Light field microscope

Abstract

The invention provides a light field microscope. The optical field microscope comprises an objective lens and a microlens array, an optical filter array is arranged in front of the microlens array, a plurality of optical filters of the optical filter array are in one-to-one correspondence with a plurality of microlenses of the microlens array in position, and the optical filter array comprises a plurality of groups of optical filters corresponding to various colors. The light passing through the plurality of channels of the plurality of groups of optical filters is received by the image sensor for respectively generating images corresponding to the plurality of channels.

Description

Technical Field

[0001] The present invention relates to the field of optical imaging, and in particular to a light field microscope. Background Art

[0002] The nervous system is composed of a large number of neurons with different morphologies and functions and complex connections between them. Recording the activity of large-scale neuronal groups is one of the keys to understanding brain function.

[0003] The green fluorescent protein calcium indicator GCaMP series is currently the most widely used single-channel fluorescent probe, with an emission peak near 510nm. When neurons are activated, calcium ion indicators can convert changes in intracellular calcium ion concentration into changes in fluorescence brightness, thereby characterizing neuronal activity. Since single-channel probes have different expression levels in each cell and imaging conditions in each experiment are different, it is impossible to accurately quantify the substrate concentration. In addition, when the observed target is moving, changes in fluorescence intensity are easily affected by the target's motion, which brings many limitations to the application scenarios of single-channel fluorescent probes.

[0004] To address these issues, dual-channel ratiometric probes can be used to characterize substrate changes using the fluorescence ratio of two channels. In recent years, modifications to fluorescent proteins have led to the development of the red calcium fluorescent protein jRGECo probe, with an emission peak near 585 nm. In addition to calcium indicators, fluorescent probes targeting different neurotransmitters in the brain are also under development. An increasing number of experiments are using two or more probe indicators to validate neuronal function, for example, to simultaneously monitor neuronal activity and dopamine signaling in the brain. Furthermore, a major type of dual-channel ratiometric probe is based on the FRET (Fluorescence Resonance Energy Transfer) principle. These probes exhibit a corresponding change in FRET efficiency when the substrate changes, and the ratio of the fluorescence intensity of the FRET channel to the fluorescence intensity of the donor channel is used to characterize substrate changes. Another type of dual-channel ratiometric probe is a fusion of a single-channel probe with a fluorescent protein expressing a different color. This fluorescent protein in the second channel is often unresponsive to substrate changes, allowing correction for differences in protein expression between cells and the effects of motility bands.

[0005] Multi-channel ratios enable quantitative measurements, eliminating the errors introduced by single-channel probes. Therefore, simultaneous multi-channel measurements are crucial for a complete understanding of brain functional signaling.

[0006] In the field of optical imaging, multi-channel optical imaging is achieved by simultaneously recording and separating fluorescent probes of different wavelengths. Whether using multiple single-channel probes to record the emission signals of different substances, or using dual-channel probes to record the changing proportions of the same substance, multi-channel optical imaging technology is required to simultaneously record the signals of these probes and clearly separate and analyze each signal.

[0007] Furthermore, conventional optical imaging techniques are often limited in imaging throughput, with low instantaneous imaging throughput, making it difficult to simultaneously achieve high-speed imaging and large-scale imaging. Light-field microscopy offers highly parallelized imaging capabilities, enabling rapid volumetric imaging by simultaneously capturing light signals at different depths with a single camera exposure.

[0008] Prior art multi-channel signal acquisition requires parallel acquisition of dual-channel images, requiring the addition of multiple optical paths and devices to the optical system to synchronously capture data from different channels. Serial acquisition of multi-channel signals requires a turntable or other device to replace the corresponding filter elements in the optical system. However, this introduces a fixed delay between acquisition paths, resulting in misalignment of the acquired time information and hindering the recording of in vivo signals during experiments.

[0009] In order to overcome the above-mentioned defects of the prior art, there is an urgent need in the art for a light field microscope that can obtain multi-channel three-dimensional dynamic imaging without adding image sensors and optical paths. Summary of the Invention

[0010] The following is a brief summary of one or more aspects to provide a basic understanding of these aspects. This summary is not an exhaustive overview of all conceivable aspects and is neither intended to identify key or critical elements of all aspects nor to define the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that will be provided later.

[0011] In order to overcome the above-mentioned defects in the prior art, the present invention provides a light field microscope capable of obtaining multi-channel three-dimensional dynamic imaging without adding image sensors and optical paths.

[0012] Specifically, the light field microscope provided according to the first aspect of the present invention includes an objective lens and a microlens array. A filter array is provided before the microlens array. The multiple filters of the filter array correspond one-to-one with the positions of the multiple microlenses of the microlens array. The filter array includes multiple groups of filters corresponding to multiple colors. Light from multiple channels passing through the multiple groups of filters is received by a single image sensor to generate images corresponding to the multiple channels respectively.

