We use cookies to enhance your experience. By continuing to browse this site you agree to our use of cookies. More info.
In a study published in the journal Photonics, researchers have demonstrated a four-camera structured illumination microscope (SIM) to image various focal planes simultaneously, capturing 3D fluorescent images with no axial movement of the sample.
Study: Flexible multiplane structured illumination microscope with a four-camera detector. Image Credit: peterschreiber.media/Shutterstock.com
In a conventional microscope, visible light is used to illuminate and produce a sample's magnified images.
In contrast, fluorescence microscopy has a more intense light source exciting a fluorescent species in a sample. This fluorescent species emits a longer wavelength of lower energy, producing magnified images.
Fluorescent microscopy can visualize specific micro-specimens details and enhance 3D features at tiny scales.
Structured illumination microscopy (SIM) is one of several techniques used in fluorescence microscopy.
In structured illumination microscopy, shifting illumination patterns provide different images that can produce optical sectioning and super-resolution (resolution past diffraction limit).
Structured illumination microscopy emerged two decades ago and has evolved with several methods for generating structured illumination microscopy patterns and processing image data. Compared to spinning disc confocal microscopy, structured illumination microscopy possesses better optical sectioning capabilities, higher resolution, and less implementation cost.
High signal-to-noise ratio (SNR), speed, and low excitation light intensities make structured illumination microscopy ideal for 3D imaging of live samples.
In fluorescence microscopy, information about intensity throughout the sample is acquired and localized to a particular location in a 3D space to produce 3D images. However, with structured illumination microscopy, 3D images are traditionally created by collecting a sequence of optically sectioned images while moving the sample axially through a Z-stack. This technique produces distinguished XY slices of the sample that can be joined to create a 3D image.
While this technique is still widely used in fluorescence microscopy, it has some drawbacks, including extensive light exposure, extended acquisition time, and possible sample disturbance.
Multifocal plane microscopy is prominent among alternative approaches, in which the need for sample movement is removed by simultaneous imaging through multiple focus-shifted image planes.
This can be achieved using various methods, for instance, using an array of beam-splitters for imaging multiple focal planes on a solitary detector. This method is compatible with various microscope objectives. However, the required construction of a z-splitter assembly and image planes' separation due to the optical system is not adjustable without altering the critical dimensions of the z-splitter.
A multidetector method was first used with two cameras, where each camera placed in different focal positions relative to the microscope's tube lens could capture images at different focal planes. Other researchers extended this idea to 3D localization microscopy by increasing the number of detectors. However, the high cost of scientific cameras limits the feasibility of multicamera imaging.
This study identifies the adverse effects of focal plane multiplexing in a 2D structured illumination microscopy system and offers techniques to tackle these issues.
Putting the detection plane's positions defocused from the illumination plane of a 2D structured illumination microscopy pattern complicates the usability of multicamera imaging. The increased defocusing of the detection planes causes the contrast of high-spatial-frequency illumination patterns to deteriorate rapidly.
Increased defocus distances may also produce unwanted artifacts, such as fluctuating magnification and defocus distances dependent on the refractive index of the medium.
The researchers demonstrated that four distinguished imaging modes are achievable through a four-camera detection system using the structured illumination microscopy method.
The study establishes the technique in which no physical manipulation of the optical system is required for switching from one mode to another, allowing versatility in imaging since different samples need specific modes for the best images.
This also provides the best solution for moving live cells by taking a sequence of 3D images over time and producing a 3D movie.
The researchers imaged mitochondrial motions in living cells, neuronal structure in Drosophila larvae, and up to 130 m-deep in mouse brain tissue.
A Z-stack of 3D images is acquired for static samples with a substantial axial extent, producing a high-axial-resolution 3D image while simultaneously reducing the amount of sample movement by four times.
A mosaic of 3D images is taken for a large sample, producing images with a large field of view (FOV) and valuable axial information. The same setup can also perform multicolor imaging.
Structured illumination microscopy processing significantly improved the cameras' resolution from 357 nm to 253 nm when using a 30 /1.05 NA objective. The researchers have also provided an open-source software platform for image processing for all these imaging modes, along with MATLAB and ImageJ tools.
Johnson, K. A., Noble, D., Machado, R., & Hagen, G. M. (2022). Flexible multiplane structured illumination microscope with a four-camera detector. Photonics. https://www.mdpi.com/2304-6732/9/7/501
Disclaimer: The views expressed here are those of the author expressed in their private capacity and do not necessarily represent the views of AZoM.com Limited T/A AZoNetwork the owner and operator of this website. This disclaimer forms part of the Terms and conditions of use of this website.
Taha graduated from HITEC University Taxila with a Bachelors in Mechanical Engineering. During his studies, he worked on several research projects related to Mechanics of Materials, Machine Design, Heat and Mass Transfer, and Robotics. After graduating, Taha worked as a Research Executive for 2 years at an IT company (Immentia). He has also worked as a freelance content creator at Lancerhop. In the meantime, Taha did his NEBOSH IGC certification and expanded his career opportunities.
Please use one of the following formats to cite this article in your essay, paper or report:
Khan, Taha. (2022, July 22). Seeing Biological Samples Clearly with a Four-Camera Structured Illumination Microscope. AZoOptics. Retrieved on July 22, 2022 from https://www.azooptics.com/News.aspx?newsID=27708.
Khan, Taha. "Seeing Biological Samples Clearly with a Four-Camera Structured Illumination Microscope". AZoOptics. 22 July 2022. <https://www.azooptics.com/News.aspx?newsID=27708>.
Khan, Taha. "Seeing Biological Samples Clearly with a Four-Camera Structured Illumination Microscope". AZoOptics. https://www.azooptics.com/News.aspx?newsID=27708. (accessed July 22, 2022).
Khan, Taha. 2022. Seeing Biological Samples Clearly with a Four-Camera Structured Illumination Microscope. AZoOptics, viewed 22 July 2022, https://www.azooptics.com/News.aspx?newsID=27708.
Do you have a review, update or anything you would like to add to this news story?
The Thermo Scientific™ ARL™ EQUINOX 3000 X-ray Diffractometer for research enables accurate measurements.
KLA’s Filmetrics F40 allows you to transform your benchtop microscope into an instrument to measure thickness and refractive index.
This product profile describes the properties and applications of the ProMetric® I-SC Solution Imaging Colorimeter.
We spoke with University of Bonn spin-off Midel Photonics, a start-up company whose laser beam shaping technology is hoping to sharpen up the laser industry.
Following Laser World of Photonics 2022, we spoke with Matthias Sachsenhauser from Hamamatsu Photonics about the role of laser-driven light sources in the future of the photonics sector.
AZoOptics speaks to Dr. Keith Paulsen about the importance of breast cancer detection and the introduction of his team's deep-learning algorithm that associates spatial images of tissue optical properties with optical signal patterns measured during an imaging experiment or patient exam.
AZoOptics.com - An AZoNetwork Site
Owned and operated by AZoNetwork, © 2000-2022