell

Rat tongue taste bud

Human colon crypt

visual interneurons ofswallowtail butterfly

3-D IMAGES

Mitosis

Tobacco BY2 cells in telophase

Tobacco leaf epidermal cells expressing

neurocapsule

Metaphase: tobacco BY2 chromosomes

• In a laser scanning confocal microscope a laser beam passes a light source aperture

and then is focused by an objective lens into a small focal volume within a fluorescent specimen.

Differences of Fluorescent and Confocal Microscopy

• Confocal microscopy is a form of fluorescent microscopy

BUT it uses a computer, rather than projected through an

eyepiece.

gives higher resolution

laser light source is used to excite the fluorophore resulting in enhanced contrast.

Fluorescent Microscope

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

Objective

Laser

Emission Pinhole

Excitation Pinhole

PMT

EmissionFilter

Excitation Filter

Confocal Microscope

Conventional fluorescence microscope Laser scanning microscope

specimen

full field illumination

full field detection

Arc lamp + excitation filter

point scan illumination

point scan detection

laser light source

Differences of non-confocal

and confocal images

Rabbit Muscle Fiber

(Magnification:100X)

• Fava bean mitosis

(Magnification:100X)

APPLICATIONS

• Resonance or flourescence energy transfer, • Stem cell research, • Lifetime imaging, • Total internal reflection, • Immuno-gold labeling,• DNA hybridization, • Membrane and ion probes,• Bioluminescent proteins, • and epitope tagging.

• studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues

…applications

It is particularly useful for,

• evaluation of various eye diseases. Imaging, qualitative analysis and quantitification of endothelial cells of the cornea.

Colocalization of Proteins

•It provides colocalization of up to 4 different proteins

•By using this function to analyze multi-colorspecimens, it is possible to discover whether different labeled substances arepresent in the same region

•Decreased cross talk with multitracking feature

…applications

-Advantages-Why is Confocal Microscopy Better?

1. More Color PossibilitiesBecause the images are detected by a computer rather than by eye, it is possible to detect more color differences.

Why is Confocal Microscopy Better?

2. Less Cross Talk If the fluorochromes have distinct excitation spectra, the fluorochromes can be excited sequentially using one excitation wavelength at a time. This is only possible with confocal systems that offer the multitracking feature.

Why is Confocal Microscopy Better?

3. Optical Sectioning of Objects Without Physical Contact

Zebra fish embryo wholemount Neurons (green) Cell adhesion molecule (red)

Why is Confocal Microscopy Better?4. Three-Dimensional Reconstruction of Specimen

3D shadow projection Tight junctions (red) Cytoskeletal structures (green)

Animated 3-Dimensional Reconstruction

Laser Scanning MicroscopyLSM5103D for LSMwww.Zeiss.com

Why is Confocal Microscopy Better?5. Improved Resolution

Rat Cerebellum

DisadvantagesDisadvantages

• Limited primarily to the limited number of excitation wavelengths available with common lasers, which occur over very narrow bands and are expensive to produce in the ultraviolet region.

• The harmful nature of high-intensity laser irradiation to living cells and tissues.

• The high cost of purchasing and operating multi-user confocal microscope systems.

Advantages:• + More clear• + Increase in effective

resolution.• + Bold net examination of

samples • + 3.D Reviews • + Depth measurement • + Advanced Signals • + Electronic zoom

Disadvantages:• - Image control

complicated • - Low tissue penetration • -Fading• - High-intensity laser can

damage tissue • - Expensive! • - Too many educated

people need it !!!!

Briefly…

THE END

References 1)Confocal ımaging animation.htm2)Confocal laser scanning microscopy-wikipedia the free enclopedia.htm3)Green flouresence protein.htm4)Nikon microscopy ıntroduction to confocal microscopy basic concepts.htm5) http://www.loci.wisc.edu/optical-sectioning/confocal-imaging6) http://www.crp-sante.lu/en/project/1209?template=description#immuno7) http://doube.org/3dnuclei.html#animation8) F:\chotwo01.html9) Fluoview1000_high.wmv10) http://www.hi.helsinki.fi/amu/AMU%20Cf_tut/cf_tut_part1-4.htm11) http://www.centrallab.metu.edu.tr/