Systems and methods for microlaser particles

Abstract

Photonic particles and methods of using particles in biological samples are disclosed. The particles are configured to emit laser light when stimulated by, for example, pump source energy. The particles may include: a gain medium having an inorganic material, an optical cavity having a high refractive index, and a coating having an organic material. The particles may be less than 3 microns along their longest axis. The particles may be attached to each other to form, for example, doublets and triplets. The particles may be injection locked by coupling an injection beam into the particles during pumping, so that the injection seeds are amplified to develop into laser oscillations. The microscope system may include a pump source, a beam scanner, a spectrometer having a resolution of less than 1 nanometer and an acquisition rate of greater than 1 kilohertz, and a spectrum analyzer configured to distinguish the spectral peaks output by the laser from a broadband background.

Description

[0001] This application is a divisional application of an invention patent application with an international application date of 2017 / 06 / 05, an international application number of PCT / US2017 / 035923, an application number of 201780046509.1 entering the Chinese national phase, and entitled “Systems and Methods for Microlaser Particles”.

[0002] Cross-references to related applications

[0003] This application is based on, claims priority to, and is incorporated herein by reference in its entirety by reference to, U.S. Provisional Patent Application Serial No. 62 / 345,070, filed on June 3, 2016, and entitled “Laser Micro-Particles.” The references cited in the above-mentioned provisional patent application are also incorporated herein by reference.

[0004] A note about federally funded research

[0005] This invention was made with government support under Grants ECCS-1505569 from the National Science Foundation and DP1-DB024242 from the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0006] This document relates to inventions that generally relate to microlasers that can be embedded, implanted, or injected into samples such as biological cells and tissues, and more specifically to optically excitable laser particles made of organic and inorganic materials, and to the fabrication, functionalization, delivery, and imaging of laser particles, and their use as probes, sensors, and assays for massively parallel imaging. Background Art

[0007] Fluorescent probes (such as dyes, fluorescent proteins and quantum dots) have become indispensable tools in biomedical imaging, cell sorting, immunohistology, high-throughput screening and many other biochemical measurements. Although these luminescent probes are very useful, their relatively wide emission spectra (usually 30-100nm) limit the number of probes that can be used simultaneously without blurring, and often make their spectra indistinguishable from the background emission of endogenous molecules in tissues. Conventional fluorescence microscopes are equipped to distinguish 3 to 4 dyes, and cell counting in the prior art is limited to 11 channels. Multiplexing four different dyes can give 16 (=24) combinations. The simultaneous expression of three genes encoded as blue, green and red fluorescent proteins in cells in different proportions (such as in Brainbow and RGB markers) can generate hundreds of colors. However, transfection is random, and the fidelity of color readings is prone to noise. So far, the number of fluorescent colors used for imaging has been limited to less than a dozen.

[0008] Designing fluorophores for narrower emission linewidths is fundamentally challenging due to quantum mechanical broadening at the electronic level in molecules. Irregular shapes and thermodynamic fluctuations lead to spectral broadening of the emission from semiconductor quantum dots. The decay of plasmon electron oscillations in metal nanoparticles leads to emission widths >50-100nm. In contrast to these electronic resonances, optical resonances provide an effective way to generate narrow emission lines. Lasers are a good example. By placing fluorophores and semiconductor materials in an optical cavity, extremely narrow spectral lines can be generated. The wavelength of the output of a single-frequency laser can be a millionth of a nanometer and can be tuned across the gain width by changing the cavity resonance. Summary of the invention

[0009] The present disclosure provides an example photonic particle comprising at least one gain medium and at least one optical cavity, and in a preferred embodiment such that the maximum dimension of the photonic particle is not significantly greater than 3 μm, or in other preferred embodiments not greater than 2 μm. The gain medium contains a sufficient number of gain elements (such as fluorophores and electron-hole pairs), and the cavity has sufficiently low optical losses so that when the gain medium is excited or stimulated by pump light at a sufficiently strong intensity level, the gain elements emit light exhibiting spectral characteristics defined by the optical resonant mode of the cavity. The particle can be made of a semiconductor material having an appropriate shape and structure (such as a quantum well structure, a spherical shape, or a multilayer Bragg reflector). Alternatively, the particle can be configured with fluorescent gain molecules, high reflection index dielectric resonators, and potentially, metals.

[0010] The example laser particles have an output emission spectrum with one or more peaks, wherein each line width is narrower than 5 nm in preferred embodiments, and typically less than 1 nm or even less than 0.3 nm in other preferred embodiments. The peaks are primarily determined by the resonance of the optical cavity in the particle. The central wavelength of the output spectrum can cover the entire visible and near infrared range, for example, from 400 to 1900 nm.

[0011] In one aspect, the present disclosure provides an optical system comprising at least one pump light source for exciting laser particles and at least one detection arrangement. The detection arrangement comprises a spectrally resolving element, such as a diffraction grating and a dichroic filter. The pump source comprises a pulsed laser with a pulse width in the range of 100 fs to 10 ns.

[0012] Particularly preferred embodiments of laser particles include quantum well microdisk lasers, independent surface emitting Bragg reflector semiconductor lasers, semiconductor spheres, all of which have a diameter or length in the range of 500 nm to 3 μm.

[0013] The foregoing and other advantages of the present disclosure will become apparent from the following description. In this description, reference is made to the accompanying drawings, which form a part hereof, and in which preferred embodiments of the present disclosure are shown by way of illustration. However, such embodiments do not necessarily represent the full scope of the present disclosure, and therefore reference is made to the claims and are used herein to interpret the scope of the present disclosure.

[0014] The foregoing and other aspects and advantages of the present disclosure will be presented according to the following description. In this description, reference is made to the accompanying drawings which form a part hereof, and in which one or more example versions are shown by way of illustration. These versions do not necessarily represent the full scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present disclosure will now be described with reference to the drawings, wherein like reference numerals refer to like elements.

[0016] Figure 1 is an illustration of a common use of lasers, where the laser is placed outside a biological system.

[0017] Figure 2 Illustration of the biomedical use of laser particles. Small biocompatible laser particles are injected into the system. Such lasers can provide new methods in imaging, diagnosis, and treatment, and allow two-way interaction with biological systems. As an extension of the concept, biological samples can be incorporated into the laser so that changes in the specimen are reflected in the output emission of the laser. In addition, the laser can be made entirely of biological matter to achieve a "living" laser that is self-powered, operated, and maintained.

[0018] Figure 3A The fluorescence emission from the pumped gain medium of the laser particle is shown. The red arrows indicate the fluorescence.

[0019] Figure 3B The optical cavity that forms the laser particle is shown. Figure 3A gain medium.

[0020] Figure 3C Shows from Figure 3B Spontaneous and stimulated light emitted from the gain medium in the cavity shown in Figure 1. The red lines in the cavity represent the cavity modes.

[0021] Figure 3D An exemplary energy diagram of a gain element (yellow circle) having two sets of energy levels or energy bands is depicted. Pumping excites the gain element to a higher electronic state from which it relaxes to the ground state, emitting spontaneous or stimulated emission. Although two electronic states are depicted in the figure, the gain medium forms a quasi-four level system taking into account nonradiative transitions within each band of electronic states.

[0022] Figure 4A The laser mode P1 and the spontaneous emission P in the steady state are depicted. f Numerical calculation of the output rate, where β = 0.01. The laser output increases nonlinearly near the threshold; the sharpness of the threshold increases as β decreases. Numerical calculation of steady state. (a) In steady state, the laser mode P1 and the spontaneous emission P f The output rate of the laser is , where β = 0.01. The laser output increases nonlinearly near the threshold; the sharpness of the threshold increases as β decreases.

[0023] Figure 4B The number of gain elements is shown, which is expressed as N1=P according to the following equation (2): th τ s q / (1+q), or equivalently, according to equation (5) N1=P f τ s · / (1-β).

[0024] Figure 5A Numerical simulations for a pump duration of 20 ns are shown. The simulation parameters are: τ s =3ns, τ c =30ps, β=0.01, and P th =3.54x10 12 s -1 According to equation (11), the pulse rate is set to twice the threshold rate.

[0025] Figure 5B Numerical simulations for a pump duration of 100 ps are shown. The simulation parameters are: τ s =3ns, τ c =30ps, β=0.01, and P th =3.54x10 12 s -1 According to equation (11), the pulse rate is set to twice the threshold rate.

[0026] Fig. 6A Molecular energy levels related to dye lasers, showing the Franck-Condon principle energy diagram for fluorophore dyes. Since electronic transitions are very fast compared to nuclear motions, transitions occur between vibrational levels in the same nuclear coordinates.

[0027] Figure 6B Absorption (dashed line) and autofluorescence (solid line) spectra of organic fluorescent dye molecules are shown.

[0028] Figure 6C A 4-level energy level diagram is shown for the pump and indicated signal wavelengths.

[0029] Fig. 7A Referring to fluorescent proteins, the chemical structure of GFP is shown. The fluorophore (insert) is protected within a beta barrel that provides many desirable properties such as stability to the environment.

[0030] Figure 7B Fluorescence intensity of aqueous eGFP solutions with different concentrations (□) and thin films of solid eGFP (at 40 mM, ■) are shown. Fluorescence of synthetic pyrromethene dye (○). All data are normalized to sample thickness and corrected for excitation depletion. Black dashed line: linear fit at low concentration (with no concentration quench). Green and pink lines: linear fit for eGFP, respectively. type quenching model and an aggregation-induced quenching model for pyrromethene dyes.

[0031] Fig. 8A An energy-momentum diagram (or Ek diagram) for a direct semiconductor lattice involving the potential energy and momentum of the carriers is shown. Excitation of electrons to the conduction band by pumping creates holes in the valence band.