[0013] Preferably, in an embodiment of the present invention, the number of the channels depends on the number of fluorescent colors of the targets marked by the sample to be imaged.

[0014] Preferably, in an embodiment of the present invention, each group of filters is evenly distributed in the filter array to cover the entire field of view.

[0015] Preferably, in an embodiment of the present invention, the plurality of groups of filters include a group of green bandpass filters and a group of red bandpass filters.

[0016] Preferably, in one embodiment of the present invention, the light field microscope further includes the image sensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above features and advantages of the present invention will be better understood after reading the detailed description of the embodiments of the present disclosure in conjunction with the following drawings. In the drawings, the components are not necessarily drawn to scale, and components with similar related properties or characteristics may have the same or similar reference numerals.

[0018] Figure 1 shows a schematic diagram of a light field microscope provided according to some embodiments of the present invention;

[0019] Figure 2 A schematic diagram of the optical path of a light field microscope provided according to some embodiments of the present invention is shown;

[0020] Figure 3 A schematic diagram showing a filter array provided according to some embodiments of the present invention; and

[0021] Figures 4A to 4C Schematic diagrams of imaging provided according to some embodiments of the present invention are shown.

[0022] Reference numerals:

[0023] 100: Light field microscopy;

[0024] 110: objective lens;

[0025] 120: microlens array;

[0026] 130: Image sensor;

[0027] 200: Two-color sample; and

[0028] 300: Filter array. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Note that the various aspects described below with reference to the accompanying drawings and specific embodiments are merely exemplary and should not be construed as limiting the scope of protection of the present invention.

[0030] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0031] Furthermore, the terms "upper," "lower," "left," "right," "top," "bottom," "horizontal," and "vertical" used in the following description should be understood to refer to the orientations depicted in that section and the accompanying drawings. These relative terms are used solely for convenience of description and do not necessarily imply that the devices described herein must be manufactured or operated in a specific orientation. Therefore, they should not be construed as limiting the present invention.

[0032] It will be understood that although the terms "first," "second," "third," etc. may be used herein to describe various components, regions, layers, and / or portions, these components, regions, layers, and / or portions should not be limited by these terms, and these terms are merely used to distinguish different components, regions, layers, and / or portions. Thus, a first component, region, layer, and / or portion discussed below may be referred to as a second component, region, layer, and / or portion without departing from some embodiments of the present invention.

[0033] As mentioned above, in the prior art, when acquiring multi-channel signals, if a parallel dual-channel image is employed, multiple collection optical paths and devices must be added to the entire optical system to synchronously capture data from different channels. If serial acquisition of multi-channel signals is employed, a turntable or other device is required to replace the corresponding filter elements in the optical system. However, there is a fixed delay between acquisition paths, resulting in misalignment of the acquired time information, which is detrimental to recording in vivo signals during experiments.

[0034] In order to overcome the above-mentioned defects in the prior art, the present invention provides a light field microscope capable of obtaining multi-channel three-dimensional dynamic imaging without adding image sensors and optical paths.

[0035] Please refer to Figure 1 and Figure 2 , Figure 1 shows a schematic diagram of a light field microscope provided according to some embodiments of the present invention, Figure 2 A schematic diagram of the optical path of a light field microscope provided according to some embodiments of the present invention is shown.

[0036] like Figure 1 As shown in FIG. 1 , in one embodiment, a light-field microscope 100 is used to collect signals from a two-color sample 200 (e.g., a red-green transgenic zebrafish). The light-field microscope 100 may include an objective lens 110 and a microlens array 120. Furthermore, a filter array 300 may be placed in front of the microlens array 120, having the same number of microlenses as the microlens array 120 and being positionally coupled thereto.

[0037] like Figure 2 As shown, a filter array 300 may be provided before the microlens array 120. The multiple filters of the filter array 300 correspond one-to-one to the positions of the multiple microlenses of the microlens array 120. Each filter is strictly aligned one-to-one with the corresponding microlens to ensure the synchronization of multi-channel signal acquisition.

[0038] exist Figure 1 The light field microscope 100 shown may further include an image sensor 130 .

[0039] Light entering each microlens first passes through the corresponding filter for splitting, and then each microlens of the microlens array 120 generates images with different viewing angles on different areas of the same image sensor 130 for light of different wavelengths.

[0040] Please refer to Figure 3 , Figure 3 Schematic diagram of a filter array provided according to some embodiments of the present invention is shown.

[0041] like Figure 3 As shown, filter array 300 may include multiple sets of filters corresponding to multiple colors. Image sensor 130 may receive light from multiple channels passing through the multiple sets of filters to generate corresponding multi-channel images during image reconstruction. The number of channels depends on the number of fluorescent colors of the targets marked on the sample to be imaged.

