Video rate mid-infrared photothermal microscopy system using synchronized laser scanning

Abstract

A mid-infrared photothermal microscopy system images a sample. The mid-infrared light source generates a mid-infrared light beam that is directed along a first light path to reach the first face of the substrate and heat the sample. The probe light source generates probe light that is directed along a second light path to reach the second face of the substrate and illuminate the sample. A first laser scanner is positioned along a first optical path and is arranged to rotate to redirect light and scan a sample with a mid-infrared beam. A second laser scanner is positioned along a second optical path and is arranged to rotate to redirect light and scan the sample with probe light. The laser scanners each include at least one mirror driven to rotate such that the mid-infrared beam and the probe light synchronously scan the sample.

Description

[0001] Government support

[0002] This invention was supported in whole or in part by government support under Grant No. R33 CA261726 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 398,017, filed on August 15, 2022, entitled “Video-Rate Mid-Infrared Photothermal Microscopy and Uses Thereof,” the contents of which are incorporated herein by reference as if fully set forth herein. Technical Field

[0005] The present disclosure relates to a mid-infrared photothermal (MIP) imaging system, and more particularly, to a mid-infrared photothermal microscopy system that provides synchronous scanning of a sample with a mid-infrared beam and a probe light. Background Art

[0006] Vibrational spectroscopy presents the molecular structure of samples through infrared (IR) spectroscopy and Raman spectroscopy, revealing rich compositional information of samples. Infrared microscopy and Raman microscopy with spatial fingerprinting capabilities have been widely used to visualize the chemical composition of biological systems and materials at the microscopic scale. Although both infrared and Raman methods measure molecular vibrations, their different physical contrast mechanisms make them suitable for different specific applications and express complementary information: dipole-based infrared absorption is sensitive to asymmetric vibration bonds such as CO and C=O; at the same time, Raman scattering, which is proportional to the molecular polarizability, is sensitive to CH and C=C. Fourier transform infrared spectroscopic microscopy has been widely used by measuring the infrared attenuation caused by absorption. Infrared absorption has a large cross-section (about 10 -22 cm 2 sr -1 ), which is larger than Raman scattering (about 10 -30 cm 2 sr -1 ) is 100 million times larger and has a broad spectral coverage for different molecular features, making it attractive as an imaging method with higher sensitivity. However, the application of infrared absorption in imaging biological systems is hampered by its low spatial resolution (several micrometers) when compared to Raman microscopy and by the presence of a strong water background at the OH bonds.

[0007] To improve the spatial resolution of infrared imaging, near-field scanning methods have been developed. Scattering-based scanning nearfield optical microscopy (s-SNOM) first improved the resolution of infrared imaging to less than 20 nm by directly measuring infrared scattering from the tip. However, due to its complex source of contrast, s-SNOM provides almost no quantitative absorption information. To compensate for the distorted absorption spectrum, atomic force microscope-infrared (AFM-IR) devices were invented. Chemical imaging with a spatial resolution of 20 nm has been achieved by measuring the photothermal process caused by absorption, and then the tapping mode method was introduced to further improve the spatial resolution to 10 nm. Although successful in characterizing material properties at the nanoscale, all near-field methods are limited to in vitro surface characterization for in vitro studies. In addition, the imaging speed of one hour makes it almost impossible to use it to visualize any faster dynamic or high-throughput characterization.

[0008] Mid-infrared photothermal (MIP) microscopy has been developed as a new approach to achieve far-field super-resolution infrared imaging by optically sensing the mid-infrared-induced photothermal effect in the far field. MIP uses a probe beam to detect the transient temperature increase caused by short-pulse infrared excitation. This local temperature modulation causes thermal expansion and refractive index changes. Those changes can be jointly revealed by detecting the scattered intensity modulation using a shorter wavelength probe beam probe, which enables infrared spectral imaging on the submicron scale. The probe beam can include visible wavelengths and / or near-infrared and ultraviolet wavelengths. In addition, the indirect measurement of the absorption effect bypasses the water background problem that hinders infrared imaging for biological systems. Since the first high-quality imaging demonstration of biological systems in 2016, the method has been rapidly expanded with different recent innovations and has achieved a wide range of applications.

[0009] Photothermal imaging is essentially the measurement of small modulations on a larger scattering background. Traditionally, this task is accomplished by heterodyne detection at the infrared pulse rate using a lock-in amplifier. Due to the nature of the photothermal process, the demodulation frequency is below megahertz, where the laser noise is typically large. Therefore, a pixel acquisition time of several milliseconds is required to extract the signal above the laser noise and shot noise. To improve detection efficiency, spatially multiplexed photothermal detection using CMOS cameras has been invented. In this system, the photothermal contrast is obtained by subtracting multiple frames acquired by the camera between the infrared on and off states. However, CMOS sensors have a limited number of tens of thousands of electrons. - ) level. To resolve a modulation depth of about 1%, thousands of frames must be averaged. In addition, the excitation fluence of weakly focused infrared decreases significantly with the field of view. A high-energy mid-IR laser source must be used to compensate. Current biological imaging speeds for photothermal microscopes are limited to minutes per frame, which is insufficient for capturing dynamics within biological systems or within large regions with high-throughput imaging.