[0032] Figure 8B Stimulated emission by recombination of free electrons in the conduction band and holes in the valence band is shown.

[0033] Fig.9AAn optical resonator for an independent laser particle in the form of a Fabry-Perot cavity with end reflection is shown.

[0034] Fig. 9B Disk-shaped, ring-shaped, and spherical whispering gallery mode (WGM) resonators are shown, respectively.

[0035] Fig. 9C Photonic crystal resonators with linear, planar, and radial grating structures are shown, respectively.

[0036] Fig.9D Shown are random microresonators formed by clusters of scattering particles.

[0037] Fig. 10A A firefly luciferase-luciferin chemically initiated electron exchange luminescence (CIEEL) reaction is provided.

[0038] Fig. 10B Provides alkaline phosphatase (AP)-chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) CIEEL reaction.

[0039] Fig. 10C A peroxyoxalate-dye CIEEL reaction is provided. Here, bis(2,4,6-trichlorophenyl)oxalate and 9,10-diphenylanthracene (DPA) are used as examples of peroxyoxalate and luminescent dye, respectively.

[0040] Fig. 10D A peroxidase-luminol CIEEL reaction is provided.

[0041] Fig.11 Thermodynamic processes during pulsed operation of laser particles are shown.

[0042] Fig.12 Shown are the temperature profile of a sphere with radius R in a medium with relaxation time τ, the temperature distribution at different times (left), the time profile of the temperature inside the sphere at r=0.5*R (middle), and the accumulated temperature rise after 100 pump pulses as a function of the pulse repetition period (right).

[0043] Fig.13A bioluminescence-driven microresonator is depicted. When luciferin reacts with luciferase attached to the surface of the bead, it generates light, part of which is coupled into the whispering gallery cavity modes (left). Light emission from the luciferase-coated bead is shown (center). The optical modes are clearly observed in the spectrum of the emitted light (right).

[0044] Fig.14 The principle of high-resolution optical sectioning is shown by detecting stimulated emission from laser particles. At the top left, a microscopic laser particle (circle) is excited by a tightly focused optical pump beam (grey). Comparison of different modalities: brightfield imaging (top right); two-photon microscopy (bottom left); laser particle stimulated emission (LASE) microscopy (bottom right).

[0045] Fig.15 Schematic diagram of the setup is shown. L1, L2, L3: spherical lenses, λ / 2: half-wave plate, CL: cylindrical lens, DM: dichroic mirror, M: mirror, Obj: objective lens (NA=0.8, water immersion), LF: long-pass filter, and BS: beam splitter. SEM: scanning electron microscope image of a typical lead iodide perovskite nanowire. Insets (from left to right): a typical fluorescence image of a perovskite nanowire below threshold, an image of stimulated emission of the nanowire above threshold, and a pump beam profile recorded in a charge-coupled device camera.

[0046] Fig.16A A typical laser output spectrum from a perovskite nanowire is shown (circles). Curves: curve fit of the fluorescence background (grey) and the laser output spectrum of stimulated emission (magenta) calculated by subtracting the fluorescence background from the measured spectrum.

[0047] Fig. 16B Shown are the stimulated emission output powers (squared) measured as a function of the pump pulse energy intensity level. Line: curve fit based on equation (6). Inset: same graph on a log-log scale.

[0048] Fig. 16C The nanowires are shown in Figure 2. th )<1 (green dashed line) and three pump-beam scan profiles of stimulated emission at p=1 (gold) and p=1.8 (cyan).

[0049] Fig.16D Measured FWHM values ​​of the lasing profile (blue circles) and the fluorescence profile (green circles) are shown. Dashed curve: simulation results.

[0050] Fig.17ADepicted is an exemplary hyperspectral microscope for measuring laser emissions from laser particles and thereby identifying and locating them in accordance with one or more embodiments.

[0051] Fig. 17B Depicted is an example multimodal microscope combining hyperspectral and multiphoton imaging in accordance with one or more embodiments.

[0052] Fig.18A The principles and applications of injection-locked laser particles are depicted, showing that injection locking requires coupling of an injection beam into the laser particle when the pump is turned on, so that the injected seed is amplified and developed into laser oscillations.

[0053] Fig.18B The injection locked laser output is shown, which is typically different from the output spectrum in the free running mode output without injected seed light. The injection locked output has a high degree of coherence with respect to the injected beam. The coherence allows the laser output to be detected by exploiting the interference of the output of the injected laser.

[0054] Fig.18C Schematic illustration of wavefront shaping guided by narrow-band light from laser particles embedded in a scattering medium such as biological tissue.

[0055] Fig.19A Depicted is a schematic diagram of an example microfluidic chip for loading laser particles into cells in a controlled manner according to one or more embodiments.

[0056] Fig.19B Depicted is a device for reading the laser particles in each cell according to one or more embodiments. A sorting device can also be added to sort cells based on the readout of the laser emission spectrum.

[0057] Fig. 20 Depicted is an example schematic diagram of a flow cytometer based on laser particles as luminescent probes targeting specific cell surface markers in accordance with one or more embodiments.

[0058] Fig.21A Scanning electron microscope (SEM) images of an example near-infrared InGaAsP microlaser (200 nm x 1.8 μm, on a partially removed sacrificial pillar) according to one or more embodiments are provided (on the left); an example microlaser wafer after partial etching according to one or more embodiments, showing the feasibility of generating multiple laser particles from a single wafer; output spectra from a 200 nm x 1.8 μm disk in water and air, where the threshold pump energy is about 5 pJ (10 ns, λ p=980nm) (right). In the right figure, the line width is only 1 / 1000 of the gain bandwidth. According to one or more embodiments, thermal analysis predicts that by 2 Heating up to 10 nJ of deposited energy is negligible.

[0059] Fig.21B Scanning electron micrographs of a microdisk laser with three InAlGaAs quantum well layers according to one or more embodiments are provided: after electron beam lithography and ion beam etching (left); after partial wet etching, leaving pillars (middle); and after complete etching (right). The isolated disk-shaped laser particle shown (right) has a thickness of 200 nm and a diameter of about 2.3 μm.

[0060] Fig. 21C On the left is shown laser light emitted from a separate disk on a plastic dish in accordance with one or more embodiments. On the right is provided the output spectrum from another disk laser collected through mouse ear skin (thickness approximately 500 μm) in accordance with one or more embodiments.

[0061] Fig.22A Depicted is a method for producing laser particles with different spectral barcode characteristics according to one or more embodiments. At the top, microdisk lasers with incrementally varying diameters are shown. At the bottom, distinctly different laser mode wavelengths depending on size are shown.

[0062] Fig. 22B Various semiconductor microdisks produced by electron beam lithography are shown for different disk diameters.

[0063] Fig.23 Shown is a histogram of output wavelengths of 512 laser disk particles made from four different semiconductor wafers with different alloy compositions indicated by color, according to one or more embodiments. The bin size is 1 nm.

[0064] Fig.24A Depicted are preferred embodiments for surface treatment of laser particles to achieve biocompatibility and strategies for targeted delivery: semiconductor disk laser (left) and encapsulation with a passivation layer (e.g., SiO2) (right).

[0065] Fig. 24B Depicted are a polymer coating (eg, poly(ethylene glycol)) (left) and optional attachment of functional molecules (right).

[0066] Fig.24C Schematic diagram depicting liposomal encapsulation of laser particles with liposomal carriers (left) and targeted liposomal carriers (right) for systemic delivery.

[0067] Fig.25 SEM images of two exemplary microdisks coated with silicon dioxide layers of different thicknesses according to one or more embodiments are provided.

[0068] Fig.26 Optical images of cells containing example laser particles according to one or more embodiments are provided. Bright field and fluorescence images of cells expressing GFP are shown. The laser disk is nearly flat along the viewing plane in the first column and tilted from the viewing plane in the second column. In the third column, the bright spot in the microscope image is due to the pump laser beam being focused on that area.

[0069] Fig. 27 Depicted are characteristics of the laser output from an example laser disk particle in a cell in an exemplary embodiment. At the top left, the number of photons measured as a function of pump energy is shown. The kink in the curve indicates the onset of lasing at a threshold pump energy of about 20 pJ. At the top right, output spectra from three laser particles L1, L2, and L3 measured at time intervals of about 1 hour are shown. At the bottom left, the full-width half-maximum (FWHM) of the measured spectra from three cells as a function of pump energy level normalized to their respective lasing thresholds are shown. At the bottom right, output spectra from 10 different intracellular laser disk particles measured 1 day after loading the laser disk particles into different cells are shown.

[0070] Fig.28 The principle of cell labeling and tracking based on wavelength division multiplexing of laser particles (squares) according to one or more embodiments is shown. Cells containing different numbers of lasers and their output laser lines are depicted. The number of combinations of single laser particles (S; singlet), two-particle doublets (D; doublets), and triplets (T) are shown. The numbers in brackets correspond to the number of elements per cell, m. The division of cells (circles) is tracked by tracking the laser probe.

[0071] Fig.29A An example process for constructing doublet and triplet particles is shown, depicting the bond of two arrays of laser particles, in accordance with one or more implementations.

[0072] Fig.29BDepicted are two types of doublet particles: with a biocompatible coating on the doublet particles (two schematics on the left), and with beads as optically insulating spacers (two schematics on the right). The doublets can be produced by assembly or directly from multilayer wafers and further coated with materials such as biocompatible polymers.

[0073] Fig.29C Depicted is a schematic diagram of a triplet laser particle made from three lasers with different diameters and different output wavelengths (right), according to one or more embodiments. DETAILED DESCRIPTION

[0074] Before describing the present invention in further detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting. The scope of the present invention will be limited only by the claims. As used herein, the singular forms "a", "an" and "the" include plural embodiments unless the context clearly indicates otherwise.

