Light sheetmicroscope price

More information on light-sheet microscopes can be found here: https://www.microscopyu.com/techniques/light-sheet/light-sheet-fluorescence-microscopy

Different types of sample mounting for LSFM: embryo embedded in hanging gel cylinder, plant growing in supported gel cylinder, adherent cells on glass, liquid sample in a sample bag

Light sheet fluorescence microscopyuses

The Lattice Light-sheet microscope was developed in 2014 by Eric Betzig while working at Janelia Research Campus. The original design was published in Science and is available here.

Since LSFM inherently produces a high contrast single play image, it may be utilised to image a single 2D plane at extremely high temporal resolution, limited only by the sample brightness, sensitivity and maximum frame rate of the camera, with typical speeds of 50 to 100fps at 4Mp for a sCMOS camera.  However to produce a 3D volume either the sample must be moved through the lightsheet (which is centred on the focal plane of the detection objective) or the lighsheet must be moved synchronously with the focal plane of the detection objective.  Both methods are utilised in commercial systems and there are many Pro's and Cons to each method, including disturbance of the sample and loss of detection resolution as two of the main Cons.  However the end result may produce a high contrast 3D volume anywhere from one volume a minute up to hundreds of volumes a second.

Light sheet fluorescence microscopyprice

Light sheet microscopy

LSFM has also been combined with two-photon (2P) excitation, which improves the penetration into thick and scattering samples.[17] Use of 2P excitation in near-infrared wavelengths has been used to replace 1P excitation in blue-visible wavelengths in brain imaging experiments involving response to visual stimuli.[18]

Comparison of different microscopy illumination modalities (LSFM: lightsheet fluorescence microscopy, WF: widefield microscopy, CF: confocal microscopy). LSFM combines good z-sectioning (as confocal) and only illuminates the observed plane

Light sheet microscopyvs confocal

Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high[1] optical resolution, but good optical sectioning capabilities and high speed. 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. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction (e.g. using a cylindrical lens). A second method uses a circular beam scanned in one direction to create the lightsheet. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because LSFM scans samples by using a plane of light instead of a point (as in confocal microscopy), it can acquire images at speeds 100 to 1000 times faster than those offered by point-scanning methods.

Light sheet fluorescence microscopycost

Some LSFMs have been developed where the sample is mounted as in standard microscopy (e.g. cells grow horizontally on the bottom of a petri dish) and the excitation and detection optics are constructed in an upright plane from above. This also allows combining a LSFM with a standard inverted microscope and avoids the requirement for specialized sample mounting procedures.[19][29][30][31]

In comparison to conventional laser microscopes, light sheet microscopes generally illuminate a plane (sheet) of laser light through the sample which is detected via a lens positioned perpendicular to the illumination sheet (Santi, 2011). Initially light sheet microscopes utilised cylindrical lenses to form a relatively thick sheet of light (>3 mm) that does not uniformly illuminate across a 10 mm focal point, meaning subcellular structures and dynamics cannot be resolved (Huisken et al., 2004). The LLSM overcomes this resolution issue through the generation of a structured light pattern (lattice) that allows for subcellular resolution (Heddleston and Chew, 2016; Huisken et al., 2004).

Light sheet fluorescence microscopyPDF

The separation of the illumination and detection beampaths in LSFM (except in oblique plane microscopy) creates a need for specialized sample mounting methods. To date most LSFMs are built in such a way that the illumination and detection beampath lie in a horizontal plane (see illustrations above), thus the sample is usually hanging from the top into the sample chamber or is resting on a vertical support inside the sample chamber. Several methods have been developed to mount all sorts of samples:

Illustration of different LSFM implementations. See text for details. Legend: CAM=camera, TL=tube lens, F=filter, DO=detection objective, S=sample, SC=sample chamber, PO=projection objective, CL=cylindrical lens, SM=scanning mirror

The Lattice Light Sheet Microscope (LLSM) is a custom microscope developed by Nobel Laureate Eric Betzig and released in late 2014 (Chen et al., 2014). Built upon light sheet imaging technologies, LLSM has improved sample penetration and allows for very high speed, 4-dimensional image acquisition (Chen et al., 2014). Importantly and uniquely, LLSM induces almost no photobleaching or photodamage, which means live samples can be imaged for very long periods of time (hours or even days).

The animation below depicts the objective orientation of the LLSM whereby the light-sheet extends from the Illumination objective through the sample at an angle of 31.8 degrees. The detection objective captures the single illuminated plane and projects it onto one of the two cameras.

https://caic.bio.cam.ac.uk/microscopes/light-sheet-microscopes/light-sheet/examples/comparison-single-vs-multi-photon-light-sheet-imaging