FIELD OF THE INVENTION

The present disclosure relates to microscopy, specifically selective plane illumination microscopy.

BACKGROUND OF THE DISCLOSURE

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Selective plane illumination microscopy (SPIM) is suitable for fast, three-dimensional imaging. By confining the excitation light to a sheet, SPIM combines axial sectioning capability with minimal light exposure and fast, camera-based image acquisition. SPIM typically uses two (objective) lenses arranged perpendicular to each other. One lens is used for light detection, while the focal plane of that lens is illuminated with a sheet of light generated via the other lens. To generate the light sheet, cylindrical optics can be used. Alternatively, the beam can be rapidly scanned across the field of view of the detection lens to generate the sheet illumination. However, the arrangement of two objective lenses perpendicular to each other provides a number of challenges in terms of instrument design and sample geometry. For example, SPIM requires specific sample preparation, typically embedding the sample in a hydrogel such as agarose. This excludes the use of conventional sample mounts, such as coverslips, culture dishes and multi well plates.

To overcome this limitation, a popular approach is to dip into the sample container from the top, with both lenses typically at a 45° angle with respect to the sample plane. Such a system can be mounted on top of an inverted microscope or implemented as an independent instrument. The drawbacks of this geometry include the requirement of a large sample container to accommodate both lenses resulting in a large immersion volume. This can cause sample disturbance due to flow/convection and increases the amount of reagents needed. Further, there is no isolation of optics and sample which is problematic when dealing with hazardous samples (toxic, cancerous, infectious, etc.). Finally, since the observation plane is at an angle with respect to the sample container, the field of view for flat samples, such as a monolayer of cells, is limited, i.e., the full field of view of the detector cannot be utilized.

Thus there remains a need in the art for new devices and methods for devices and apparatuses with improved imaging capabilities.

SUMMARY OF THE DISCLOSURE

Various embodiments include a device comprising a side illumination unit, a two window sample chamber, and a refractive index matching. In another embodiment, the side illumination unit comprises components to generate a light sheet illuminating on a sample. In another embodiment, the two window sample chamber comprises two optically transparent windows perpendicular to each other. In another embodiment, the two window sample chamber further comprises a magnetic attachment to ensure easy to handle, stable, and reproducible mounting of a sample. In another embodiment, the refractive index matching comprises raising a sample in the two window sample chamber using an optically transparent material with a refractive index identical to the sample immersion fluid. In another embodiment, the refractive index matching allows imaging of flat samples such as a monolayer of cells. In another embodiment, the device is coupled with a microfluidic device. In another embodiment, the device is coupled with high throughput 3D imaging of multiple samples.

Other embodiments include a imaging apparatus comprising a microscope, and a device comprising a side illumination unit, a two window sample chamber, and a refractive index matching. In another embodiment, the apparatus is capable of imaging thick samples, such as cells, tissues, or small organisms embedded in a hydrogel. In another embodiment, the apparatus is capable of imaging a monolayer of cells. In another embodiment, the imaging apparatus further comprises a fluorescence lifetime measurement capability. In another embodiment, the side illumination unit comprises components to generate a light sheet illuminating on a sample. In another embodiment, the two window sample chamber comprises two optically transparent windows perpendicular to each other. In another embodiment, the two window sample chamber further comprises a magnetic attachment to ensure easy to handle, stable, and reproducible mounting of a sample. In another embodiment, the refractive index matching comprises raising a sample in the two window sample chamber using an optically transparent material with a refractive index identical to the sample immersion fluid. In another embodiment, the refractive index matching allows imaging of flat samples such as a monolayer of cells. In another embodiment, the observation plane is parallel to the sample surface, maximizing field of view for flat samples. In another embodiment, the microscope is an inverted or standard research microscope. In another embodiment, the imaging apparatus is coupled with a microfluidic device. In another embodiment, the imaging apparatus is coupled with high throughput 3D imaging of multiple samples.

Other embodiments include a method of imaging a sample comprising providing an apparatus comprising a microscope and a device comprising a side illumination unit, a two window sample chamber, and a refractive index matching, and imaging the sample in the apparatus. In another embodiment, the sample is a thick sample, such as cells, tissues, or small organisms embedded in a hydrogel. In another embodiment, the sample is a flat sample, such as a monolayer of cells. In another embodiment, no dipping into a container containing the sample is required to image the sample. In another embodiment, the imaging may be a video imaging. In another embodiment, the video image maps diffusion of molecular domains. In another embodiment, the imaging is a high throughput three-dimensional time course imaging.

Various embodiments include a method of improving an imaging device, comprising providing an imaging device, and illuminating a sample of the imaging device from the side with an accessory. In another embodiment, the imaging device is a microscope. In another embodiment, the imaging device is a standard research or regular inverted microscope. In another embodiment, the accessory comprises a side illumination unit. In another embodiment, the accessory comprises a side illumination unit, a two window sample chamber, and a refractive index matching.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.