[0075] It should be apparent to those skilled in the art that many additional modifications other than those described are possible without departing from the inventive concepts herein. When interpreting the present disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. The variant of the term "comprising" should be interpreted as citing elements, parts or steps in a non-exclusive manner, so the quoted elements, parts or steps can be combined with other elements, parts or steps that are not clearly mentioned. Embodiments that are referred to as "comprising" certain elements are also considered to be "essentially composed of these elements" and "consisting of these elements".

[0076] like Figure 1 As depicted in , the standard paradigm of using light to characterize or manipulate samples in biomedical applications introduces several practical and conceptual limitations. For example, light undergoes scattering and absorption as it propagates through biological tissue, so the 1 / e penetration depth of light in the visible and near-infrared range does not exceed 3 mm. This leads to difficulties in delivering light into tissue beyond optical penetration and limits the clinical usefulness of light. Moreover, when the laser is used only as an illuminator, the interaction between the light source and the biological system is unidirectional; the laser affects the sample, but not vice versa.

[0077] Lasers that are biocompatible and miniaturized to the size of a cell (or smaller) can be used inside living systems by being implanted in the body for a certain duration, or injected into tissue and operated remotely. This new paradigm involves physically delivering the light source to the target. Figure 2, such laser "particles" can be used as internal light sources, which can solve at least some of the practical problems of using lasers for external light delivery (such as penetration depth), as well as enable new ways of using light for phototherapy. In addition, laser particles embedded in tissue can be made to interact with their local environment in various ways, allowing two-way interaction. Not only can lasers affect the biomedical environment, but living systems can also affect lasers, changing their output characteristics. This ability can be harnessed to improve diagnostics and health monitoring.

[0078] A laser consists of three elements: a gain medium, a cavity, and a pump energy source. Optical amplification in the gain medium is necessary to generate stimulated emission. An amplifier with sufficient gain and a long propagation length can generate amplified spontaneous emission. Although this process can generate an output with laser-like properties such as spectral narrowing, the narrow definition of the laser requires optical feedback from the cavity. The cavity confines and resonates the light.

[0079] In addition to optical or photonic cavities, other types of cavities of quasiparticles, such as polaritons and plasmas, can be used to make lasers possible. Polariton lasers based on exciton polaritons are representative examples of "non-photonic" lasers. The mechanism of polariton lasers is usually described in the context of Bose-Einstein condensation of polaritons. Surface plasmon amplification or "spaser" by stimulated emission of radiation usually involves a metal cavity for free electron or plasma resonance.

[0080] Consider a gain medium containing active gain elements such as dye molecules in aqueous solution or semiconductor atoms assembled in a solid-state crystal. Figure 3A A gain medium is shown in which the gain elements are excited by a pump source (such as an excitation laser) to a higher energy state from which they emit fluorescence when returning to the original ground state. Figure 3B and 3C In the example above, the same gain medium is now placed inside an optical cavity and pumped at a rate of P, part of the fluorescence is captured in the cavity, and the intracavity light is amplified by stimulated emission. If there is sufficient single-pass gain and sufficient passage through the gain medium, the intracavity light will develop into laser emission. While the spontaneous fluorescence emission radiates in all directions, the laser emission leaves the cavity with a specific intensity pattern determined by the cavity. However, for small cavities, where the size is comparable to the optical wavelength, the directionality of the output coupling is less pronounced, and the laser will behave as a point source for both spontaneous and stimulated emission.

[0081] Figure 3DThe pumping and emission processes in the gain medium are depicted using energy diagrams of the electronic states of gain elements (such as fluorophores, electrons in semiconductors, or ions in crystals). The gain elements are initially populated in a ground state, which is typically composed of many vibrational states, which are either discrete or continuous. Gain elements that absorb pump energy are excited to excited states, then accumulate in lower energy levels of the excited states through non-radiative intraband transitions, and then undergo spontaneous or stimulated emission when interacting with intracavity photons.

[0082] The simplified rate equation describing the laser particle in this type of four-level gain medium can be written as:

[0083]

[0084]

[0085] Here, N1(t) is the number of excited fluorophores in the gain medium, q(t) is the number of photons in the laser cavity mode, P(t) is the pump rate, and τ s is the spontaneous emission (fluorescence) lifetime of the gain molecule, and the spontaneous emission factor β describes the fraction of spontaneous emission that is captured by the laser mode. 1 / τ s corresponds to the spontaneous emission rate for all possible radiation modes at all frequencies and directions; therefore, β / τ s represents the spontaneous emission rate coupled to the laser mode. The stimulated transition rate is proportional to the number of cavity modes and is equal to the spontaneous transition rate of the cavity mode multiplied by the number of photons: βN1(t)q(t) / τ s .

[0086] For optical pumping, the pump rate can be expressed as:

[0087]

[0088] where QY is the quantum yield that specifies the probability that an absorbed photon will generate a photon via spontaneous or stimulated emission, σ a is the absorption cross section of the gain element, and N g (t) is the number of gain elements in the ground state. Figure 3D In most cases, N g The sum of N1(t) and N1(t) equals the total number of gain elements N 总 (t), N 总 (t) can be constant or vary with time.

[0089] In equation (3), the intensity of the pump light is assumed to be spatially invariant. This assumption applies to laser particles whose size is much smaller than the size of the pump beam. For larger lasers with non-uniform pumping, the pump rate is determined not only by the peak pump power but also by the overlap between the pump intensity profile and the laser mode profile.

[0090] The output emission rate of the cavity mode P l (t) and the output emission rate P of the spontaneous fluorescence emission f (t) is given by the following equation:

[0091]

[0092]

[0093] where q l (t) and q f (t) are the number of emitted photons of the laser mode and fluorescence emission, respectively. It follows that P(t) = P l (t)+P f (t), which states the conservation of the number of photons (given by the definition of P(t)).

[0094] Steady state (t>>τ s ): When the laser is in a steady state (ie, dN1 / dt = dq l / dt=0), we get

[0095]

[0096]

[0097] in

[0098] P th =(βτ c ) -1 . (8)

[0099] Figure 4A and 4B A plot of the stimulated and spontaneous output emission rates as a function of the pump rate is shown. From the plot, it is evident that P th corresponds to the pump rate of the laser mode at threshold.

[0100] Laser oscillation enhances the "recycling" rate of the gain element. Below the laser threshold, unless gain saturation occurs; that is, N1 becomes equal to N 总 The number of excited elements N1 increases linearly with the pump rate ( Figure 4B). Above threshold, N1 is clamped to a constant level regardless of the pump rate, so that the optical gain is equal to the cavity losses; i.e., the net gain of the laser is unity. As pumping increases, the intensity of the light within the cavity increases, and so the rate of stimulated emission also increases. This process brings the excited gain elements to the ground state at an increased rate. This enhanced relaxation rate is balanced by the pumping rate, resulting in a constant number of gain elements in the excited state and a constant optical gain. As a result, the effective lifetime of the excited state decreases as the pumping rate increases above threshold.

[0101] Transient build-up state: The governing equations (1) and (2) describe the dynamics of the laser oscillation. The necessary condition for the build-up of the laser mode is dq(t) / dt>0 (at t=0; q=0), where the minimum number of gain elements in the excited state should satisfy:

[0102] N1(t)>τ s / βτ c (9)

[0103] Consider supplying pump energy at a uniform rate for a duration of τ p For q = 0 (below threshold) and a square-profile pump pulse, the solution to equation (1) is:

[0104] N1(t=0)=Pτ s [1-exp(-τ p / τ s )] (10)

[0105] The pump rate required to achieve the threshold condition in equation (9) is:

[0106] P th,τp =(βτ c ) -1 ·[1-exp(-τ p / τ s )] -1 (11)

[0107] For the case of quasi-continuous pumping (τ p >τ s ), we confirm equation (8):P th =(βτ c ) -1 ; The pump power at the lasing threshold is independent of the pulse duration, and the threshold pulse energy is proportional to the pulse duration. On the other hand, for the case of short pulse pumping (τ p <τ s ), we found that P th =(βτ c ) -1·τ s / τ p ; When the pulse width decreases, the pump power increases, and the threshold pump energy P th τ p Independent of pump pulse duration.

[0108] Figure 5A and 5B Numerical results for two schemes are shown: Figure 5A In, τ p >τ s , and in Figure 5B In, τ p <τ s , where τ s =3ns and τ c =30ps. First, for τ p = 20 ns, after the start of pumping at t = 0, the number of excited gain elements N1 and the fluorescence emission rate P f Increase( Figure 5A ). The initial overshoot and rapid ringing are called relaxation oscillations, which are typical for square pulses but less pronounced for Gaussian pulses. s The laser reaches steady state when t = 20 ns. In this simulation, the pump rate is set to twice the threshold according to equation (11). After the pumping ends at t = 20 ns, the fluorescence emission starts with a relaxation time equal to τ. s =3ns exponential decay, while stimulated emission takes the cavity lifetime 1 as τ c =30ps decreases exponentially. When using τ p = 100ps short pump pulse, a short laser pulse is generated ( Figure 5B ). The output pulse duration is of the same order of magnitude as the peak width of the relaxation oscillations. The population of excited state gain elements peaks at the end of the pump pulse, decays rapidly with the emission of the laser pulse, and decays exponentially once it drops below a threshold level.

