BACKGROUND OF THE INVENTION

A fluorescence microscope is an optical microscope that uses a fluorescence signal to study the properties of organic or inorganic substances. A sample, e.g., the organic or inorganic substance under observation, is illuminated with light having a wavelength spectrum. The light is absorbed by the sample, such as being absorbed by the fluorophores in the sample, causing the fluorophores to emit light of longer wavelengths. The emitted light has a color in the longer wavelength portion of the visible light spectrum. For example, a green fluorescence protein can emit a green light after being irradiated with a blue light.

FIG. 1illustrates a schematic of a fluorescence microscope according to some embodiments. A light source130can generate light131and131* in the visible or ultraviolet spectrum which is directed toward a beamsplitter150. A fluorescence excitation filter135is disposed in the path of the light, to filter out light131* having undesirable wavelengths, such as light having wavelengths longer than a threshold wavelength. The threshold wavelength can be the longest wavelength that can generate fluorescence excitation on the sample. After the fluorescence excitation filter135, only light131within a desirable wavelength range can reach the beamsplitter150, and can be reflected toward the sample120.

The fluorophores in the sample, after absorbing the fluorescence excitation light131, can emit fluorescence light111, which can pass through an emission filter115to reach the camera110. The emission filter115is also functioned to filter out the reflective portion111* of the illumination light131, which can be much stronger than the emitted fluorescence. The beamsplitter150can also be a dichroic beamsplitter, which can act as a wavelength specific filter, transmitting the fluorescence111through to the camera, and reflecting the reflective excitation light131. Thus, the wavelength ranges, e.g., the band pass filter characteristics, of the fluorescence excitation filter, the emission filter, and the dichroic beamsplitter are chosen to match the spectral excitation and emission characteristics of the fluorophores in the sample.

To use the fluorescence microscope, the sample must be a fluorescent sample, such as labeling the sample with fluorescent stains or a fluorescent protein.

Often, it is beneficial to excite and detect more than one type of fluorescent emission, e.g., more than one color, or a set of optical wavelengths. Detecting more than one fluorescence color is helpful for acquiring more information about a specimen of interest, or for ratiometric fluorescence imaging in which the intensity of one fluorescence emitter is divided by the intensity of another. Nearly all current fluorescent microscope arrangements detect multiple fluorescence emission wavelengths over time, by mechanically shifting between more than one emission filter between subsequent snapshots. Unfortunately, these setups are not able to accurately image more than one fluorescence emission spectrum in quickly moving specimens, such as freely moving organisms.

There are also setups that use more than one image sensor placed behind a common objective lens, along with arrangement of beam-splitters, to image more than one fluorescent emitter simultaneously. Unfortunately, these arrangements are limited to a small field-of-view when imaging at high resolution, restricted by the field-of-view of the employed objective lens for imaging. They also require a complex optical arrangement.

To circumvent the use of multiple detectors and beamsplitters, it is also possible to rely on a spatial light modulator to alter the point-spread function of each fluorescence emission wavelength. In either of these strategies, due to lens aberrations and limited availability of large-format digital detectors with more than a few hundred megapixels, it is difficult to design a single-lens fluorescence microscope that offers high resolution over a wide field-of-view.

SUMMARY OF THE EMBODIMENTS

In some embodiments, the present invention discloses systems and methods for a fluorescence microscope with the ability to synchronize the captured fluorescence signals through the process of multi-channel imaging.

In some embodiments, the present invention discloses a multi-channel computational fluorescence microscope system, which can capture simultaneously fluorescence caused by multiple fluorescence excitation sources and can analyze the data, including synchronizing and correlating the interaction between various fluorescence elements and also with the surrounding morphologies. The multi-channel fluorescence capability of the microscope can be achieved by partitioning the camera array and the illumination source into multiple imaging channels, with each channel observing a similar area of the sample but designed to generate and obtain different fluorescence excitation, emission and interaction.

In some embodiments, the multi-channel computational fluorescence microscope system can include a camera array and an illumination source, together with a controller having a post processing algorithm to analyze the captured data. The camera array can include multiple groups of camera units, with each group of camera units having one or more camera channels configured to simultaneously capture multiple channels of information. The illumination source can include multiple groups of radiation source units, with each group of radiation source units having one or more types of radiation source units configured to provide corresponding radiation to the camera channels.

A benefit of the multi-channel computational fluorescence microscope system is a new ability to accurately correlate fluorescence measurements from one or more fluorescence markers at high resolution across an unbound field of view. This capability can open up new directions for scientific discovery, such as allowing for temporal correlations to be drawn over ever increasing sample sizes, more than just several square centimeters. It can also enable experiments to be performed on freely moving organisms, as well as large bacteria colonies that might behave differently than if they were constrained to a small volume.

