What is Fluorescence Microscopy?
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.
What is Structured Illumination Microscopy (SIM)?
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.
How 3D Images are Produced in Conventional Structured Illumination Microscopy (SIM)
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.
Alternatives to Conventional Structured Illumination Microscopy (SIM) Technique
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.
Detection Planes Positions Defocused from the Illumination Plane
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.
What is Being Achieved by this Study?
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