[0109] From equation (9), the minimum number of excitation gain elements in the laser should be greater than τ s / (βτ c ). In order to have so many elements in a gain medium of volume V, the absolute minimum concentration is greater than τ s / (βτ c V). For example, when τ s =3ns, τ c = 300fs, and β = 0.05, the gain medium should have at least 2x10 5 gain element. For V = 10 μm 2 , the theoretical minimum concentration is 330 nM, and for V = 1 μm 2, requiring a higher concentration of more than 330 μM. In practice, most lasers require N much larger than N1 due to limited absorption of pump energy (discussed further below). 总 Therefore, the minimum concentration of gain elements is significantly higher than the above estimates by one or two orders of magnitude.

[0110] Semiconductor and solid-state lasers are more compact and easier to maintain than dye lasers. However, fluorescent dyes are an important and viable option as gain materials for biomedical laser particles. A variety of fluorescent dyes with improved brightness and photostability compared to earlier laser dyes and optimized biosensitivity are available for fluorescence-based bioimaging and biochemical assays.

[0111] Fig. 6A Depicted are the electronic and vibrational energy levels of a typical fluorophore molecule as a function of the atomic separation in the molecular structure. Upon absorption of a pump photon, the molecule undergoes an electronic transition from the ground state (g) in the highest energy occupied molecular orbital or HOMO band to an excited state (1') in the lowest energy unoccupied molecular orbital or LUMO band. The initially excited electron migrates to the lowest energy level (1) in the LUMO by nonradiative (vibrational) relaxation, from which the electron decays to the ground state (g') in the HOMO, thereby emitting light. This energy diagram is generally applicable to any molecule, including isolated fluorescent dyes in solution, fluorescent probes (at low concentrations such that the dyes interact weakly with each other), and protected fluorophore units within protein structures (i.e., fluorescent proteins).

[0112] Figure 6B Typical absorption and emission spectra of fluorophores are shown. The absorption and emission spectra partially overlap each other, which can be explained by Fig. 6A In this example, when pumped by blue light (λ≈430-490 nm), the molecule forms an effective 4-level system of laser operation with negligible fluorescence emission. The 4-level system consists of: a base level g; a pump level 1'; a metastable upper laser level 1; and a lower laser level g' ( Figure 6C For most organic molecules, the lifetime of this upper state is typically 1-10 ns, but can be amplified by external perturbations such as quenching or The transition from the upper laser level to the lower laser level can be by both spontaneous emission or stimulated emission.

[0113] Typical dye molecules have peak extinction coefficients (ε) in the range of 10,000-100,000 M -1 cm -1 For example, let us consider a network with ε = 56,000M -1 cm -1If half of the molecules in the gain medium are excited to the upper level at a given time, we can obtain up to 16,800 M -1 cm -1 Stimulated emission (gain) of 1 μM in a 1-cm-long cuvette can generate 10 0.0168 = 1.039 single pass optical gain; that is, the optical intensity is amplified by 3.9% or 0.168 dB. The same amount of single pass gain is achieved by a single mammalian cell containing a concentration of 1 mM of the molecule in a cytoplasm with a diameter of 10 μm.

[0114] Fluorescent proteins are proteins that are able to emit fluorescence. Green fluorescent protein (GFP) was the first type discovered in the jellyfish Aequorea victoria. The increasing popularity of fluorescent proteins in biomedical science has led to the continuous discovery of new wild-type proteins from various organisms, and to the development of mutant variants with improved fluorescence properties. Several effective fluorescent proteins have been derived from jellyfish (eGFP, mCFP, eYFP, etc.), coral reefs (DsRed, tdTomoto), and bubble tip sea anemones (TurboRFP). FPs have high QY and large absorption and emission cross sections, comparable to or even better than typical fluorescent dyes, and may be very suitable for use as gain media for biological lasers. The most unique and significant advantage of FPs compared to small molecule organic dyes is that they can be genetically encoded. This allows the expression of FPs in specific target cell types by genetic targeting. This property of FPs can be used to achieve genetic specificity for the application of laser particles based on FPs as gain materials.

[0115] Reference Fig. 7A , FPs have a common pot-type cylindrical shape with a fluorophore in the center. GFP has 11 strands of a conventional β-barrel surrounding the actual fluorophore 4-(p-hydroxybenzylidene)-imidazolidin-5-one at the center of the molecule. The β-pot structure is essential for the fluorescence of GFP because it forces the Ser-Tyr-Gly sequence into its emitting conformation. The unique protective molecular shell prevents concentration quenching of fluorescence. GFP maintains bright fluorescence. This is in contrast to small synthetic fluorescent dyes, which lose their fluorescence at high concentrations by quenching at high concentrations (see Figure 7B ). The low quenching of GFP at high concentrations results in a very high quenching of 96 dB cm for the dried enhanced GFP film. -1 The gain factor of .

[0116] Inorganic semiconductors can be used as gain materials for lasers. Semiconductors are crystalline or amorphous solids with a filled valence band and an empty conduction band. Semiconductors differ from insulators in that the band energy gap E g Typically less than 4 eV, so that some electrons can transition to the empty conduction band by optical or thermal excitation, and the materials can be doped with impurities to change their electronic properties. The most well-known semiconductor materials are crystalline inorganic solids. They include: Group IV materials such as silicon (Si) and germanium (Ge), Group III-V compounds such as GaAs and InP, and their alloys such as AlGaAs and InGaAsP, and II-VI compounds such as CdS and ZnO. Oxides such as TiO2, and 2-dimensional (2D) transition metal dichalcogenides such as MoS2 and WS2 are also semiconductors. Since laser action relies on an efficient radiation process, indirect gap materials such as silicon and germanium may not be suitable, and direct gap III-V and II-VI compound semiconductors are preferred.

[0117] Regarding the physics of laser action in these systems, Fig. 8A and 8B A simple energy diagram for an intrinsic semiconductor material with a direct bandgap is shown. Because the valence band is completely filled in the absence of pumping, this semiconductor laser behaves like a three-level laser system. A finite population of excited states is necessary to obtain net gain in the material; the carrier density that achieves this condition is called the transparent population and is typically on the order of 10 18 cm -3 The stimulated emission of photons in these materials results from the radiative recombination of free charge carriers, namely electrons in the conduction band and holes in the valence band.

[0118] To achieve finite gain, population inversion is required, although lasing schemes without inversion exist. The number of electronic states in the conduction and valence bands is easily determined from the density of carrier states, ρ c To calculate, it is:

[0119]

[0120] For bulk (3D) semiconductors, where m * is the effective mass of the carriers. c The number of electrons with energy up to ΔE, N1, is given by the following equation:

[0121]

[0122] For example, for bulk GaAs, the effective electron mass is m e * =0.61x10 -31 kg; when T = 0K and ΔE = 0.1eV, we get N1 / V = 2.5×10 18 Electron / cm 3 , which is 4.15 mM in terms of electron concentration, comparable to the highest density of organic fluorophores without quenching (see Figure 7B ). In practice, semiconductors can achieve very large gains of about 1% per micron.

[0123] Semiconductors efficiently absorb all wavelengths of light shorter than their band edge. The absorption coefficient (α, on a natural logarithmic basis) is about 10 in optical frequencies just above the band gap. 4 cm -1 The radiative lifetime of the emitting transition (electron-hole combination) is τ s The particle number density of holes and the radiation recombination coefficient B (B≈10 -10 cm 3 s -1 ) is proportional to: approximately,

[0124] τ s =(BN1 / V) -1 . (14)

[0125] For a typical particle number density at the laser emission location (N1 / V≈10 18 –10 19 cm -3 ), the radiation lifetime is 1-10ns, which is of the same order of magnitude as the fluorescence lifetime of the dye.

[0126] Early semiconductor lasers exploited population inversion obtained at the interface region of a pn homojunction. Due to diffusion of minority carriers, the actual active region is relatively thick (about 1 μm), requiring high threshold pump energy. A more efficient design is a double heterostructure, where the active region is defined as a thin layer (about 100 nm) of a different semiconductor with a lower bandgap and a higher refractive index. With this architecture, the charge carriers are confined in a thin active layer, increasing the population density. Higher N1 can be achieved by further limiting the size of the charge to << 100 nm where quantum effects become dominant (such as quantum wells (QWs) with confinement in one dimension, quantum wires with 2D confinement, and quantum dots (QDs) in 3D confinement).

[0127] The density of states of the QW is corrected to

[0128] The gain spectral range of the semiconductor can be tuned to some extent by changing the stoichiometry of the tuning compound and inducing mechanical stress. The emission spectra of 720-850 nm for AlGaAs and 900-1100 nm for InGaAs are relevant for biomedical applications because they match well with the optical windows for light penetration in tissues. For emission in the visible, the InGaAlP system works in the red (630-700 nm), while for the blue-green region (400-530 nm), group III nitrides (like GaN and InGaN) are usually used.

[0129] Semiconductor materials can be grown via epitaxial processes on wafers, whereby the final device is manufactured via photolithographic processes. Nanoscale inorganic semiconductors can also be grown in the form of colloidal nanocrystals by chemical synthesis in solution. Various colloidal nanocrystals are widely used in biological applications, mainly as fluorescent labels for bioimaging and biosensing. Due to the high quantum confinement in the nanoscale size, the band gap and therefore the emission wavelength can be easily tuned by controlling the size of the particles. Typical nanocrystals are in the shape of spherical core / shell quantum dots, in which the core is the emitting material, and the shell is usually used as a passivation layer to reduce the non-radiative recombination from surface defects. In addition, the core / shell structure has been shown to be important for obtaining optical gain using these materials. More complex shapes, like rods or branched nanocrystals, have also been developed, which have the advantages of increased radiative transition lifetime, controlled polarization and multiple emission bands.