[0010] In order to achieve higher sensitivity detection at faster speed, the imaging system requires not only a large detection photon capacity for resolving small modulations above the shot noise, but also non-diluted IR excitation at higher repetition rates. Therefore, a MIP system with improved speed while maintaining image quality is needed. As described in this paper, the above problems are solved by a newly designed photothermal microscope structure. Summary of the invention

[0011] In brief summary, based on the above needs, the present disclosure includes a single-pulse laser scanning mid-infrared photothermal (MIP) microscope that allows for higher sensitivity and faster imaging at video rates. First, the present disclosure describes a phase-locked demodulation scheme using high-speed digitization that improves the system response to nanoseconds to resolve the contrast caused by a single infrared pulse. To achieve video-rate, scanning-based imaging, a sub-microsecond system response per pixel is provided. Unlike coherent Raman microscopy, the modulation rate of photothermal microscopy is limited to 1 MHz or less to avoid heat accumulation in photothermal microscopy. In this case, the photothermal contrast at each pixel needs to be extracted within a single infrared excitation cycle, which makes phase-locked filtering ineffective in picking up signals from a noisy background. The disclosed system overcomes this difficulty by replacing phase-locked narrowband detection with a broadband amplifier and a megahertz digitizer for time-gated detection. Using this method, the photothermal modulation caused by a single infrared pulse can be resolved in the time domain. With improved system response, a faster imaging scheme is needed to replace sample scanning to match the expected pixel dwell time of several microseconds. To address this problem, the disclosed system can include two laser scanning mechanisms, such as galvanometer mirrors for synchronous scanning of both the focused mid-IR light and the probe beam to achieve line rates exceeding 2.5kHz. These synergistic innovations allow for the first time microsecond acquisition of photothermal signals from a single IR pulse at each pixel.

[0012] The system provides video-rate (25 Hz) imaging (150 pixels x 100 pixels) of chemical dynamics within living cells. In addition, synchronized scanning of the infrared and probe beams allows uniform illumination of a larger field of view (over 400 microns) to achieve chemical precision inspections with a higher throughput. With this capability, the present application captures rapid lipid dynamics within living fungal cells. Video-rate imaging further allows spectral decomposition of individual cell walls. The ultrastructure of the cell wall is revealed based on the unique infrared absorption of the cell wall (exine wall, inner wall, and membrane). Compared to previous phase-locked sample scanning MIP microscopy, the system of the present disclosure increases the speed by three orders of magnitude (from milliseconds per pixel to microseconds per pixel). The following shows a wide range of applications to a wide range of living systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to make it easier for a person of ordinary skill in the art to understand how to make and use the system of the present disclosure, reference may be made to the following drawings.

[0014] Figure 1A is a schematic diagram of a MIP microscopy system using a laser scanning mirror according to the subject technology.

[0015] Figure 1Byes Figure 1A Schematic diagram of the system shown, Figure 1B A laser scanning scheme according to the subject technology is shown.

[0016] Figure 1C According to the subject technology, Figure 1A Electronics diagram of the system shown.

[0017] Figure 2A is a graphical representation of the position and velocity of a galvanometer during a unidirectional scan according to the subject technology.

[0018] Figure 2B is a graphical representation of the position and velocity of a galvanometer during bidirectional scanning in accordance with the subject technology.

[0019] Figure 2C is a diagram of an image reconstruction method based on the position and velocity of a galvanometer during bidirectional scanning in accordance with the subject technology.

[0020] Figure 2D is an exemplary image made by bidirectional galvanometer scanning of a target without feedback in accordance with the subject technology.

[0021] Figure 2E is an exemplary image made by bidirectional galvanometer scanning of a target with feedback in accordance with the subject technology.

[0022] Figure 3A is a graphical representation of the raw signal detected across a single polymethylmethacrylate (PMMA) particle in accordance with the subject technology.

[0023] Figure 3B According to the subject technology, Figure 3A Graphical representation of the signal shown after signal filtering and contrast extraction.

[0024] Figure 3C According to the subject technology, Figure 3A Graph of the signal shown after intensity extraction.

[0025] Figure 3D is a single-pulse MIP image of a PMMA particle according to the subject technology.

[0026] FIG. 3E to FIG. 3H is an image of MIP imaging of PMMA particles in a larger field of view (FOV) according to the subject technology.

[0027] Figure 4A is a hyperspectral image of a laser recorded at video rate according to the subject technology.

[0028] Figure 4B According to the subject technology, Figure 4AGraphical representation of the estimated spectrum of a PMMA particle at the particle shown.

[0029] Figure 5A is a transmission image of a living fungal cell sample according to the subject technology.

[0030] Figure 5B According to the subject technology, it is shown that there is infrared absorption, Figure 5A Images of the lipid components of the samples shown.

[0031] Figure 5C According to the subject technology, Figure 5A and Figure 5B The merged image.

[0032] FIG. 5D to FIG. 5F According to the subject technology, Figure 5A Sample reconstructed image of volume imaging of the fungal cell sample shown.

[0033] Fig. 6A is a MIP image of a fungal cell wall studied in vivo according to the subject technology.

[0034] Figure 6B According to the subject technology, Fig. 6A Line drawing of the fungal cell wall shown.

[0035] Figure 6C According to the subject technology, from 900 cm -1 Up to 1200 cm -1 Spectral image of fungal cells generated by laser frequency scanning.

