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EP-4271978-B1 - MICROSCOPE WITH SPATIAL IMAGING AND BEAM HOMOGENIZER

EP4271978B1EP 4271978 B1EP4271978 B1EP 4271978B1EP-4271978-B1

Inventors

  • WERLEY, CHRISTOPHER
  • LU, YANG
  • MOHAN, ARVIND
  • LIU, Pin
  • DEMPSEY, GRAHAM T.
  • BREMMER, NATE
  • AMAR, WILLIAM
  • ZHANG, Hongkang

Dates

Publication Date
20260506
Application Date
20211228

Claims (12)

  1. A microscope comprising: a stage configured to hold a multi-well plate; a light source for emitting a beam of light mounted within the microscope; and an optical system that directs the beam towards the stage from beneath, wherein the optical system comprises a homogenizer for spatially homogenizing the beam, characterized in that the optical system further comprises an opaque screen having a plurality of apertures, the screen being positionable to select an aperture for beam passage, and the homogenizer comprises two microlens arrays, and the optical system includes microlens array position stops for fixing a distance between the two microlens arrays at predetermined spacings to thereby shape the beam to match each of the apertures.
  2. The microscope of claim 1, wherein the stage comprises a motorized xy translational stage.
  3. The microscope of claim 2, further comprising a control system comprising memory connected to a processor operable to move the translational stage to position individual wells of the multi-well plate in the path of the beam.
  4. The microscope of claim 1, wherein the optical system includes a prism immediately beneath the stage, whereby the beam enters a side of the prism and passes into a well of the plate.
  5. The microscope of claim 4, wherein when a well of the plate containing an aqueous sample is positioned above the prism, the prism directs the beam into the sample at angle that avoids total internal reflection within the bottom of the plate.
  6. The microscope of claim 4, wherein when a well of the plate containing an aqueous sample is positioned above the prism, the prism directs the beam into the aqueous sample at an angle of refraction that restricts light to about the bottom 10 microns of the well.
  7. The microscope of claim 1, comprising at least three light sources for emitting three beams at three distinct wavelengths, wherein the optical system comprises one or more dichroic mirrors to join the three beams in space and pass the three beams through the homogenizer.
  8. The microscope of claim 1, wherein the homogenizer forms the beam into a substantially uniform and rectangular region of illumination.
  9. The microscope of claim 1, further comprising a stimulation light source that emits a stimulation beam, wherein the optical system comprises digital micromirror device (DMD) and the stimulation beam reflects off the DMD to illuminate a bottom of a well of the plate with a pattern defined by the DMD.
  10. The microscope of claim 9, wherein the beam is at an excitation wavelength of a fluorophore, and the stimulation beam is at a second wavelength.
  11. The microscope of claim 1, further comprising an imaging lens beneath the stage to direct light from a sample in a well of the plate onto an image sensor mounted within the microscope.
  12. The microscope of claim 11, wherein the optical system includes a prism immediately beneath the stage, whereby the beam enters a side of the prism and the prism directs the beam into an aqueous sample in a well of the plate at an angle of refraction that restricts light to about the bottom ten microns of the well, the microscope further comprising a stimulation light source that emits a stimulation beam, wherein the optical system comprises digital micromirror device (DMD) and the stimulation beam reflects off the DMD to illuminate a bottom of a well of the plate with a pattern defined by the DMD.

Description

TECHNICAL FIELD The disclosure relates to microscopes. BACKGROUND There are numerous diseases that involve electrically-active cells, such as neurons and cardiomyocytes. As a result, there is a significant push to study characteristics and interactions of those cells. Fluorescence microscopy, a technique in which fluorophores are bound to the specimen to detect phenomena such as cell surface binding, neurotransmitter release, or specific DNA sequences is often used to study cellular behavior. However, fluorophores and other compounds in the surrounding medium and even in the optical components of the microscope can exhibit autofluorescence and overwhelm fluorescence from the sample. Attempts have been made to reduce background fluorescence by using total internal reflection fluorescence (TIRF) microscopy. A TIRF microscope illuminates only a thin region of the sample so that fluorophores in the surrounding medium do not receive the excitation energy needed for fluorescence. However, existing TIRF microscopes require a prism to be pressed down onto the sample in a configuration that severely limits what conditions are allowable for the sample. For example, the TIRF prism occludes any culture media, as would be required for living cells, and prevents any physical access to the sample. Thus, fluorescence microscopy has not proven satisfactory for studying fine details of living, electrically active cells. Other light microscopy techniques have failed to bring about results that are not similarly limited. Thus, there is a need in the art for improved techniques for optical measurement of cellular activity. Publication WO 01/01112 A1 discloses a method and apparatus for the measurement of radiation, especially fluorescence from samples in assays. SUMMARY According to the present invention there is provided a microscope according to claim 1. The invention provides microscopes for imaging samples within wells of multi-well plates. Microscopes of the disclosure include a beam homogenizer system that shapes a beam from a light source into a shape with excellent uniformity to the bottom of a well of a multi-well plate. In particular, microscopes of the disclosure illuminate wells for imaging by passing light through a prism beneath the sample. The light enters the prism from the side and is refracted into the well at an angle such that the light only illuminates about a bottom ten microns of the well. The beam homogenizer shapes the light from the light source so that, instead of hitting the prism as a spot with an irregular shape, the light enters the prism in a substantially rectangular pattern with homogeneous optical power level over the pattern. Thus, cells at the bottom of well are illuminated uniformly with good optical power for imaging. Moreover, the microscope can include an adjustable optical system that allows the homogenizer components to be positioned and an aperture to be selected so that the homogenized illumination beam can be matched to wells of a particular multi-well plate or a particular imaging lens and its associated field of view. This allows different types of multi-well plates to be loaded onto the microscope for imaging. Each well can include a living sample culture with multiple living cells in each well that are imaged by the microscope. The cells can include fluorescent reporter proteins that emit light in response to cellular electrical activity. The beam homogenizer ensures that the entire sample in each well receives strong and uniform illumination while avoiding off-target light that causes autofluorescence. Using the beam homogenizer, the microscope can successfully image living cells in multiple wells of a multi-well plate and record movies of electrical activity useful to show, for example, action potentials propagating within living neurons. For example, the microscope may be operable with two preselected imaging tube lenses and three types of multi-well plates, such as 96-, 384-, or 1536- well plates. The beam homogenizer may use a pair of a microlens arrays that are repositionable to one of six pairs of pre-set stops and the optical system may include a screen or block with six pre-defined apertures. For each of the six possible combinations of imaging tube lens and well-plate, the appropriate homogenizer microlens array spacing and aperture may be set so that the homogenized illumination beam enters the side of the prism as a homogeneous rectangle of illumination that gets refracted into about the bottom 10 microns of the well across the relevant field of view. Because only the relevant part of a sample (e.g., cells grown on a bottom of the well) is illuminated, excess autofluorescence is avoided. The light is restricted to the lower portion of the well by sending the beam into the prism at angle that promotes near-total-internal-reflection (TIR) within a glass bottom of the well. The light does not undergo TIR, but instead passes across only the bottom ten or so microns of th