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Function ofstagein microscope
In addition to complicated sample preparation procedures, imaging the fluorescent proteins within small animals requires specialized instrumentation consisting of an excitation light source, a high-performance camera, emission filters, and associated software.
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High powerobjective microscope function
Analytik Jena US. 2020. In Vivo Fluorescence Imaging with GFP and RFP. News-Medical, viewed 22 November 2024, https://www.news-medical.net/whitepaper/20200930/In-Vivo-Fluorescence-Imaging-with-GFP-and-RFP.aspx.
The UVP GelSolo was employed to image a fluorescent silicone mouse embedded with both red and green round plastic fluorescent rods. The fluorescent rods were selected because red fluorescent protein (RFP) and green fluorescent protein (GFP) are frequently used for in vivo imaging.
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Analytik Jena is a provider of instruments and products in the areas of analytical measuring technology and life science. Its portfolio includes the most modern analytical technology and complete systems for bioanalytical applications in the life science area.
This article will show how in vivo fluorescent imaging of a typical mouse model employed in research labs is carried out, substituting a fluorescent silicone mouse.
Stage clipsmicroscope function
The fast and simple tracking of primary and metastatic cancer development research in live animals is enabled within vivo imaging using fluorescent proteins. Discovered decades ago in the jellyfish Aequorea victoria as a primary feature of bioluminescence1, a green fluorescent protein (GFP) is a commonly utilized fluorescent dye for in vivo imaging.
The UVP GelSolo imager was used to image the silicone mouse. Selections for exposure time, light sources, and emission filter are shown in Table 1.
School laboratories can utilize these instruments for teaching purposes and can use this article to supplement STEM training, particularly students aiming for advanced degrees and research careers.
With more than 25 years of market experience, Analytik Jena with its CyBio® Product Line is a leading provider for high quality liquid handling and automation technologies. In the pharmaceutical and life science industries, our products enjoy the highest reputation for precision, reliability, robustness and simplicity.
The silicone mouse and the individual GFP and RFP signals are clearly detected with the UVP GelSolo imaging system, as demonstrated in Figure 2a-c. Figure 2d shows the location of each signal in a composite image.
Usually, depending on the spectrum of the fluorescent protein as shown in Figure 2b and c, the fluorescent signal will be pseudo-colored. Compositing the colored fluorescent images with a white light image helps to locate the fluorescent signals on the animal.
An easy-to-use entry-level imager, the UVP GelSolo, is an ideal option for multi-user laboratories, school laboratories, and practical training where users may not have access to relevant samples or experience with in vivo imaging.
The intensity of the signal can be analyzed using the Area Density feature in the software for a more in-depth analysis. As an easy-to-use system, the UVP GelSolo is designed for imaging and analysis without the requirement for any additional training.
Analytik Jena has a long tradition in developing high-performance precision analytical systems which dates back to the inventions made by Ernst Abbe and Carl Zeiss. We have grown to become one of the most innovative manufacturers of analytical measuring technology worldwide.
The structure of the objective lens is complicated and the production is precise. It is usually composed of lens groups, and the lenses are separated from each other by a certain distance to reduce the phase difference. Each group of lenses is made of one or several lenses of different materials and parameters. The objective lens has many specific requirements, such as coaxial and parfocal. The modern microscope objective lens has reached a high degree of perfection, its numerical aperture is close to the limit, and the difference between the resolution at the center of the field of view and the theoretical value is negligible. However, the possibility of continuing to increase the field of view of the microscope objective lens and improving the imaging quality at the edge of the field of view still exists, and this kind of research work is still in progress. Parfocality means that during the microscope inspection, when the image is observed clearly with an objective lens of a certain magnification, when the objective lens of another magnification is switched, the image should be basically clear, and the center deviation of the image should also be within a certain range. , that is, the degree of alignment. The pros and cons of parfocal performance and the degree of coaxiality are an important indicator of the quality of the microscope, which is related to the quality of the objective lens itself and the precision of the objective lens converter.
Analytik Jena US. "In Vivo Fluorescence Imaging with GFP and RFP". News-Medical. https://www.news-medical.net/whitepaper/20200930/In-Vivo-Fluorescence-Imaging-with-GFP-and-RFP.aspx. (accessed November 22, 2024).
Application of in vivo imaging can be seen over a multitude of disciplines from medicine and drug discovery to cell biology and physiology. Yet, there is usually extensive training and specific experience involved working with live animals when preparing in vivo samples with fluorescent-labeled proteins.
Aberrations related to wide beams are spherical aberration, coma, and positional chromatic aberration; aberrations related to field of view are astigmatism, field curvature, distortion, and magnification envelope aberration. The difference between the microscope objective lens and the eyepiece lens is that it participates in imaging. The objective lens is the most complex and important part of the microscope, working in wide beams (large aperture), but the inclination angle of these beams to the optical axis is small (viewing angle small field); the eyepiece works in a narrow beam, but its inclination angle is large (large field of view). When calculating the objective lens and eyepiece, there is a big difference in eliminating aberrations.
The emission filter blocks unwanted wavelengths and permits only the chosen wavelengths to pass through, as seen in Figure 2b and c. The RFP emission filter permits signals with 575 nm to 640 nm to pass through and the GFP emission filter permits fluorescent signals with wavelengths of 510 nm to 523 nm to pass through.
