Oblique Single Plane Illumination Microscopy (oSPIM)

Author: Dan Croucher, Applications Team Manager, Photometrics


Conventional fluorescence microscopy uses high intensity light to illuminate the sample but this excites all fluorophores in the light path, not just the plane of interest. The result is that light emitted from outside the focal plane contributes to the image. Confocal microscopy overcomes this problem by using pinholes to selectively collect light only from the plane of interest. However, high intensity light still penetrates through the entire sample which causes photobleaching and photodamage.

Light Sheet Fluorescence Microscopy (LSFM) or Selective Plane Illumination Microscopy (SPIM) only illuminates the plane of interest which allows us to collect information from a single plane while also minimizing photobleaching and photodamage to the rest of the sample. By eliminating out-of-focus light in this way, lower light intensities can be used to excite the sample which further contributes to the reduction in photobleaching and photodamage, allowing us to image for extended periods of time. More exposures can therefore be taken using LSFM than any other form of fluorescence microscopy.

Conventional LSFM is performed with two objectives oriented orthogonally to each other so that one objective introduces the light sheet and the other detects the fluorescence signal. However, this orthogonal orientation requires the detection objective to be placed slightly away from the sample to prevent the two objectives colliding in space. Therefore, a long working distance detection objective is necessary which means that high NA, oil-immersion objectives are incompatible with the conventional LSFM design.

This presents a problem for the detection of cellular or subcellular structures which require a high NA detection objective for the superior resolution and light collection efficiency.

Oblique Single Plane Illumination Microscopy (oSPIM) overcomes this problem by changing the objective orthogonal orientation and instead, generates the light sheet at an oblique angle using an oil-immersion objective below the sample. A high NA, water dipping, detection objective sits above the sample and is tilted 60 degrees perpendicular to the light sheet which also provides an ideal geometry for cell and tissue culture samples.

oSPIM Principle

oSPIM is a platform for high resolution light sheet microscopy combining the low photobleaching and photodamage of LSFM with high magnification, high NA objectives for cellular and subcellular imaging. It’s currently manufactured and distributed by Applied Scientific Instrumentation (ASI).

Figure 1

Figure 1. ASI oSPIM implementation. Left: oSPIM objective geometry, Right: oSPIM implemented on the frame

The light sheet is formed by the illumination objective below the sample which generates the light sheet at an oblique angle, 30 degrees from the lens. The water dipping detection objective is tilted 60 degrees from the illumination objective, resulting in a 90 degree angle between the light sheet and detection objective. This provides the perfect arrangement for positioning a 35 mm or larger glass-bottom dish to hold the sample. By positioning the objectives in this orientation instead of orthogonally, the issue of high NA objectives colliding in space is overcome.

The high NA oil-immersion objective below the sample can also be used for conventional fluorescence imaging such as widefield, confocal or TIRF. In this way, the oSPIM system is two microscopes in one, combining high resolution fluorescence microscopy with high resolution light sheet fluorescence microscopy in one system.

The oSPIM does not give isotropic resolution as the light sheet is only introduced from one side. However, a modified version of the oSPIM has been created to replace the illumination objective with a tilted objective below the sample, providing dual-view oblique SPIM (doSPIM).

doSPIM Principle

Figure 2

Figure 2. ASI doSPIM implementation. Left: doSPIM objective geometry, Right: doSPIM implemented on the frame

In doSPIM, the objectives are tilted at an angle greater than 90 degrees from each other. This ensures that we can take advantage of the high NA without the objectives colliding in space. Like in oSPIM, the light sheet is generated at an oblique angle, 30 degrees from the lens. However, the light sheet can now be generated through both objectives sequentially.

doSPIM is similar in operation to diSPIM where the objectives alternate between illumination and detection. For the first frame of data, the bottom objective generates the light sheet and the top objective detects the fluorescence signal, then for the second frame of data, the top objective generates the light sheet and the bottom objective detects the fluorescence signal. This cycling of illumination/detection allows us to generate an image from two sides of the sample simultaneously, providing isotropic resolution.

The big advantage of doSPIM is that it’s capable of providing isotropic resolution using high NA objectives for cellular and subcellular imaging which isn’t possible with conventional LSFM. However, the ability to use the system for conventional fluorescence microscopy is lost as this is not compatible with the tilted bottom objective.

oSPIM and doSPIM Advantages

The primary advantage of oSPIM and doSPIM is applying the benefits of LSFM to imaging cellular and subcellular structures. Conventional and confocal microscopy are invaluable tools for cell biology but with light sheet microscopy, far longer acquisitions are possible due to greatly reduced photobleaching and photodamage. Higher NA objectives are also more light efficient than conventional LSFM objectives which means that an even lower light intensity can be used, further reducing photobleaching and photodamage. This allows cell biologists to follow processes over much longer time scales than previously possible.

The system is a standalone microscope which means that it isn’t necessary to have an existing microscope to use it. Neither does the system require any special camera modes or extra features, making the system very simple to implement. The oSPIM has the added advantage of being able to perform conventional widefield, confocal and TIRF microscopy, increasing the versatility of the system. However, if isotropic resolution is more important, the doSPIM is an attractive alternative for high resolution light sheet microscopy.

oSPIM and doSPIM Camera Choice

Any camera can be used with oSPIM and doSPIM but we believe that the best performance can be achieved with the 95% quantum efficient, back-illuminated Scientific CMOS camera, the Photometrics Prime 95B.

The almost perfect, 95% quantum efficient (QE) sensor allows light intensity to be reduced even further while maintaining a high level of signal detection. This is ideal for samples that need to be monitored over very long time scales without photobleaching as well as samples that are particularly sensitive to photodamage. Compared to standard sCMOS devices, the exposure time on the Prime 95B could be reduced by up to four times and still give equivalent detection.

The Prime 95B also has the large field of view (18.66 mm diagonal) and high speed (82 fps, full frame) expected of CMOS devices. This allows for large samples to be imaged without requiring any stitching. If a larger field of view is required, a version of the Prime 95B with a 25mm diagonal is also available.

The large, 11x11 µm pixels provide additional sensitivity and have a large 80,000 e- full well capacity with a low 1.6 e- read noise, giving the 95B a very high dynamic range, ideal for performing high contrast imaging. Larger pixels also fit perfectly with high magnification objectives, achieving Nyquist sampling without the need for any additional optics with 100x magnification.

The Prime 95B fits all functions of the oSPIM and doSPIM; high resolution light sheet microscopy can be performed with even lower exposure times and it’s also the perfect camera for confocal and TIRF microscopy to take advantage of the extra functionality of the oSPIM design.

We would encourage anyone considering using oSPIM and doSPIM to request a demonstration of the Prime 95B to see the advantages it provides.