ZEISS Apotome 3

Optical sectioning in fluorescence imaging for your widefield microscope

Create optical sections of your fluorescent samples - with structured Illumination, removal of out-of-focus light becomes simple and efficient, allowing you to fully focus on your research. ZEISS Apotome 3 recognizes the magnification and moves the appropriate grid into the beampath. The system then calculates your optical section from a number of images with different grid positions. It’s a totally reliable way to remove out-of-focus light, even in thicker specimens. Yet your system remains just as easy to operate as always. You get images with high contrast in the best possible resolution – simply brilliant optical sections.

Brilliant Optical Sections: Apotome 3 comes with three grids of different geometries, giving you the best resolution no matter which magnification you choose.
Free Choice of Light Source and Dyes: Apotome 3 adapts to your fluorophores and lights source. So you stay flexible when your experiments evolve in complexity and requirements.

More Structural Information: Your created images are improved even more by deconvolution, using a patented algorithm for structured illumination. Better recognize important structures of your examined objects.

Highlights

Brilliant Optical Sections

To image structures of sizes ranging from hundreds of micrometers to the nanometer range, you typically use objectives with different magnifications. Apotome 3 comes with three grids of different geometries, giving you the best resolution for each objective. You can fully focus on your experiment as the ideal grid is automatically selected, always resulting in high-contrast optical sections. Apotome 3 significantly increases the axial resolution compared to conventional fluorescence microscopy: you obtain brilliant optical sections that allow 3D-rendering, even from thick specimens.

Free Choice of Light Source and Dyes

Your experiments often evolve over time in complexity and requirements. That’s why you need equipment which is not only performant but also flexible. Use Apotome 3 with conventional metal halide lamps, economic white light LEDs, or the gentle, multi-color Colibri illumination system. Simply change the filter and the system automatically moves the grid into the correct position. It’s your decision, not the technology’s: Whether you work with DAPI, Alexa488, Rhodamin, Cy5, or with vital dyes such as GFP or mCherry – Apotome 3 adapts to your fluorophores and light source, creating the sharp and brilliant images you expect.

Courtesy of L. Behrendt, Leibniz-Institute on Aging – Fritz-Lipmann-Institut e.V. (FLI), Germany.

More Structural Information

Courtesy of L. Behrendt, Leibniz-Institute on Aging – Fritz-Lipmann-Institut e.V. (FLI), Germany.

Improve the images you created with Apotome 3 even more by deconvolution, using a patented algorithm for structured illumination. While retaining all raw data, the system allows you to switch between widefield, optical section and deconvolved images for maximum flexibility and best comparability. The fast and robust deconvolution algorithms are easy to use and improve both lateral and axial resolution of your images. Thanks to the improved contrast, higher optical resolution and suppression of existing noise, you can better recognize the structure of the examined objects.

Working Principle

Three Grids for Optimal Optical Section Thickness

Emission light from areas outside of the focal plane is detected by your camera. Contrast and resolution are reduced, depending on the thickness or the volume of the specimen (Figure A: Acquisition with conventional epifluorescence illumination).

No matter which magnification you are using – Apotome 3 automatically places the optimum grid in the beampath of your microscope. Reduction of unwanted background fluorescence increases with the grid frequency and the optical sections become thinner. Image information from outside of the focal plane is suppressed (Figure B, C, and D). This improves contrast and resolution of the optical section. “Low grid” delivers the optimal section thickness in our example (Figure D). Images of this type are particularly suitable for 3D analyses and the processing of your image data with rendering software.

ZEISS Apotome 3 - Optimal Optical Section Thickness

C. elegans, whole mount, green: GFP, blue: DAPI Objective: Plan-Apochromat 20 ×/0.8 Courtesy of Prof. Schnabel, T.U. Braunschweig, Germany.

Scanning Mechanism

Apotome 3 projects a grid structure into the focal plane of your specimen, then moves it into different positions using a scanning mechanism. At each grid position, Apotome 3 auto matically acquires a digital image. The system processes all images into one optical section with improved contrast and increased resolution using a patented algorithm. The resulting image is free from grid structures.