Analytik Jena US. (2020, September 30). In Vivo Fluorescence Imaging with GFP and RFP. News-Medical. Retrieved on November 22, 2024 from https://www.news-medical.net/whitepaper/20200930/In-Vivo-Fluorescence-Imaging-with-GFP-and-RFP.aspx.
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The enhanced properties and unique characteristics of fluorescent proteins have since permitted the non-invasive visualization of dynamic processes and structural organization in living cells and organisms by in vivo imaging2. This technology enables subjects to be monitored longitudinally over the course of a study to supply real-time information on internal changes.
What isobjective lens in microscope
With a peak wavelength at 450nm, the Blue LED light source is a commonly employed excitation source for green and red fluorescent protein labels utilized for in vivo imaging.
The results exhibit the distinct imaging of fluorescent signals within an organism in an easily reproducible and non-invasive manner. In addition to the in vivo image acquisition method, researchers and students will also be able to learn post-processing for captured in vivo images.
Analytik Jena US. "In Vivo Fluorescence Imaging with GFP and RFP". News-Medical. 22 November 2024. .
Students at non-research institutions who are interested in learning in vivo imaging but might not have access to animal models can use the UVP GelSolo to practice the in vivo imaging method.
The Life Science product area demonstrates the biotechnological competence of Analytik Jena AG. We provide a wide product spectrum for automated total, as well as individual solutions for molecular diagnostics. Our products are focused to offer you a quality and the reproducibility of your laboratory results.
In order to practice in vivo imaging methods and get a taste of how in vivo imaging works in a laboratory setting, researchers are quickly able to work with the UVP GelSolo and a silicone mouse sample.
Function ofcondenserin microscope
The straightforward features and user-friendly automation make the UVP GelSolo an ideal entry-level imager for multi-user laboratories, school laboratories, and practical training.
The emission filter for GFP has been selected as 510 nm to 523 nm with a peak wavelength of 513 nm. The emission filter designed for RFP has a wavelength range of 575 nm to 640 nm and a peak wavelength of 605 nm.
Our business unit Analytical Instrumentation offers excellent competencies in the fields of optical spectroscopy, sum parameters and elemental analysis. Being proud of our core competency we grant all our customers a long-term warranty of 10 years for our high-performance optics.
This will surely ease your daily work and speed up your work processes in a certain way. All together we support you through the complete process of the lab work. Besides we offer customized solutions and are able to adapt our products to your needs. Automated high-throughput screening systems for the pharmaceutical sector are also part of this segment’s extensive portfolio.
These requirements for acquiring images and preparing samples largely limits opportunities for entry-level researchers or students to participate in research involving in vivo applications.
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Upon determining its novel utility for fluorescence labeling in other living organisms, the development of new molecules has expanded the range of available fluorescent proteins to encompass almost the complete visible spectrum from 350 nm (UV) to 750 nm (Infrared).
Figure 1 shows their spectral profiles, blue LED Light, having a wavelength of 445 nm to 470 nm and a peak wavelength near 456 nm, which can excite these fluorescent proteins which lie in the GFP excitation spectrum.
Objective lensmagnification
Typesof objectivelenses
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High powerobjective lens
These images confirm that the emission filter blocks unwanted signals efficiently and provide a clean image. However, it is difficult to establish where the signal is located. In this instance, the compositing and pseudocolor functions in the software become useful.
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Two pieces of 1 cm red round plastic fluorescent rods were inserted into the head of the silicone mouse and two pieces of 1 cm green-yellow round plastic fluorescent rods were inserted into the back of the mouse before the silicone model was completely solidified to visually mimic tumors. The silicone rubber mouse then set for four hours and was subsequently imaged.
The objective lens is the most important optical component of the microscope. It uses light to make the object under inspection image for the first time. Therefore, it directly affects and affects the quality of the image and various optical technical parameters. It is the primary standard to measure the quality of a microscope.
The microscope objective lens is an aplanatic system. This means that: in terms of a pair of conjugate points on the axis, when the spherical aberration is eliminated and the sinusoidal condition is realized, each objective lens has only two such aplanatic points. Therefore, any change in the calculated positions of objects and images results in larger aberrations. Installed on the rotator at the lower end of the lens barrel, there are generally 3-4 objective lenses, among which the shortest one engraved with the symbol "10×" is a low-magnification lens, and the longer one engraved with the symbol "40×" is a high-power lens. The long one engraved with the "100×" symbol is an oil lens. In addition, a circle of lines of different colors is often added to the high-power lens and the oil lens to show the difference.
Figure 2. a) White image of the silicone mouse. b) GFP signal in the silicone mouse. c) RFP signal in the silicone mouse. d) Composited white channel, GFP channel, and RFP channel to help visualize the GFP and RFP signal.
The mouse model which was utilized for this experiment is created from colored silicone rubber. In order to mimic the look of the true nude mouse model commonly used in fluorescent in vivo imaging white and red and acrylic paints were applied.
Equipped with a 5.0-megapixel camera, UV transilluminator, 8-48mm f/1.2 manual zoom lens, white and blue overhead LED light sources, the UVP GelSolo will be employed to image the silicone mouse.