Laser Scanning Microscopy

1. Objective
To image a sample of rhodamine-doped polystyrene etched with various shapes, using a Confocal Laser Scanning Microscope and to analyse the sample topography by 2D and 3D reconstruction of the sample image.
2. Introduction

This CLSM design combined the laser scanning method with the 3D detection of biological objects labeled with fluorescent markers for the first time…..laser scanning microscopy, the object or specimen surface is scanned point by point by a focused laser beam. The image or other characteristic of the object is then generated by an electronic system. In a scanning laser microscope detecting fluorescent light from the specimen, the depth-discriminating property of confocal scanning has been used to carry out optical slicing of a thick specimen. The recorded digital images constitute a three-dimensional raster covering a volume of the specimen. The specimen has been visualized in stereo and rotation by making look-through projections of the digital data in different directions.
3. Materials and Devices
A rhodamine-doped polystyrene sample etched with different shapes mounted onto a glass slide has already been prepared. A Zeiss LSM 510 META Axiovert 200M confocal microscope is used for imaging the sample.
Fig. 1 A Zeiss LSM 510 META laser scanning confocal microscope [].
4. CLSM-principles,working of the technique,
Confocal Laser Scanning Microscopy is a novel optical imaging technique that merges the technology of a Laser Scanning Microscope and a Confocal Microscope resulting in high resolution images CLSM enables to form images from selected depths from within the sample (z stacks) and generate a 3D image of a specimen by stacking 2D images from successive depths []. In this technique, a laser beam is passed through an aperture and focused by an objective lens onto a small area of the specimen. The reflected light (laser light and fluorescent light from illumination of the sample) is collected by the objective lens, and passed through a pinhole which removes ‘out of focus‘light and only allows light from the plane of focus to reach the detector which is a photo multiplier tube enabled with a filter that blocks the original laser light thus allowing the detection of the excited fluorescent wavelengths alone []. The laser beam scans the specimen point by point thus resulting in the formation of the images pixel by pixel, which can be viewed on a screen [][].
Since the scanning process in a CLSM is in a point wise fashion, in order to obtain data from, the entire sample, the specimen is moved relative to the laser beam or the laser beam is guided across the sample and CLSMs are as such also referred to as point-probing scanners [][].
The advantages of this imaging technique over other techniques are numerous such as obtaining high-resolution optical images with depth selectivity, higher level of sensitivity, less invasive form of imaging, ability to acquire in-focus images from selected depths ( a process known as optical sectioning) and thereby reconstructed with a computer to obtain three-dimensional reconstructions of samples.
For the study of the rhodamine-doped polystyrene sample, a 40x C-Apochromat water-based lens was used and the CLSM imaging specifications were set to a frame size of 512?512 pixels, the depth was set to 8 bit, scan direction was set to a single direction. By varying the pinhole diameter, the degree of confocality can be varied and here the pinhole was set to 1 Airy unit. Thus only the first order of the diffraction pattern reaches the detector while the higher orders are blocked, thus improves the resolution but also results in a slight decrease in brightness. Also the laser wavelength required to successfully image the rhodamine doped polystyrene sample is xxxxx [].
5. Principle of Imaging in 3 Dimensions
Plan how to experimentally characterise the surface topography (height of the geometric structures) 5. Preparation of sample and Imaging and Characterisation of Surface Topography with astd errors
Imaging in 3 DimensionsThis is carried out by altering the level at which the sample is observed ie by altering the plane of focus. Thus by changing this, a series of images at different positions can be produced that ps through the through the sample thickness. Thus resulting in a series of X-Y images at different Z axis positions. Therefore by optical sectioning a series of images are obtained which are then digitally reconstructed by computer softwares to give 3D representations of the sample. In this experiment, the surface topography f the rhodamine doped polystyrene sample is carried out by performing a z- stack [][]. The boundaries of the scan are set by using the focus control and marking the top and bottom of the sample. Thus between these boundaries a series of images are taken at different z- axis and a projection of the images are performed. The parameters taken for the projection are- Initial Angle of 0°, number of projections is 64 and a Difference Angle of 6 degrees.
2 D and 3 D images of a rhodamine doped polystyrene sample etched with various shapes were taken. The images show that the surface of the polystyrene sample consists of circular, triangular and square micropillars with varying heights. With the help of 3D reconstruction of the ample topography, the heights of the micropillars were calculated. A series of three z stacks were performed for each kind of micropillar (circular, square and triangular respectively) and their heights were determined.
The errors associated with the measurement of the height of each micropillar has been calculated by using standard deviation method and the following bar graphs have been plotted with the error bars denoting the standard deviation.
The images obtained from the of rhodamine-doped polystyrene
The consequences of quenching and photobleaching are suffered in practically all forms of fluorescence microscopy,
The two phenomena are distinct in that quenching is often reversible whereas photobleaching is not .
Most quenching processes act to reduce the excited state lifetime and the quantum yield of the affected fluorophore.
photobleaching (also termed fading) occurs when a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification An important class of photobleaching events is represented by events that are photodynamic, meaning they involve the interaction of the fluorophore with a combination of light and oxygen (158-161).Reactions between fluorophores and molecular oxygen permanently destroy fluorescence and yield a free radical singlet oxygen species that can chemically modify other molecules in living cell
Comparison of LSM with atomic force microscopy (AFM) in view of application to biology
Confocal Laser Scanning Microscopy (CLSM) has found tremendous application in the field of biology ranging from cell biology and genetics to microbiology and developmental biology. It allows imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 micrometers. advantages, including the ability to control depth of field, 3D reconstrucyion of images, non-invasive nature, enables study of both living and fixed specimens with enhanced clarity.. Additional advantages of scanning confocal microscopy include the ability to adjust magnification electronically by varying the area scanned by the laser without having to change objectives (zoom factor).
CLSM has the advantage of not requiring a probe to be suspended nanometers from the surface, as in an AFM for example, where the image is obtained by scanning with a fine tip over a surface.
tomic Force Microscopy (AFM) is a powerful form of scanning probe microscopy (SPM) that performs its imaging function by measuring a local property of the surface being inspected, such as its height, optical absorption, or magnetic properties. AFM employs a probe or tip that’s positioned very close to surface to get these measurements
The ability to monitor this deflection allows the AFM to create an image of the sample non-destructively even if the tip is continuously in contact with the sample. To prevent the cantilever tip from damaging the surface of the sample, it is maintained at a constant angular deflection so that the force applied by the tip on the surface is also kept constant. Contact mode imaging employs a soft cantilevered beam that has a sharp tip at its end, which is brought in contact with the surface of the sample. The force between the tip and the sample causes the cantilever to deflect in accordance with Hooke’s Law, exhibiting a spring constant th
The advantages of AFM include the following: 1) it generates true, 3-dimensional surface images; 2) it does not require special sample treatments that can result in the sample’s destruction or alteration; and 3) it does not require a vacuum environment in order to operate (it can operate in both air and liquid). On the other hand, itsdisadvantages include the following: it is slow in scanning an image
A stand-alone atomic force microscope (AFM) has been developed, which features a large scan area and which allows operation under liquid. This system was combined with a confocal laser scanning microscope (CLSM). Information about cell structures, obtained by CLSM, can be complemented with images of the cell surface obtained with the AFM.
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