Light sheet fluorescence microscopy

In contrast to epifluorescence microscopy only a thin slice (usually a few hundred nanometers to a few micrometers) of the sample is illuminated perpendicularly to the direction of observation.

Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy.

Some studies also use a selective plane illumination microscope to image only one slice of the sample, but at much higher temporal resolution.

[24] As the illumination typically penetrates the sample from one side, obstacles lying in the way of the lightsheet can disturb its quality by scattering and/or absorbing the light.

If parts of the samples have a significantly higher refractive index (e.g. lipid vesicles in cells), they can also lead to a focussing effect resulting in bright stripes behind these structures.

That means that the lightsheet's direction of incidence is changed rapidly (~1 kHz rate) by a few degrees (~10°), so light also hits the regions behind the obstacles.

[8] Alternatively, the Variational Stationary Noise Remover (VSNR) algorithm has been developed and is available as a free Fiji plugin.

This setup allowed the observation of particles with sizes smaller than the microscope's resolution and led to a Nobel prize for Zsigmondy in 1925.

[5] After this publication by Huisken et al. the technique found wide application and is still adapted to new measurement situations today (see above).

[39] During 2012 also open source projects have started to appear that freely publish complete construction plans for light sheet fluorescence microscopes and also the required software suites.

[40][41][42][43] Selective plane illumination microscopy/light sheet fluorescence microscopy is often used in developmental biology, where it enables long-time (several days) observations of embryonic development (even with full lineage tree reconstruction).

[20][21] Strongly scattering biological tissue such as brain or kidney has to be chemically fixed and cleared before it can be imaged in a selective plane illumination microscope.

Depending on the index of refraction of the cleared sample, matching immersion fluids and special long-distance objectives must be used during imaging.