[0036] Fig.6D According to the subject technology, Figure 6C Illustration of the spectral positions of the indicated fungal cells. DETAILED DESCRIPTION

[0037] For those of ordinary skill in the art, the advantages and other features of the systems and methods disclosed herein will become more readily apparent from the following detailed description of some preferred embodiments in conjunction with the accompanying drawings, which illustrate representative embodiments of the invention. The same reference numerals are used herein to represent the same components. In addition, terms indicating orientation, such as "upper", "lower", "far" and "near", are used only to assist in describing the position of components relative to each other. For example, the "upper" surface of a component is only used to describe the surface separated from the "lower" surface of the same component. Terms indicating orientation are not used to describe absolute orientation (i.e., the "upper" component must always be at a higher altitude).

[0038] As discussed in more detail below, the system described herein may include software and may be implemented by one or more designated computers and / or servers that are configured to execute software instructions and perform a plurality of functions in accordance with the teachings herein. It should be understood that as part of the present system, commercially available hardware components may be used to run the software described herein. However, the hardware must be specifically configured to execute the software instructions in accordance with the teachings herein.

[0039] More specifically, the functionality of the systems and methods described herein can be implemented by digital electronic circuits, computer hardware, computer firmware, computer software, and combinations thereof. As described herein, computer programs can be provided in any form of programming language, including compiled and / or interpreted languages, and computer programs can be deployed in any form, including as stand-alone programs or as subroutines, elements, and / or other units suitable for use in a computing environment. Computer programs can be deployed to execute on one computer or on multiple computers at one location.

[0040] The method steps and functions may be implemented by one or more programmable processors executing a computer program to perform the functions of the invention by operating on input data and generating outputs. The method steps may also be performed by a device that may be implemented as a dedicated logic circuit. For example, the circuit may be an FPGA (field programmable gate array) and / or an ASIC (application specific integrated circuit). Subroutines and software agents may refer to portions of a computer program, processor, dedicated circuit, software, and / or hardware that implement the function.

[0041] Processors suitable for the execution of computer programs include, by way of example, both general-purpose and special-purpose microprocessors, and any one or more processors of any type of digital computer. In general, a processor receives instructions and data from a read-only memory or a random access memory or both. The essential elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. In general, a computer may include a computer operatively coupled to receive data from and / or transfer data to one or more mass storage devices (e.g., magnetic disks, magneto-optical disks, or optical disks) for storing data.

[0042] Data transfer and instructions may also be carried out over a communications network. Suitable information carriers for carrying computer program instructions and data include all forms of non-volatile memory, including, for example, semiconductor memory devices. The information carrier may be, for example, an Erasable Programmable Read-Only Memory (EPROM), an Electrically Erasable Programmable Read-Only Memory (EEPROM), a flash memory device, a magnetic disk, an internal hard disk, a removable disk, a magneto-optical disk, a Compact Disc Read-Only Memory (CD-ROM) disk, and / or a Digital Versatile Disc Read-Only Memory (DVD-ROM) disk. The processor and memory may be supplemented by and / or incorporated into a dedicated logic circuit.

[0043] To provide interaction with a user, a computer with a display device can be used to implement the systems and methods described herein. For example, the display device can be a cathode ray tube (CRT) display and / or a liquid crystal display (LCD). For example, interaction with a user can be displaying information to the user, as well as a keyboard and pointing device (e.g., a mouse), through which the user can provide input to the computer (e.g., interact with user interface elements). Other types of devices can also be used to provide interaction with a user. For example, other devices can be feedback provided to the user in any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback). For example, input can be received from a user in any form including sound, voice, and / or touch input.

[0044] The system described herein may include a distributed computing system including a back-end component. The back-end component may be, for example, a data server, a middle-tier component, and / or an application server. The system described herein may include a distributed computing system including a front-end component. The front-end component may be, for example, a client computer with a graphical user interface, a web browser through which a user can interact with an example embodiment, and / or other graphical user interfaces for a transmission device. The components of the system may be interconnected by digital data communication (e.g., a communication network) in any form or any medium. Examples of communication networks include a local area network (LAN), a wide area network (WAN), the Internet, a wired network, and / or a wireless network.

[0045] The system may include clients and servers for data transmission. The client and server are essentially remote from each other and usually interact through a communication network. The relationship of client and server is implemented by multiple computer programs running on respective computers and having a client and server relationship relative to each other.

[0046] Packet-based networks may include, for example, the Internet, carrier internet protocol networks (e.g., local area networks (LAN), wide area networks (WAN), campus area networks (CAN), metropolitan area networks (MAN), home area networks (HAN)), private IP networks, IP private branch exchanges (IPBX), wireless networks (e.g., radio access networks (RAN), 802.11 networks, 802.16 networks, general packet radio service (GPRS) networks, high-performance wireless local area networks (HiperLAN)), and / or other packet-based networks. Circuit-based networks may include, for example, a public switched telephone network (PSTN), a private branch exchange (PBX), a wireless network (e.g., a Radio Access Network (RAN), Bluetooth, a code-division multiple access (CDMA) network, a time division multiple access (TDMA) network, a global system for mobile communications (GSM) network), and / or other circuit-based networks.

[0047] Devices for sending or receiving signals may include, for example, computers, computers with browsing devices, phones, IP phones, mobile devices (e.g., cell phones, personal digital assistant (PDA) devices, laptops, email devices), and / or other communication devices.