[0042] In this way, light from multiple channels passing through multiple groups of filters on the filter array 300 can be received by the same image sensor 130, and multi-channel acquisition can be completed without the need for multiple image sensors.

[0043] In a preferred embodiment, the dual-color sample 200 may be a red and green dual-color transgenic zebrafish. Thus, the multiple groups of filters included in the filter array 300 may be a group of green bandpass filters and a group of red bandpass filters.

[0044] As shown in Table 1, the diameter and thickness of the green and red bandpass filters are the same: 2.5 mm in diameter and 1.1 mm in thickness. The green bandpass filter has a passband of 500 to 560 nm and a transmittance greater than 90%. The green bandpass filter has stopbands of 300 to 500 nm and 560 to 650 nm, with a transmittance less than 5%. The red bandpass filter has a passband of 560 to 750 nm and a transmittance greater than 90%. The red bandpass filter has a stopband of 300 to 560 nm and a transmittance less than 5%.

[0045]

[0046] Please continue to refer to Figure 1 Filter array 300 separates the two fluorescence signals from dual-color sample 200 into two channels. Light from each channel is exposed and enters the microlenses for imaging simultaneously, without any time difference or exposure deviation between the two channels. In other words, during a single imaging exposure, light from the sample within the depth of field is simultaneously split by each filter in the filter array and incident on the corresponding microlens, forming an image on the image sensor.

[0047] like Figure 3 As shown, each group of filters is evenly distributed in the filter array 300 to cover the entire field of view, thereby avoiding the segmentation of the field of view and ensuring the integrity of the two-channel imaging.

[0048] Please refer to Figures 4A to 4C , Figures 4A to 4C Schematic diagrams of imaging provided according to some embodiments of the present invention are shown.

[0049] like Figure 4A As shown, by separating the signals of the two channels through the filter array 300 , images of different channels are respectively imaged on different areas of the image sensor 130 , thereby achieving synchronous capture of multi-channel three-dimensional structures.

[0050] like Figure 4B and Figure 4C As shown, Figure 4B 1 shows an image generated by a group of channels of light received by the image sensor 130 during the image reconstruction phase. Figure 4C The image generated during the image reconstruction phase by light from another set of channels received by the image sensor 130 is shown. Since each set of filters is evenly distributed in the filter array 300, the field of view of the image formed by each channel during the image reconstruction phase is complete.

[0051] In summary, the light-field microscope provided by the present invention adopts multi-viewing angles and dual wavelength encoding. While ensuring the resolution and field of view of the light-field microscope, through strict alignment between the filter array, microlens array and different channels, the purpose of dual-channel or even multi-channel simultaneous acquisition is achieved without adding additional equipment, while also reducing the computational cost of subsequent image reconstruction.

[0052] Furthermore, the light-field microscope provided by the present invention can accurately capture the temporal characteristics of multi-channel three-dimensional dynamic changes, which is extremely critical for many biological in vivo imaging applications. The light-field microscope provided by the present invention has unique advantages in application scenarios requiring synchronous acquisition of multi-channel three-dimensional in vivo imaging, such as synchronous monitoring of neuronal activity and neurotransmitters in living small animals, and monitoring of neurons in freely moving animals. The use of multiple channels allows for simultaneous observation of multiple components of different labeled targets, thereby reflecting different aspects of biological processes, suggesting the dynamic linkage of multiple processes, and improving the specificity and sensitivity of detection.

[0053] The previous description of the disclosure is provided to enable any person skilled in the art to make or use the disclosure. Various modifications to the disclosure will be apparent to those skilled in the art, and the general principles defined herein may be applied to other variations without departing from the spirit or scope of the disclosure. Thus, the disclosure is not intended to be limited to the examples and designs described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A light field microscope comprising an objective lens and a microlens array, characterized in that: A filter array is provided before the microlens array. The multiple filters of the filter array correspond one-to-one with the positions of the multiple microlenses of the microlens array. The filter array includes multiple groups of filters corresponding to multiple colors. Light of multiple channels passing through the multiple groups of filters is received by a single image sensor to generate images corresponding to the multiple channels respectively.

2. The light field microscope according to claim 1, wherein The number of channels depends on the number of fluorescent colors of the targets labeled in the sample to be imaged.

3. The light field microscope according to claim 1, wherein Each group of filters is evenly distributed in the filter array to cover the entire field of view.

4. The light field microscope according to claim 1, wherein The multiple groups of filters include a group of green bandpass filters and a group of red bandpass filters.

5. The light field microscope according to claim 1, wherein The light field microscope also includes the image sensor.

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