[0130] Organic semiconductors are formed in a concentrated state, and they are incorporated into fluorescent small molecules, oligomers or polymers at very high concentrations, so that each element is coupled and jointly forms a π-conjugated system with delocalized electrons. The π-conjugated system gives them strong electronic transitions and the ability to conduct charge in visible light. The typical structure of an organic semiconductor is composed of a central conjugated skeleton that determines the main optoelectronic properties, modified with a side group (side group) (such as an alkyl chain, to improve solubility) that can be customized to give the molecule a specific function. Organic semiconductors usually have a fairly wide gain spectrum, particularly in the case of macromolecular conjugated polymers, which allows their emission to be fine-tuned without changing the material. Laser emission from these materials usually occurs in the visible range. Conjugated polymers with a band gap in the near infrared do not usually have effective luminescent emission.

[0131] Laser emission in organic materials involves the radiative recombination of singlet excitons. The lifetime of excitons is hundreds of picoseconds to hundreds of nanoseconds. As in the case of fluorescent dyes and proteins, this short lifetime requires high pumping rates to reach the laser threshold, and organic semiconductor lasers have only been demonstrated in pulsed regimes. Compared to crystalline inorganic semiconductors, organic semiconductors have lower charge mobility, and polarons (electron-phonon coupled charge carriers) have significant absorption, resulting in high losses at high carrier densities. For these reasons, electrical pumping of organic semiconductors has not been successful to date.

[0132] The stability of organic semiconductors is an important factor to be considered for use in biological environments. These materials are usually very sensitive to physicochemical interactions with oxygen and water, which are particularly unfavorable for charge transport and fluorescence emission. Nevertheless, organic semiconductors provide several properties that are potentially attractive for laser particles in biological environments. One is that compared with inorganic materials, they usually have excellent biocompatibility due to their organic composition, more compliant mechanical properties, and cell-friendly surface roughness. Some organic materials also allow ion and electron conduction, making them suitable for engagement with biological tissues, where electrical signals are usually transmitted by ion movement. In addition, organic materials can be chemically customized using functional groups that are sensitive to specific analytes. Chemical and biological sensing based on fluorescence modulation and even laser emission from conjugated materials has been reported.

[0133] Ions of rare earth or transition metals included as impurities in a transparent solid host are well-established gain elements for lasers. The first laser demonstrated by TH Maiman in 1960 used a Cr-doped Al2O3 crystal. 3+ The host material can be an oxide crystal (for example, Al2O3 and Y3Al5O 12 Amorphous materials can be made into micrometer-sized materials more easily than crystalline hosts, and their optical attenuation and thermal impedance (usually higher than crystals) are ideal for small lasers. The most common dopant ions for gain elements include the trivalent lanthanide neodymium (Nd 3+ ), Erbium 3+ ) and ytterbium (Yr 3+ ), and transition metal titanium (Ti 3+ ) and chromium (Cr 2+ Cr 3+ Cr 4+). They usually have multiple electronic transitions that can support lasing, and their emission properties also depend on the specific host material. In most cases, lasing is in the near-infrared region between 0.9 and 1.6 μm, but there are also some systems at shorter (like ruby ​​at 694 nm) and longer (like Er:YAG at 2.9 μm) wavelengths. A common characteristic of ion-doped solid-state lasers is that the electronic transition from the upper state to the ground state is much weaker, making the relaxation time τ s Can be very long, in the order of μs to 10ms. Long relaxation times are well suited for continuous operation.

[0134] In view of the long lifetime, rare earths are suitable for upconversion to generate emission at a higher energy than the pump photon. The process involves absorbing two photons in sequence to promote the electron to a metastable state, then to an excited state, from which the electron decays to the ground state, thereby emitting a single photon with about twice the energy of the pump photon. Since the intermediate state has a long lifetime, the process is very efficient. Upconversion is different from typical nonlinear two-photon absorption, because the intermediate state (and similarly, the final upper state) of the typical nonlinear two-photon absorption is a virtual state with a lifetime of only a few femtoseconds, so only ultrafast pulses with high peak intensity can be used to achieve effective excitation. Detailed photophysical approaches have been extensively reviewed elsewhere. The first laser based on upconversion was demonstrated in 1971, and the process has attracted significant interest because it allows the use of near-infrared pumps to obtain visible light emission, especially for biomedical applications as luminescent nanoparticles. Such materials are attractive candidates for laser particles, with the major advantage of near-infrared pump light, which has a deeper penetration depth and is less harmful in tissue than visible light.

[0135] An optical resonator or laser cavity provides phase-coherent optical feedback, allowing regenerative amplification in the gain medium. Various optical elements, including the gain medium, can be incorporated into the laser cavity to control and tune the optical properties of the intracavity light and the output laser properties.

[0136] Cavity modes are specific spatial (transverse) and spectral (longitudinal) optical waves that can resonantly oscillate in a cavity. Transverse modes are determined by the shape and size of the cavity along the dimension parallel to the axis of optical oscillation. Longitudinal modes are a set of frequency components that satisfy the total optical phase accumulated in one round trip equal to an integer multiple of 2π. This resonance condition is expressed as:

[0137]

[0138] Where l is a positive integer, ν lis the optical frequency of the lth longitudinal resonant mode, L c is the round trip length of the cavity (e.g., twice the length of a linear cavity or 2π times the radius of an annular cavity), and n is a l Since the minimum l is 1, the minimum length scale of the resonator is equal to the free space wavelength λ divided by the effective refractive index n of the resonator.

[0139] A high n is beneficial for small lasers because it allows a small L c Semiconductor materials are therefore attractive materials for realizing resonators (and gain media). Plasmon polaritons made of metals are another attractive approach, which exploits the fact that the real part of the refractive index is enhanced by coupling of light to plasmons, the collective motion of free electrons confined in the cavity.

[0140] The number of cavity modes scales with the volume of the cavity. As the cavity size becomes comparable to the optical wavelength, the total number of radiating modes decreases, and the spontaneous emission factor β increases (see Equation 1). When β approaches 1, most of the absorbed pump energy goes into the cavity modes. In the extreme case, when the size is less than half the optical wavelength, the number of modes becomes 1, a situation known to produce a "threshold-less" laser.

[0141] Optical cavities can affect the spontaneous emission rate of gain elements in the gain medium by coupling to the cavity modes. The enhancement of the spontaneous decay rate of confined modes, known as the Purcell effect, is a factor that can be exploited in micro- and nanoscale lasers.

[0142] When the size is comparable to or smaller than the optical vacuum wavelength λ, the laser behaves like a point source, emitting light over a wide solid angle. Subsequently, the so-called "directivity" of the laser output becomes less pronounced. As the size approaches the smallest possible size (i.e., L c =λ / n), the laser output emitted from the cavity will radiate in all directions. The laser output is emitted in all directions, just like fluorescence from a single molecule. However, some additional features such as asymmetry can be incorporated into the laser cavity to enhance the directionality.

[0143] Laser cavities can be divided into various types with respect to their geometry and optical resonance axis. Fig.9A An example is shown in -D.

[0144] Cavity life τ c (the inverse of the cavity loss rate) is related to the cavity's Q factor, which is defined as follows:

[0145] Q=2πντ c , (16)

[0146] where ν is the optical frequency. Different cavity modes may experience different cavity losses and therefore have different Q factors. In small cavities, the cavity lifetime is usually much longer than the optical lifetime τ of the spontaneous emission. s For example, for λ = 532nm, when the Q factor is 10 6 When τ c = 0.28ns, when the Q factor is 10 3 When τ c =280fs.

[0147] From equation (8), the threshold pump rate is inversely proportional to the Q factor:

[0148]

[0149] The spectral linewidth (below threshold) of a passive cavity is given by

[0150]

[0151] Above the threshold, the linewidth of the laser mode can be reduced because the cavity losses are compensated by the gain. According to the Schawlow and Townes limits, the fundamental limit on the laser linewidth can be expressed as:

[0152]

[0153] In practice, several factors contribute to broadening the laser linewidth. Thermal heating during laser operation changes the refractive index n of the cavity. eff , resulting in linewidth broadening. In semiconductor lasers, carrier-induced refractive index modulation is an important linewidth broadening factor.

[0154] In general, pulsed pumping produces a wider spectral width than continuous wave pumping. Modulation of the temperature and refractive index of the cavity causes the resonant frequency of the lasing mode to be chirped over time, thereby broadening the output spectrum. Relaxation oscillations during the laser build-up enhance the modulation. In microlasers operated in a pulsed scheme, the actual laser spectral width can be several orders of magnitude larger than the theoretical limit in equation (18). In many of these practical cases, although the cavity Q is important for reaching the lasing threshold (Equation 17), the cavity Q is not a direct determinant of the laser linewidth.

[0155] Consider a linear or concentric resonator formed by a pair of plane or spherical mirrors with reflectivities R1 and R2 separated by a distance L (L c / 2). The cavity life is expressed as:

[0156]

[0157] where n is the refractive index of the medium in the cavity and η is the fractional internal loss per pass, which may occur due to absorption or scattering losses in the cavity.

[0158] Consider the following description of a Fabry-Perot resonator ( Fig.9A ): a medium filled with a refractive index of n = 1.6, containing a fluorescent dye emitting at λ = 600 nm, and at R 1,2 (1-η) = 0.98 with a silver mirror. From equation (16), Q = 1,000 is obtained when L = 3.2λ / n = 1.2μm. The smallest resonator is given by L = λ / 2n. For n = 1.6 and R 1,2 = 0.98 for a cavity with this minimum length of 375 nm, we obtain Q = 155.5 and Δν 腔体 =3.86nm.