[0048] In brief summary, this paper discloses a novel MIP microscopy system. By optically sensing the photothermal effect caused by absorption, mid-infrared photothermal (MIP) microscopy achieves super-resolution infrared imaging of biological systems in water. However, the speed of current sample scanning MIP systems is limited to a few milliseconds per pixel, which is insufficient to capture biological dynamics. Here, by rapidly digitally detecting transient photothermal signals caused by a single infrared pulse, the present disclosure describes a laser scanning MIP microscope that increases the imaging speed by three orders of magnitude. To achieve single-pulse photothermal detection, synchronized galvanometer scanning of both mid-infrared light and the detection beam is utilized to achieve an imaging line rate of more than 2kHz. Using the system disclosed in this article, and at video rate speeds, the present application presents the dynamics of different biomolecules in vivo at multiple scales. In addition, through hyperspectral imaging, the present application chemically dissects the hierarchical ultrastructure of fungal cell walls. Finally, through more than 200×200 µm 2 Using a uniform field of view, this application maps fat storage in freely moving C. elegans and living embryos.

[0049] Reference now Figure 1A to Figure 1B , an exemplary MIP microscopy system 100 capable of performing video rate MIP imaging of a sample 102 on a substrate 104 is shown. The system 100 uses an inverted microscope frame 106. An infrared light source 108 provides a pulsed infrared excitation beam 110 for heating the sample 102. This can be a pulsed quantum cascade laser such as Daylight Solutions' MIRcat 2400 or a similar product. The infrared repetition rate is externally controlled between 500kHz and 1MHz with a duty cycle of less than 30%. A detection light source 112 provides a detection beam 114 for imaging the sample 102. For example, the detection beam 114 can be provided by a continuous wave laser having a central wavelength of 532nm. In one embodiment, the detection beam can be Cobolt's Samba 532 or a similar product. An infrared beam 110 is provided to excite the sample for imaging, while a probe light 114 is reflected from and / or transmitted through the sample 102 and collected (e.g., by photodiodes 132a, 132b) to image the sample 102. Specifically, the photodiode 134a detects light reflected from the sample 102, while the photodiode 134b detects light transmitted through the sample 102.

[0050] The system includes two laser scanners, such as two pairs of galvanometer mirrors 150a, 150b, 152a, 152b, which are rotated to deflect light within the system 100. Alternatively, other laser scanning devices that can steer the beam in at least one axis, such as fast steering mirrors, micromechanical mirrors, or other laser scanning devices, can be used. In one embodiment, a first pair of galvanometer mirrors 150a, 150b (generally represented as 150) are positioned in the path of the detection beam 114, and the rotation of the first pair of galvanometer mirrors 150 scans the detection beam 114 across the sample 102. The galvanometer mirrors 150a, 150b can be positioned to scan parallel to each other along the path of the detection beam 114. In one example, the plurality of galvanometer mirrors 150 can be Saturn IB ScannerMAX XY galvanometer mirrors or similar products. For example, the plurality of galvanometer mirrors 150 can be rotated to quickly scan with the detection beam 114 at a higher resonant frequency (e.g., 3kHz). Depending on the details of the laser scanner / galvanometer system used, other resonant frequencies may also be used for scanning. The scanning probe beam 114 is conjugated to the back focal plane of the objective 116 through a scan lens 118 (e.g., a dual lens pair with a focal length of 75 mm using Thorlabs' AC508-150-A) and a tube lens 120 (e.g., a TTL180-A with a focal length of 180 mm using Thorlabs), which introduces a 2.4x beam expansion. This essentially fills the back focal plane of the objective. The scanning probe beam 114 is focused on the sample 102 through a water immersion objective 122 (e.g., UPLSAPO 60X 1.2NA from Olympus or similar).

[0051] A second pair of galvanometers 152a, 152b (generally denoted as 152) is positioned in the path of the infrared beam 110, and the rotation of the second pair of galvanometers 152 scans the sample 102 with the infrared beam 110 to excite the sample 102. In one example, the plurality of galvanometers 152a, 152b are positioned to scan parallel to each other along the path of the infrared beam 110. The galvanometers 152 can be XY galvanometers, such as Thorlabs' GVS002 or similar products. The plurality of galvanometers are placed within 200mm of a reflective objective 140 (such as Thorlabs' LMM40X-P01 40X 0.5NA or similar products). Prior to imaging, the focus of the infrared beam 110 is aligned and coincident with the focus of the detection beam 114. During imaging, the infrared focus is scanned synchronously with the detection beam, which maintains uniform excitation and detection over a larger field of view.

[0052] As described in detail below, the light beams 110 and 114 are scanned by the galvanometers 150 and 152 in the forward and backward directions. After reaching the sample 102, the probe light can return along a path similar to the transmission path of the probe light beam 114 until it reaches the beam splitter 130a. The beam splitter 130a can be any known type of beam splitter that allows some light to be deflected while allowing some light to be transmitted therefrom (e.g., a 50 / 50 beam splitter). In the example shown, the beam splitter 130a allows some light emitted from the probe light source 112 to be transmitted therefrom along a first axis while deflecting the returned light to a second axis and passing through the detection lens 132a to the photodiode 134a that detects the light. The photodiode 134a detects the light and can convert the light into an electrical signal for further processing through optional amplification / filtering. The synchronized scanning of the galvanometers 150 and 152 generates a line scan of the sample 102, which allows photothermal detection at each pixel during the scanning process.