Fluorescence excitation light passes through two glass plates in the Apotome 3 slider. When a grid structure is applied to the first glass plate, the grid pattern is “imprinted” in the excitation light. A scanning mechanism tilts the second glass plate and the image of the grid is laterally shifted in the focal plane of the specimen.

Schematic illustration of the grid projection. A: Widefield image. B – D: Raw images with different positions of the grid. E: Resulting optical section through the sample. Out of focus light is efficiently removed by the structured illumination (arrow).

Typical Applications

Application	Task	ZEISS Apotome 3 Function
Cell Culture	2D imaging	2D single images
	Fast imaging of a 2D image	Optical section available online on the monitor
	Reliable detection of the marker even with strong background fluorescence	Automatic grid selection for optimum contrast with each objective
	Combination of multiple contrast techniques	Any combination of fluorescence channels, brightfield, DIC and phase contrast Individual configuration of each fluorescence channel as an optical section or widefield image
Live Cell Imaging	Reduction of phototoxicity	Particularly low phototoxicity in combination with LED illumination and high sensitvie cameras like ZEISS Axiocams
Live Cell Imaging	Time-lapse images	Depending on the exposure time, up to three images per second Doubling of the frame rate with “burst mode”
Vibratome Sections, Histological Samples	3D imaging	Automatic selection of the optimum grid for each objective
	Modification of the optical section thickness	Grid freely selectable depending on the specimen
	Penetration depth	Depending on the optical density of the tissue
	3D reconstruction	Rendering of the image stack via integrated software function Automatic transfer of the parameters of the individual fluorescence channels
	Quantitative analysis	Reproducible size measurements through automatic system calibration
Whole Mounts	3D imaging	Multi Channel, Z Stack and Time Lapse, Deconvolution, images in raw data mode, 3D Rendering
Whole Mounts	Large image areas	Automatic acquisition of large sections using Tiles & Positions

ZEISS Apotome 3 at Work

Drosophila Neurons

Molecular and Developmental Genetics, University of Leuven, Belgium

Drosophila neurons, blue: DAPI, yellow: GFP. Objective: Plan-Apochromat 20×/0.8. Courtesy of M. Koch, Molecular and Developmental Genetics, University of Leuven, Belgium.

Left: Conventional Fluorescence. Right: ZEISS Apotome 3

Drosophila Embryo

Institute for Neurobiology, University of Münster, Germany

Drosophila embryo, green: HRP, red: glia marker, 100 µm Z-stack. Courtesy of C. Klämbt, Institute for Neurobiology, University of Münster, Germany.

Mouse Embryo

Centre for Anatomy, University of Göttingen, Germany

Mouse embryo, tissue section, green: GFP, red: Cy3. Objective: Plan Apochromat 40×/1.3 Oil. Courtesy of N. Büttner, T. Vogel, Centre for Anatomy, University of Göttingen, Germany.

Cortical Neurons

Leibniz-Institute on Aging – Fritz-Lipmann-Institut e.V. (FLI), Germany

Comparison of a widefield image and 3D rendering of cortical neurons stained for DNA and microtubules. Courtesy of L. Behrendt, Leibniz-Institute on Aging – Fritz-Lipmann-Institut e.V. (FLI), Germany.

Widefield

ZEISS Apotome 3

Widefield

ZEISS Apotome 3

Lotus Japonicus Root Infected with Symbiotic Bacteria

University of Freiburg, Germany

Autofluorescence of a Lotus Japonicus root infected with symbiotic bacteria stained with mcherry. Courtesy of F. A. Ditengou, University of Freiburg, Germany.

Widefield

Apotome 3

Apotome 3 + Deconvolution

Transgenic Zebrafish Larvae

Leibniz-Institute on Aging – Fritz-Lipmann-Institut e.V. (FLI), Germany

Transgenic zebrafish larvae at 4 days post fertilization staining for: Glial fibrillary acidic protein, acetylated Tubulin, GFP and DNA. Embedded in 1.2% low melt agarose. Courtesy of H. Reuter, Leibniz-Institute on Aging – Fritz-Lipmann-Institut e.V. (FLI), Germany.

Widefield

Apotome 3

Apotome 3 + Deconvolution

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ZEISS Apotome 3