[0159] Photonic crystal cavities have periodic structures and defects. By having laser gain in the defects and a sufficiently high Q factor, lasing can be achieved. The periodic structure can be 1D, 2D or 3D ( Fig. 9C ). For 1D and 2D photonic crystals, light confinement in other dimensions is achieved through total internal reflection. Lasers with linear grating structures include distributed feedback lasers (DFB) and vertical cavity surface emitting lasers (VCSELs). 2D lasers are typically implemented on thin plates with internal bandgap structures. Although 3D photonic crystal lasers are more difficult to fabricate, successful lasers have been reported. Using self-assembly of liquid crystals, radial Bragg microcavity lasers (1D periods on a 3D object) with a diameter of 15μm have been demonstrated. The output emission morphology is radial and uniform in all directions.

[0160] Consider a periodic slab with m bilayers of two alternating quarter-wave-thick materials (with refractive indices n1 and n2, respectively). The Bragg wavelength λ B Given by the following formula

[0161] λ B =2(n1+n2)Λ, (21)

[0162] where Λ is the periodicity of the double layer structure. The reflectivity at the Bragg wavelength is:

[0163] For example, at least 4 bilayers of semiconductor material with n1=3.5 and low-index voids with n2=1.33 are needed to achieve 99% reflectivity. Two such stacks on each side of the optical cavity would have a total thickness equal to about 1.6 μm (8Λ). This would be small enough to be used inside a cell.

[0164] In circular resonators such as spheres, rings, cylinders, and tori ( Fig.9A -B)) in the cavity. The refractive index must be greater than that of the surrounding medium so that the modes are confined to and circulate in the cavity by total internal reflection at the external interface. The optical field of the intracavity light can evanescently extend out of the cavity. This evanescent field allows the gain medium to be physically placed outside the cavity and is critical in certain applications such as sensing molecules in the surrounding environment.

[0165] A WGM can be specified by a radial mode number q, a polar mode number l, an azimuthal mode number m, and a polarization p. For a sphere, the frequency of the WGM can be approximated using an asymptotic expansion (for l>>1):

[0166]

[0167] where n1 is the refractive index of the sphere, n2 is the refractive index of the surrounding medium, χ = 1 for the TE mode, and χ = n2 / n1 for the TM mode, a is the radius of the sphere, and α q is the negative q-th zero of the Airy function (-2.3381, -4.0879...). The above formula can be further approximated as 2πn1a / λ=l, which is consistent with equation (15) (where L c =2πa) is consistent.

[0168] When the size of a WGM resonator approaches the optical wavelength, its Q factor is limited by radiation (curvature) losses:

[0169]

[0170] in and n r =n1 / n2 is the relative refractive index, and for the TE mode, k=0, for the TM mode, k=1. For a sphere containing a few mM of a typical organic fluorescent dye and nanosecond pumping, the minimum Q factor required to achieve lasing is about 10,000. From the above equation, the following approximate formula can be obtained:

[0171]

[0172] For a given Q factor, a high n1 is required to reduce the resonator size. For example, to achieve Q = 10 in the cytoplasm (n2 = 1.37) at λ = 0.6 μm 4 , where 2a<1μm, the resonator material should have n1>2.61.

[0173] In cavities made of dielectric materials, optical energy is stored as photonic modes with length scales equal to or greater than the optical wavelength. Better confinement can be achieved with metals, for example by coating a thin metal layer on the subwavelength dielectric resonator. This configuration can support two different types of resonant modes: purely reflective cavity modes and plasmon-based resonant modes.

[0174] In the first case, the intracavity energy is stored in an optical wave, just like in a dielectric cavity. The metal layer acts as a mirror, which confines the mode inside the cavity by high reflection at the metal surface. The effective confinement provided by the reflective surface allows high Q factors, even for dimensions at the size of the optical wavelength.

[0175] The second type of mode is based on the plasmon effect generated at the metal-dielectric interface. The energy in the cavity is stored in a plasmon mode that is strongly confined at the interface between the dielectric medium and the metal, with a wavelength much shorter than the optical wavelength. Therefore, plasmon mode resonances can be achieved with cavities much smaller than the optical wavelength. Plasmon modes are usually divided into long-range surface plasmon polaritons (LR-SPP) and localized surface plasmons (LSP).

[0176] Long-range polaritons are electromagnetic waves that propagate along the interface between metal and dielectric materials. They are evanescently confined in a direction perpendicular to the surface. Typical structures include metal / insulator / semiconductor / insulator / metal waveguides and metal-insulator / semiconductor nanowires. Plasma modes are primarily confined in one or two dimensions in the insulating region, while the cavity is defined by the end facets of the structure along the propagation direction, still on the order of (at least) several wavelengths. However, it should be noted that the strong coupling of electromagnetic modes to electrons in metals leads to higher resistor-type (ohmic) losses, which are inherent to plasma modes and are therefore difficult to minimize. Therefore, their Q factors are lower than similar structures based on photon modes. So far, lasing has only been achieved at low temperatures.

[0177] Localized plasmons are collective oscillations of electrons at the surface of metal nanoparticles by coupling to strong electromagnetic fields. Surface plasmons act as resonant electric dipoles, with the resonant frequency determined by the shape of the nanoparticle and the dielectric constants of the metal and surrounding dielectric medium. Plasmonic lasers have been demonstrated by coating metal nanospheres with dye-doped dielectric shells. Upon optical pumping of the dye, the optical energy is readily transferred to the spectrally matched plasmon resonances of the metal core via FRET. Surface plasmon excitation generates strong local fields that stimulate increased coupling of emission from the dye into the plasmon mode. This feedback mechanism is known as surface plasmon amplification by stimulated emission of radiation, and devices operating on this mechanism are known as spasers.

[0178] Despite some experimental observations, the stable realization of spasers has been difficult due to low Q factors and metal heating as well as the limited number of gain elements in nanoscale volumes. 18 cm -3 ) at high concentrations, a 40 nm sphere can only contain 40 dye molecules. To date, no convincing evidence of experimental realization of spasers at the level of single particles has been reported. All experimentally demonstrated spasers have dimensions larger than a few μm including the substrate.

[0179] The conventional cavities described so far provide well-defined optical resonance paths. In the case of random lasers, light is trapped in a gain region in the disordered medium by multiple scattering ( Fig.9D ). For this medium, the transmission mean free path l t is defined as the distance after which the propagation direction of light is randomized, and l g represents the gain length, which is defined as the distance over which light is amplified by a factor of e (i.e., 4.34 dB). The scattering medium should have a critical minimum volume to have sufficient gain and scattering feedback. According to this condition, the minimum size of the random laser, L c Given by the following formula

[0180] L c ≈(l t l g / 3) 1 / 2 (25)

[0181] For example, consider a medium containing TiO2 nanoparticles as a scatterer and rhodamine 6G as a gain dye. A TiO2 nanoparticle with a diameter of 200 nm is 10 cm –3 At high concentrations, there is l t=35 μm. The gain length of the Rhodamine 6G solution at a high concentration of 10 m M is about 10 μm. This random laser cavity provides L c ≈10μm critical size. That is, when the strong pump light is used to illuminate the 3 In principle, laser emission can be generated when the volume is smaller than 100 nm. Typical skin tissue has a t =50 μm, and applying 10 mM rhodamine 6G to the tissue can obtain L c =13μm.

[0182] The emission spectrum of a random laser usually contains several narrowband peaks with different intensities at irregular wavelengths. The spatial profile of the output emission is very complex, similar to a spot morphology. In principle, the laser spectrum and spatial profile are determined according to the specific distribution of scatterers. However, it is difficult to analyze light scattering from multiple scatterers. Nevertheless, the sensitivity of the laser output to the scattering medium may be useful for sensing.

[0183] For a laser, some form of energy needs to be supplied in order to operate and generate light. The input energy absorbed by the gain medium should be sufficient to produce a population inversion in the gain medium and generate enough amplification to overcome the losses in the cavity. There are many different ways to pump a laser particle. Optical pumping provides the most convenient way and is well suited for situations where light is easily accessible to the laser particle; for example, for laser particles placed in cells in culture or implanted in tissue at shallow depth.

[0184] Current injection is an established method for driving conventional semiconductor lasers. Electrical pumping may be possible when semiconductor particles are embedded in biological systems, but requires clever methods to pump enough current and voltage across the semiconductor medium to produce a net gain. Wireless energy transfer is an option that has been shown to be able to drive millimeter-sized light-emitting diodes (LEDs) embedded in animals. As devices are further miniaturized, the corresponding antennas become smaller, increasing the resonant frequencies required for effective wireless transfer. When the size approaches the micrometer or submicrometer scale, the optimal frequency range approaches optical frequencies. This makes wireless pumping equivalent to optical pumping. In this section, we describe the basic principles of different pumping methods. In addition, we discuss the exciting but elusive potential of using biological energy extracted from living systems to operate laser particles.

[0185] Optical pumping is a convenient way to provide the energy used to operate the laser particle. Let the absorption coefficient of the gain medium be ε. It is related to the absorption cross section σ a Related: ε=ln(10) -1 σ a N g / V[see equation (3)] Absorption efficiency η吸收 or the fraction of the incident pump energy absorbed by the laser particle, roughly determined by ε times the particle diameter 2a:

[0186]

[0187] where ν p is the optical frequency of the pump light. For example, consider a molar extinction coefficient of 65,000 M -1 cm -1 The gain medium including the dye at a concentration of 1 mM has ε = 65 cm -1 A film of dye with a thickness of 10 μm absorbs 14% of the pump beam (η 吸收 =0.86).

[0188] Absorption of semiconductors and carrier density ρ c The absorption coefficient of a bulk semiconductor with a direct band gap is expressed as ε = A(ν p / v g -1) 0.5 , where A = 10 in most bulk semiconductors 4 -10 5 cm -1 , and ν g is the band gap frequency. For example, for GaAs(hν g =1.42eV), ν p / v g =1.2, ε is 1.5x10 4 cm -1 A thin GaAs slab with a thickness of 250 nm can absorb 58% of the pump energy (η 吸收 =10 -1.5*0.25 =0.42).