[0053] Similarly, the probe light may also be allowed to transmit through the sample 102. The probe light that transmits the sample 102 may travel along the return path to the transmission path of the infrared beam 110 until the beam splitter 130b, where the returned probe light is split toward the detection lens 132b and the photodiode 134b that detects the light. The beam splitter 130b may be a dichroic mirror, which allows the infrared beam 110 to transmit therethrough while blocking the probe light directed toward the photodiode. The photodiodes 134a and 134b may be, for example, silicon photodiodes (e.g., Thorlabs' DET100A or similar) connected to a homemade preamplifier and filter circuit.

[0054] It should be understood that other typical optical components (e.g., lenses, mirrors) and / or electrical components (power supplies, amplifiers, processing devices) may be included to allow the system to function as described herein. For example, Figure 1A An exemplary system is shown having an additional mirror 136 in the infrared beam path for redirecting the emitted infrared beam 110 and the probe beam returning along the infrared beam path. Another additional mirror 138 is positioned in the probe light path to redirect the probe light within the system.

[0055] In some cases, the system 100 may include an additional weak light detector 142, such as a photomultiplier tube (PMT), a high gain photodiode (APD), or the like. The additional weak light detector 142 collects and measures light, including effects of modulated fluorescence originating from the sample 102. The system may include a dichroic mirror 144 that deflects light returning from the detection light path 114 and transmits it to the PMT 142 through a filter 146. This combination enables simultaneous measurement of infrared absorption and sample fluorescence.

[0056] Reference now Figure 1C , an electronics diagram 180 of the system 100 is shown. In general, the functions of the system can be controlled by a processor 184, which interfaces with all other components of the system. The processor 184 can include typical hardware and software components to perform the functions described herein, including being configured to provide communication with other components of the system 100. A high-speed digitizer 186 is included to process analog signals detected by other components and convert them to digital form. The digitizer can then transmit the digital signal to the processor 184.

[0057] To drive the two pairs of galvanometers 150, 152, four control signals are generated, for example, analog signals can be generated by a digital-to-analog (DAC) device 182 (such as National Instrument's PCIe-6363 or similar products) and used for galvanometer motion control. Similar control can be provided for alternative laser scanners as needed. The two pairs of galvanometers 150, 152 are scanned synchronously with an angular scaling factor, which is calculated by the focal length of the detection objective lens and the infrared objective lens and the beam expansion rate of the relay system calibrated before use. The angular scaling factor can also be determined empirically by optimally coinciding the infrared light with the detection beam, for example, using the intensity of the detected photothermal signal as an indicator of the coincidence and synchronization efficiency.

[0058] The angular scaling factor is applied to ensure that the infrared light 110 and the detection beam 114 can be accurately focused on the same point at the same time during the scanning process. Multiple galvanometers turn the beam to an angle, and the subsequent optical device relays and focuses the angle on the spatial position on the sample. However, the relationship between the angle and the position is different for different optical devices. In this system, the present application specifically designs two independent infrared light / detection beam paths, which introduces this difference during the galvanometer scanning process. Therefore, the scaling factor will be used to compensate for optical differences (including objective lens focal length, beam expansion rate, etc.).

[0059] The signals from the photodiodes 134a, 134b (generally denoted as 134) can be sent to a corresponding amplifier 188, which can provide a scalable gain of 40dB to 96dB at a maximum bandwidth of 500MHz. The amplified signal is sent directly to a high-speed digitizer 186 (e.g., Oscar14bit from Gage applied) with a sampling rate of 50MS / s. The photodiodes 134 are also interfaced with corresponding low-pass filters 190, which are directly interfaced with the digitizer 188. In some cases, the system 100 may also include a Mercury-Cadmium-Telluride (MCT) detector 192, which can detect the returning infrared pulses and provide the detected signal to the digitizer 186.

[0060] refer to Figure 2A 2F, the laser scanning and image reconstruction scheme used in system 100 will be discussed. As described above, the two laser scanners of system 100 scan the mid-infrared light and the detection beam synchronously so that they coincide throughout the scanning process. The embodiment shown in FIG2 uses multiple pairs of orthogonally oriented galvanometer mirrors to achieve video rate raster scanning of the infrared light and the detection beam along the X-direction and the Y-direction (i.e., in a plane). In alternative embodiments, other laser scanning mechanisms may be used, such as fast steering mirrors that can be tilted / tilted along two substantially orthogonal axes. In FIG. 2A to FIG. 2B In FIG. 3 , graph 302 shows the change of the galvanometer position over time, while graph 304 shows the change of the galvanometer speed over time. Arrows 306 and 308 show the rotation period of the galvanometer in one direction. For example, arrow 306 shows the rotation of the galvanometer in a first direction, and arrow 308 shows the rotation of the galvanometer in a second direction opposite to the first direction.

[0061] Raster scanning is essentially performed in two types of methods, unidirectional scanning (shown in diagram 300a) and bidirectional scanning (shown in diagram 300b). For imaging line rates below 1kHz, unidirectional scanning is used because of its uniform scanning speed and negligible backlash error. However, when the galvanometer is operated at a rotation speed faster than a critical speed (e.g., 1kHz for some galvanometers), unidirectional scanning can have a limited imaging duty cycle of less than 50%, such as Figure 2A In this method, the system 100 may spend more than half of its time rotating the galvanometer back, which results in a slower imaging speed. To achieve a higher scan line rate, such as more than 2kHz, a bidirectional scanning and image reconstruction scheme may be used. In one embodiment, the galvanometers 150 and 152 may be driven in a sinusoidal rotation motion at their resonant frequencies exceeding 2kHz, such as Figure 2B As shown. Signal detection is performed in both directions of galvanometer movement, which provides double imaging efficiency when the duty cycle is greater than 90%. To address image distortion caused by nonlinear scanning speed and backlash error, a galvanometer position sensor is acquired as feedback. The galvanometer position sensor can also be included in the system 100, in particular to provide feedback related to the scanning galvanometer 150 and the scanning galvanometer 152.