[0189] Consider having E p The energy and beam size of the pump pulse are then 吸收 =η 吸收 E p *η 面积 to give the absorbed energy, where the overlap parameter η 面积 is equal to the ratio of the size of the laser particle to the beam size, and when the beam size is smaller than the size of the gain medium, η 面积 is 1. Part of the total absorbed energy is radiated from the cavity as light via spontaneous and stimulated emission; the rest is converted into heat and other forms of energy.

[0190] As the pump intensity increases, more and more gain elements are excited to the upper state. From equations (1) and (3) and N 总 (t) = N g(t)+N1(t), the fraction of the gain element in the transmit-ready upper level in the steady state can be expressed as:

[0191]

[0192] When the signal-induced gain saturation (i.e., the last term in the denominator) is neglected (βq<<1, close to and below the threshold), 50% inversion is achieved when the pump intensity is equal to 总 =N g )

[0193]

[0194] 50% pump intensity corresponds to one photon per absorption cross section per upper horizontal lifetime divided by the quantum yield. For high efficiency gain media, 50% population inversion can be achieved by optical pumping. For example, for λ = 488 nm (hν p =4.1x10 -19 J), the absorption cross section of eGFP is σ a =2.1x10 -16 cm 2 , and QY=0.8. When the pulse duration is equal to the lifetime (τ s =τ p ), the pump flux I required to excite half of the molecules to the upper level p,50％ τ p About 2.4mJ / cm 2 or 24 pJ / μm 2 This fluence level is 1 to 3 orders of magnitude (depending on pulse width) below typical safety limits for human skin tissue in terms of photothermal damage.

[0195] Chemical energy can be used to create a population inversion. A known example is found in hydrogen fluoride lasers, which utilize the fundamental reaction F+H2→H+HF * The reaction releases a free energy of ΔG = -31.6 kcal / mol. Part of this chemical energy is converted into photons in the spectral range of 2.7-2.9 μm (9.8-10.6 kcal / mol). However, a typical exothermic chemical reaction based on rotational vibration excitation produces a ΔG of only 1-10 kcal / mol. This is much less than the energy of a visible photon (e.g., 40-70 kcal / mol at 400-700 nm). As a result, all conventional chemical lasers based on this chemical energy operate in the infrared region.

[0196] Chemical reactions involving electronic rather than vibrational transitions can be suitable for pumping visible or near-infrared lasers. Electronic transitions are relatively rare in chemical reactions, and so-called "chemically initiated electron exchange luminescence" (CIEEL) reactions uniquely favor electronic transitions. CIEEL can be divided into two types. Type 1 is intramolecular CIEEL and utilizes energy transfer in the solvent cage and direct light emission from the reaction products: Symbolically, A+B→[C+D * ] and C+D * →C+D+hv. Type 2 is intermolecular and uses fluorescent dyes to emit light: A+B→[C+D * ]; D * +E→E * +D; and E * →E+hv, where E represents the dye.

[0197] Fig. 10A -D depicts various examples of CIEEL reactions: namely, the firefly luciferase-luciferin reaction, the alkaline phosphatase (AP) and chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) reaction, the peroxyoxalate-dye reaction (also known as the glow stick reaction), and the peroxidase-luminol reaction. Despite the different starting reactants, all of these reactions have a similar high-energy intermediate 1,2-dioxetane. 1,2-Dioxetane has unique advantages as a chemiluminescent reaction intermediate. The decomposition of 1,2-dioxetane is the reverse process of π2-π2 photocycloaddition, which is prohibited for thermal excitation, so most of its energy is released through photon energy rather than thermal energy. The energy difference between 1,2-dioxetane and its two carbonyl products is about 72.5 kcal / mol, making electronic transitions for visible light emission possible.

[0198] Bioluminescent and chemiluminescent reactions are potential candidates for pump laser particles. The high energy intermediate state can emit light as electromagnetic radiation (Type 1), or transfer photon energy to a nearby luminescent particle, such as a dye, via near-field FRET (Type 2). When FRET occurs from a bioluminescent molecule, it is often referred to as "bioluminescent resonant energy transfer," or BRET.

[0199] Kinetic conditions require that the pump reaction rate should be much faster than the decay rate. To achieve high reaction rates, conventional chemical lasers use gas phase reactions. For example, the rate-determining step of the pump reaction of a hydrogen fluoride laser is H+F2→F+HF * The kinetic constant for this reaction is 1.6 x 10 10 M -1 s -1, similar to the typical kinetic constant of 10 for diffusion-controlled bimolecular reactions at room temperature. 9 to 10 10 M -1 s -1 In contrast, liquid-phase chemiluminescent reactions have lower kinetic rates. For example, the typical kinetic rate constant for luciferase-luciferin is only 10 s. -1 For the peroxyoxalate-rubrene dye reaction, the kinetic rate constant can be even higher, at 10 5 s -1 , and for the luminol-peroxidase reaction with the phenol enhancer, the kinetic rate constant is 10 7 s -1 The latter is only 1,000 times slower than the reaction in a hydrogen fluoride laser.

[0200] The oxidation of glucose releases a large amount of Gibbs free energy: C6H 12 O6(s)+6O2(g)→6CO2(g)+6H2O(l), ΔG=-685.7kcal / mol. This chemical energy can theoretically be used to generate light, for example, via a subsequent chemiluminescent reaction. Metabolic energy is consumed to produce adenosine triphosphate (ATP) molecules, which are known as the energy currency of the cell. The process involves adding a third phosphate group to adenosine diphosphate (ADP). A total of 30 or 32 ATP molecules are generated from one glucose molecule. At any given time, there is about 1 gram of ATP in our body. When cells need energy, ATP molecules are hydrolyzed to break down into ADP and phosphate. On average, in a typical cell, 1-2 million ATP molecules are hydrolyzed per second and provide free energy for various cellular functions via the following process: ATP(aq)+H2O(l)→ADP(aq)+Pi(aq), ΔG=-7.3kcal / mol. This energy is not enough to directly generate photons in the visible and near-infrared ranges. Some bioluminescent organisms, such as fireflies and quorum sensing bacteria, require ATP as a cofactor to overcome the activation barrier in the chemiluminescent reaction. Using metabolic energy as a pump source for laser particles may be an option.

[0201] Possible methods of electrical pumping include electrochemical luminescence, current injection into semiconductors, and discharges in gases. Gas lasers such as He-Ne lasers and CO2 lasers use current emitted through a gas. Electrons are accelerated by high voltage (300-1400V) and collide with gas molecules to generate plasma. The excited molecular ions emit light directly or transfer energy to other gain molecules.

[0202] The general mechanism of electrochemiluminescence involves electron transfer reactions at the electrode surface and the annihilation of radical intermediates: (1) A → A ·+ +e - (oxidized at the electrode); (2) B+e - →B ·- (reduced at the electrode); (3) A ·+ +B ·- →A · +B (excited state formation); and (4)A · →A+light (A·→A+light). When ion annihilation is the rate-determining step, the reaction occurs near the diffusion-controlled limit. The kinetic rate of the ion annihilation step of 9,10-diphenylanthracene (DPA) is 2x10 10 M -1 s -1 Electrochemiluminescence pumping of DPA lasers was demonstrated by using a mirror electrode with 10 V.

[0203] In an electrically pumped semiconductor laser, the pn junction is forward biased with a voltage (typically 1.5 to 2.5 V). When forward biased, electrons injected into the n-type side flow to the p-type side, where electrons in the conduction band recombine with holes in the valence band. This radiative recombination process can be described by the following simplified rate equation:

[0204]

[0205]

[0206] Here, N1 is the number of carriers in the gain region, N0 is the number of carrier particles of optical transparency, q is the number of photons in the cavity, I is the injection current, η is the coupling efficiency (equivalent to the quantum yield), e is the electron charge, and g is the gain factor (≈β / τ s ). Typical semiconductor materials have N0 / V = ~10 18 cm -3 , so for V = 10 -12 cm 3 Micron-sized laser, N0 = 10 6 . Using η = 0.4, τ c =1ps, τ s = 3ns, the threshold current in the steady state is determined by I th ≈eN0 / (ητ s ) = ~130μA is given.

[0207] Although electrical pumping is commonly used in semiconductor lasers, traditional device architectures may not be directly applicable to independent laser particles. We can consider both wired and wireless approaches. It is relatively simple to use some type of narrow wire to provide the necessary bias voltage and current. Wireless power transfer is more attractive for in vivo applications. RF transmission delivers 4.1 mW of electrical power to drive a semiconductor LED at a distance of 1 m, generating 7 mW / mm 2 of optical power. However, for individual micrometer-sized laser particles, both wired and wireless approaches are less feasible. As the size of the laser decreases, the efficiency of the antenna decreases unless the wavelength of the electrical signal decreases proportionally. This requires the frequency to be in the optical frequency, making the wireless delivery equivalent to optical pumping.

[0208] Given this challenge, one might consider the potential to harvest current and voltage from surrounding biological matter. A relevant example is a biofuel cell, which converts chemical energy into electrical energy. For example, open circuit potentials of 0.5-0.8 V have been achieved in mitochondria-based anodes using pyruvate and live clam-based anodes using glucose. The electric eel is capable of generating 600 V. Artificial batteries inspired by the electric eel could enable electrical pumping of laser particles.

[0209] Other types of energy include kinetic energy generated by muscle and body movement, which can be converted into electrical energy by having piezoelectric materials, and sonoluminescence.