[0062] The image is then reconstructed based on the position of each pixel by using linear interpolation, such as Figure 2C shown. Figure 2D An exemplary image 310 reconstructed from a bidirectional scan is shown. In contrast, Figure 2E An exemplary image 312 is shown that is reconstructed from a bidirectional scan and adjusted by feedback from a galvanometer position sensor as described above.

[0063] Now refer to Figures 3A to Figure 3H , shows an example of imaging 500 nm diameter polymethyl methacrylate (PMMA) nanoparticles using video rate MIP imaging using the system 100 described herein. Specifically, Figure 3A The acquired raw signal across a line of PMMA particles is shown. Figure 3B The signal after signal filtering and contrast extraction is shown. Figure 3C Shown from Figure 3A The signal shown is the signal after intensity extraction. Figure 3D Single pulse imaging of a 500 nm diameter PMMA particle is shown. FIG. 3E to FIG. 3H Shown is the MIP imaging of PMMA particles in a larger field of view.

[0064] First, PMMA particles in solution form (e.g., MMA500 from Phosphorex) are diluted with deionized water. A drop of the solution is then applied to the surface of a 0.2 mm thick calcium fluoride (CaF2) substrate for imaging. In this embodiment, the photothermal signal is detected from backscattering (although the system described herein supports photothermal detection in both forward scattering and backscattering by detectors 134d and 134b). A quantum cascade laser (QCL) (e.g., infrared laser 108) is operated at 500 kHz and a pulse width of 600 ns. Laser scanning imaging is performed with a pixel dwell time of 2 μs, which corresponds to a single pulse measurement. By adjusting the infrared excitation to 1729 cm corresponding to the absorption peak of the C=O bond of PMMA, the absorption peak of the C=O bond of PMMA is detected. -1 , the broadband detection system of the present application can clearly distinguish the single pulse photothermal originating from each particle, e.g. Figure 3AAs shown (wherein the graph 320 in the diagram 330a shows the original signal changing over time).

[0065] The signal can be further processed by applying matched filtering and extracting the modulation amplitude as the contrast of each pixel, such as FIG. 3B to FIG. 3C As shown in Figures 330b and 330c in FIG. 330b and 330c, graph 322 represents the original signal intensity, graph 324 represents infrared excitation, graph 326 represents the signal after matched filtering, and graph 328 represents the signal after modulation amplitude extraction. The MIP intensity is extracted by subtracting the peak amplitude after heating and complete attenuation.

[0066] Then, the raster scanned photothermal image is reconstructed by applying linear interpolation using the galvanometer position feedback, as Figure 3D 358. The signal-to-noise ratio for the submicron particles shown is over 70. In addition to video rate MIP imaging, with synchronously scanned infrared lasers and detection lasers, a uniform imaging field of view (FOV) of over 200 μm can be maintained without slowing down. High speed large area imaging such as Figure 3E The image size was set to 800 × 800 with a pixel size of 150 nm. The infrared excitation was adjusted to 1729 cm -1 and 1600cm -1 , to present chemical contrast. Figure 3G and Figure 3H Show Figure 3E Magnified images 362, 364 of the image 360 ​​are shown, which show that good spatial resolution and sensitivity are maintained. As the scanning range exceeds 200 μm, and due to the beam cut at the entrance pupil of the infrared objective lens, the excitation flux decreases. This problem can be solved by a total reflection relay design, which can further expand the system FOV by more than 1 mm. In short, without the need for sample movement and stitching, the laser scanning MIP microscope structure disclosed in this article supports sub-micron resolution imaging on a FOV of more than 1 mm, which significantly promotes biological imaging applications that require both fast dynamics and high throughput.

[0067] Leveraging the significantly improved imaging throughput and fast tuning capabilities of quantum cascade lasers (QCLs), this application expands the system's high-speed mid-infrared spectral imaging capabilities to further improve chemical specificity. Conventional MIP imaging is performed by using narrow-line lasers to excite a single isolated infrared band. Although monochromatic imaging has been shown to have good chemical selectivity for distinguishing substances with different molecular compositions, monochromatic imaging cannot distinguish overlapping bands contributed by target molecules from background components, nor can it quantitatively distinguish molecular substructures.

[0068] With video-rate imaging capability, high-speed spectral imaging can be performed by sweeping the mid-IR laser during continuous video recording. Figure 4A The hyperspectral imaging results of PMMA particles with D = 500 nm are shown. Figure 4A The images 400a to 400f shown were taken at a speed of 20 frames per second, and the laser was emitted at a speed of 50 cm. -1 The speed sweep provides 2.5cm -1 The effective spectral resolution is Figure 4B The diagram 402 shown plots the underlying spectrum of a single particle, as shown in graph 404. The spectrum imaging covers 200 cm in 4 seconds. -1 Since the latest QCL sources can be operated in wavelength sweep mode at speeds up to 1000 cm⁻¹ / s, multiple combinations of spectral resolution and imaging speed can be freely selected in this imaging mode. Overall, the spectral imaging mode that fully exploits the fast sweep capability of the QCL combined with the high-speed laser scanning imaging scheme significantly enhances the chemical specificity of MIP microscopy, which enables molecularly resolved imaging in highly complex biological samples. This can be applied, for example, in the imaging of living fungal cells to resolve the chemical composition within the cell wall.

[0069] Reference now FIG. 5A to FIG. 5F , showing video-rate MIP imaging of rapid lipid dynamics in living fungal cells obtained by the system described herein. Specifically, Figure 5A A transmission image of a living fungal cell sample 502 is shown. Figure 5B The sample is shown at 1740 cm -1 The lipid component 504 has infrared absorption. Figure 5C yes Figure 5A and Figure 5B The merged image. FIG. 5D to FIG. 5F Lipid dynamics at different focal planes within sample 502 are shown.

[0070] Resolving chemical composition in highly dynamic systems is always a huge challenge, which places stringent requirements on detection sensitivity and speed. Utilizing a large infrared absorption cross section and a fast scanning scheme, the present application demonstrates chemical imaging of dynamics in living cells using a laser scanning MIP microscope system 100. Using the system 100 described herein, the present application acquires fast lipid dynamics within fungal cells at a speed of 20 Hz. By tuning the infrared excitation to 1740 cm corresponding to the absorption peak of the C=O bond in the lipid, the molecular dynamics of the lipids were analyzed. -1 , enabling specific imaging of individual lipid droplets and cell membranes. Significant lipid movements in living fungi were revealed, such as FIG. 5A to FIG. 5C As shown. This lipid component plays an important role in fungal cells as an energy source, membrane construction and synthesis precursor. Therefore, it is crucial to further study the involvement of lipid components in metabolic pathways in real time and in vivo. The technology disclosed in this article provides a new way to image metabolism using spectroscopic methods.

[0071] In addition to rapid imaging of the sample in the XY plane, 3D dynamics can also be resolved by utilizing the high axial resolution (about 700 nm) of the confocal scanning system 100. By adjusting the objective lens 116 at different focal planes, volumetric imaging of fungal cells can be performed, such as FIG. 5D to FIG. 5F The results show the unique three-dimensional distribution of lipids in living cells. This powerful sectioning capability is essential for studying the actual movement of smaller organelles and the distribution of molecules at lower concentrations.

[0072] Reference now FIG. 6A to FIG. 6D , showing the spectral MIP imaging results of the fungal cell wall in in vivo studies. The fungal cell wall plays an important role in maintaining cell function and proliferation. It is also the most common target for antifungal drugs used for pathogenic yeast. Direct studies of changes in the chemical composition and morphology of the fungal cell wall during cell-drug interactions can significantly promote the development of new therapies. However, the thickness of the fungal cell wall is extremely thin, about 100nm. This makes it extremely difficult to visualize using conventional dye-free optical microscopes. With molecular specificity, submicron resolution and high imaging speed, the disclosed system 100 is able to visualize the dynamics and chemical composition of the cell wall in in vivo studies.

[0073] The fungal cell wall is mainly composed of polysaccharides including chitin and glucan, which have extremely high infrared absorption in the CO region. When the infrared excitation is switched to 1070 cm corresponding to the absorption peak of β-1,3-glucan, -1The cell wall morphology of individual fungal cells can be clearly distinguished, such as Fig. 6A The line graph at the position shown in image 600 is Figure 6B 602, 604, and 606. Specifically, the line graph of molecule 610a is represented by line 612a, the line graph of molecule 610b is represented by line 612b, and the line graph of molecule 610c is represented by line 612c. To further provide molecular information in the cell wall components, the QCL laser was switched from 900 cm -1 Frequency sweep to 1200cm -1 For spectral imaging. The total intensity of the hyperspectral stack is Figure 6C The infrared spectrum in the CO functional group region can be clearly resolved at each pixel. In addition, through subsequent multivariate analysis of the spectrum, the unique component ratios of different cells can be revealed, such as Fig.6D This indicates a strong heterogeneous distribution among cells. Fig.6D Show Figure 6C Spectral lines at the positions shown, where 994 cm corresponding to β 1-6 glucan, β 1-4 glucan and β 1-3 glucan, respectively -1 (line 630), 1026 cm -1 (line 632), 1077 cm -1 Deconvolution of the peak is performed at (line 634).

[0074] All orientations and settings of components shown herein are used only by way of example. In addition, it will be appreciated by those of ordinary skill in the art that, in alternative embodiments, the functions of several elements may be realized by fewer elements or a single element. Similarly, in some embodiments, relative to the operations described in the illustrated embodiments, any functional element may perform fewer operations or different operations. In addition, functional elements presented as independent for illustrative purposes may be incorporated into other functional elements in a specific implementation.

[0075] Although the subject technology has been described with reference to preferred embodiments, it will be readily appreciated by those skilled in the art that various changes and / or modifications may be made to the subject technology without departing from the spirit or scope of the subject technology. For example, each claim may be subordinate to any or all claims in a multiple-dependency manner, even if not originally written as such.

[0076] The following references are background information that can be understood by those skilled in the art:

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[0079] 3. Baker, MJ et al., Analysis of biological materials using Fourier transform infrared spectroscopy, Nature Protocols 9, 1771-1791 (2014).

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Claims

1. A mid-infrared photothermal microscopy system for imaging a sample on a substrate, the system comprising: a mid-infrared light source configured to generate a mid-infrared light beam directed along a first optical path to reach the first side of the substrate and heat the sample; a detection light source configured to generate detection light that is directed along a second light path to reach a second side of the substrate and illuminate the sample, the second side being opposite to the first side; a first laser scanner comprising at least one movable mirror positioned along the first light path and configured to rotate to redirect light and scan the sample with the mid-infrared light beam; as well as a second laser scanner comprising at least one movable mirror positioned along the second light path and configured to rotate to redirect light and scan the sample with the probe light, The laser scanner is driven to rotate so that the mid-infrared light beam and the detection light scan the sample synchronously.

2. The system of claim 1, further comprising a photodiode configured to detect probe light from the sample to generate a reconstructed image of the sample.

3. The system of claim 2, wherein the mid-infrared light beam is a pulsed light beam, and wherein the mid-infrared photothermal system further comprises single pulse photothermal detection.

4. The system of claim 1, wherein: The first laser scanner comprises a first pair of scanning mirrors, the first pair of scanning mirrors comprising a first mirror and a second mirror; and The second laser scanner includes a second pair of scanning mirrors including a third mirror and a fourth mirror.

5. The system of claim 4, wherein the first pair of scanning mirrors has a plurality of orthogonal scanning axes; and the second pair of scanning mirrors has a plurality of orthogonal scanning axes. 6 . The system according to claim 1 , further comprising a digital-to-analog conversion device configured to generate four control signals to drive the laser scanner for synchronous scanning of the mid-infrared light beam and the detection light.

7. A system according to claim 6, wherein at least one of the four control signals is adjusted by an angular scaling factor based on the focal length of the detection light objective lens and the mid-infrared light objective lens and the beam expansion ratio of the relay system, and the angular scaling factor is used to determine the relative angular movement of the detection light and the mid-infrared light beam for the laser scanner.

8. The system of claim 1 wherein the relative proportions of movement of the mid-infrared and probe light laser scanning mechanisms are arranged so that the probe light and mid-infrared beams are focused at a plurality of overlapping locations on the sample during scanning.

9. The system of claim 1, further comprising: a first photodiode positioned along the first light path and configured to detect probe light transmitted through the sample; as well as A second photodiode is positioned along the second light path and is configured to detect probe light returning after reflection from the sample.

10. The system of claim 1, wherein the mid-infrared beam is pulsed at a repetition rate between 500 kHz and 1 MHz and a duty cycle of less than 30%.

11. The system of claim 1 , wherein the plurality of pairs of laser scanners are arranged such that the sample is scanned at a frequency of substantially 3 kHz.

12. The system of claim 1 , further comprising an objective lens and a scan lens, both of which are positioned on the second light path between the second pair of laser scanners and the substrate, wherein the probe light is conjugated to a back focal plane of the objective lens through the scan lens.

13. The system of claim 12, further comprising a tube lens positioned in the second light path between the objective lens and the scan lens, wherein the tube lens introduces beam expansion to substantially fill the back focal plane of the objective lens.

14. The system of claim 1, wherein a focus of the mid-infrared light beam is aligned with a focus of the probe light.

15. The system of claim 1, further comprising a reflective objective lens positioned in the first light path between the first laser scanner and the substrate.

16. The system of claim 1, wherein at least one of the laser scanners comprises at least one galvanometer.

17. The system of claim 1, wherein the probe light and the mid-infrared light beam scan across a shared focal point of the sample as each of the laser scanners rotates in both a forward direction and a backward direction, the backward direction being opposite to the forward direction.

18. The system of claim 17, wherein the rotational movement of the laser scanner follows a sinusoidal pattern.

19. The system of claim 1, further comprising a laser scanning position sensor configured to measure a position of at least one of the laser scanners during a scan, wherein a reconstructed image of the sample is adjusted based on feedback from the galvanometer position sensor.

20. The system of claim 1, wherein the laser scanner provides an effective scanning area of ​​at least 100 microns for the mid-infrared light and the probe light on one surface.

21. The system of claim 1, wherein the laser scanner provides an effective scanning area of ​​at least 200 microns for the mid-infrared light and the probe light on one surface.

22. A method of operating a photothermal infrared microscope, the method comprising: a) Illuminating the sample with a beam of mid-infrared light; b) illuminating the sample with a beam of probe light at least partially overlapping the beam of mid-infrared light at the sample; c) synchronously scanning the mid-infrared light beam and the probe light beam at a plurality of locations on the sample so that the two light beams substantially overlap during the scanning process; d) collecting probe light from the sample; e) detecting changes in probe light collected from the sample at a plurality of locations on the sample in response to emission from mid-infrared light absorbed by the sample.

23. The method of claim 22, further comprising extracting a signal from the detected changes in the collected probe light to produce an image representative of the infrared absorption of the sample.

24. The method of claim 22, further comprising collecting an image of a portion of the sample that is mid-infrared absorbing and covers at least 10 microns on one face in 0.1 seconds or less.

25. The method of claim 23, further comprising collecting a plurality of images at a plurality of mid-infrared wavelengths.

26. The method of claim 23, further comprising measuring multiple images at different times to record dynamic changes in the sample.

27. The method of claim 22, further comprising measuring cellular dynamics within the biological cell.

28. The method of claim 23, wherein the image representing the infrared absorption of the sample has a signal-to-noise ratio greater than 50.

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