[0210] When a pump pulse is absorbed, part of its energy is converted into heat and increases the temperature of the gain medium. For pump pulses shorter than <10 ns, the heat dissipation to the surrounding water medium is negligible during the pulse duration. In this case, immediately after the absorption of the pump energy E 吸收 The subsequent peak temperature increase ΔT is given by:

[0211] ρVc V ΔT=(1-QY)·E 吸收 (31)

[0212] Where ρ is the mass density, V is the volume, c V is the specific heat capacity. The factor (1-QY) represents the fraction of the absorbed energy that is converted into heat.

[0213] Let us consider three examples. Case I: For a particle with a size of 20 μm (3.8 ng) and a specific heat (c p ) is 1.8 J g - 1 K -1 The oil droplet has a flux of 10 nJ (i.e., 3.1 mJ / cm 2) generates 4.5 nJ of heat energy and causes a temperature increase of ΔT = 0.7°C. Case II: For a cell with a size of 10 μm (0.52 ng) and c p =1.4 J g -1 K -1 polystyrene beads, 1 nJ on the beads (i.e., a flux of 1.3 mJ / cm 2 ) produces 0.45 nJ of heat energy and a temperature rise of ΔT = 0.6°C. Case III: For a 1 μm diameter and 250 nm thickness (about 1.1 pg = 5.8 kg / m 3 (ρ)*0.2x10 - 12 cm 3 ) and c p =0.33 J g -1 K -1 GaAs disk, 5pJ on the disk (fluence is 0.6mJ / cm 2 ) generates 2.2 pJ of heat and ΔT = 6.1°C.

[0214] Excessive temperature increases can have a detrimental effect on the surrounding medium. A typical effect is protein denaturation, which induces the unfolding of the protein structure and leads to the loss of protein activity. The change in free energy of a typical protein when unfolding in water is 10-20 kcal mol -1 This is consistent with the activation energy E of 0.4-0.9 eV per molecule. a According to the Arrhenius model, the degradation rate is Proportional.

[0215] For pulse heating, the magnitude of thermal damage represented by protein denaturation depends not only on the temperature but also on the duration of exposure to the elevated temperature. e The amount of denaturation or magnitude of thermal damage is given by:

[0216]

[0217] If the heating time is reduced, denaturation requires considerably higher temperatures. For example, when exposed to heat for a short duration of 10 ns, protein denaturation occurs at a peak temperature of 90°C (ΔT = 53°C), whereas for a long exposure of 1 s, protein denaturation is induced at a temperature of 60°C (ΔT = 23°C).

[0218] The change in internal pressure due to a temperature rise is given by

[0219] p1-p0=α V K B(T1-T ∞ ) (33)

[0220] where α V is the coefficient of thermal expansion (2.1x10 -4 K -1 , soft tissue is about 4x10 -4 K -1 , and GaAs is about 1.7x10 -5 K -1 ), K B is the (isothermal) bulk modulus (2.2×10 9 Pa, and GaAs is 8.6×10 10 Pa), p0 and p1 are the pressures before and after the pump pulse. For incompressible solids and liquids, a 1°C increase in temperature will result in an increase of 0.4 to 1.5 MPa.

[0221] Pressure and thermal expansion create forces. Part of this energy causes mechanical (acoustic) waves.

[0222] Sound energy is expressed as:

[0223]

[0224] The efficiency of photoacoustic generation (defined as the ratio of acoustic energy to absorbed thermal energy) is given by:

[0225]

[0226] Here, Γ = α V K B / (ρc V ) is the Grüneisen parameter. For water and cells, its value is about 0.11, and for soft tissues, its value is 0.7-0.8. For water, the coefficient Γα V 2.3x10 -5 (For GaAs, 1.3x10 -5 ). In biomedical applications, T1–T ∞ Usually limited to <10℃, so only a small part (<10 -4 )'s absorbed energy is radiated as sound waves.

[0227] The conversion efficiency is not constant, but rather scales with the temperature rise. It is interesting to realize that the acoustic energy is proportional to the square of the absorbed energy, rather than being linearly proportional to the input energy itself. This relationship may seem counterintuitive, but is due to the fact that the initial pressure increase given in equation (34) is actually proportional to the subsequent (adiabatic) volume expansion ( Fig.11) decreases with time. Photoacoustic generation can be considered a heat engine. In solids or liquids, the efficiency is low. In gases, α V = ~1 / T1 (where T is given in K), and for a Carnot heat engine we obtain the familiar expression: efficiency ≈ (T1 – T ∞ ) / T1. In this case, a higher photoacoustic conversion efficiency can be obtained.

[0228] Heat conduction in radially symmetric geometries can be described as follows:

[0229]

[0230] where k is the thermal conductivity, and is the rate of heat generation per unit volume. Blackbody thermal radiation is neglected.

[0231] Case I: Short pulse pumping: Let us consider a spherical particle with radius R, which is initially connected to T = T ∞ The medium at t is in thermal equilibrium. The particle is then pumped by a short optical pulse at t<0 and heats the particle to temperature T1 at t=0. For simplicity, we assume that the particle and the medium have the same material properties including thermal conductivity. Convection originating from the motion of molecules in the surrounding medium is neglected during short time scales. The boundary conditions can be rewritten as:

[0232]

[0233]

[0234] The Green's function for the differential equation is

[0235]

[0236] where α = k / ρc p is the thermal diffusion coefficient of the medium. At a given t, the Green's function is r = (4αt) 1 / 2 It has a half-width of 1 / e.

[0237] Therefore, the time it takes for heat to diffuse through a sphere of diameter 2R becomes:

[0238] τ=R 2 / α (40)

[0239] This time τ is called the thermal relaxation time of the spherical particle.

[0240] Using Green's functions and the above boundary conditions, the solution can be shown to be:

[0241]

[0242] Where ΔT 归一化 ≡(T(r, t)-T ∞ )(T1-T ∞ ) is the normalized temperature profile, and erfc=1-erf represents the complementary error function. Fig.12 On the left, we plot ΔT at several time delays. 归一化 The temperature gradient within the particle is due to the finite thermal conductivity and thermal diffusivity of the particle. Fig.12 The middle figure shows how the temperature of the particle at r = 0.5R decreases with time. It follows an exponential decay, where it is 1 / e times 4τ when t < 0.2τ and T ∝ t when t > τ. -3 / 2 The finite relaxation time implies heating due to repeated pumping. Fig.12 The cumulative temperature rise after 100 pulses with a uniform period Δt between pulses is plotted in the right figure of . When Δt=0.3τ, the steady-state temperature increase is about twice the temperature rise by a single pulse. This sets a limit on the maximum repetition rate for pumping, such as 500kHz in this example.

[0243] When the pulse width is shorter than the thermal relaxation time, all the heat accumulated in the particle during the pulse absorption cannot diffuse out to the medium. When the medium is tissue or cells, its thermal properties are similar to those of water: therefore, we can use k = 0.6 Wm -1 K -1 and α = 0.14x10 -6 m 2 s -1 The relaxation time τ is the fundamental time constant governing the cooling of a particle by heat dissipation into the medium. The time constant is proportional to the inverse of the thermal diffusivity. Aqueous media, such as cytoplasm and tissue, are better conductors of heat than air but more insulators than semiconductors. Therefore, heat dissipation in biological media is generally slower than in on-chip semiconductor lasers. Note that τ is proportional to R 2 Or proportional to the surface area of ​​the particle. The larger (smaller) the particle, the longer (shorter) the thermal relaxation time; for R = 10 μm, τ = 700 μs; for R = 1 μm, τ = 7 μs; and for R = 100 nm, τ = 70 ns.

[0244] The above analysis is valid for homogeneous materials and is for particles with high water content or particles composed of liquid and polymer (such as oil (k = 0.15 Wm -1 K -1 ), polystyrene (k = 0.14Wm -1 K -1 ) and silicon dioxide (k = 1.4Wm -1 K -1)) is a reasonable approximation of .

[0245] Laser particles made of semiconductors and metals have thermal conductivity 2-3 orders of magnitude greater than that of dielectrics: for example, GaAs (k = 52 Wm -1 K -1 ), silicon (k = 150Wm -1 K -1 ), silver (k = 410Wm -1 K -1 ), and diamond (k = ~1000Wm -1 K -1 ). In this case, the temperature inside the particle is almost constant. This condition is usually described in terms of the Biot number (Bi = hR / k1), where h is the heat transfer coefficient and k1 is the thermal conductivity of the particle. For short time scales comparable to the characteristic time (e.g., t<0.2τ), h is time-varying but can be approximated as h≈k2 / R, where k2 is the thermal conductivity of the medium, and therefore, Bi≈k2 / k1. The Biot number corresponds to the ratio of convection at the surface of the particle to conduction in the bulk. A small Biot number indicates a small resistance to heat conduction and a small temperature gradient within the particle.

[0246] Case II: Continuous Pumping: We now consider The pump energy is continuously supplied to the particle at a rate of , and the temperature of the particle is calculated when the particle reaches a steady state. According to the heat equation in the medium (i.e. ) and boundary conditions The surface temperature T1 of the particle is:

[0247]

[0248] The energy rate per unit volume Depend on The temperature rise is proportional to the relaxation time of the medium.

[0249] The requirements for optical radiation are quantified in terms of maximum permissible exposure (MPE), which is roughly defined as one-tenth of the damage threshold for harmful photothermal and photochemical effects. The ANSI Z136.1 standard classifies lasers according to their biohazards to the retina and skin. According to the standard guidelines, the laser exposure limit for the skin is given by: For τ p =1-100ns, 0.02*C A J / cm 2 , and for τ p = 100ns to 10s, 1.1*C A τ p 0.25 J / cm 2 , where CA is a wavelength-dependent empirical coefficient: For the spectral range of 400-700 nm, C A =1, for 700-1050nm, C A =10 0.002(λ-700) , and for 1050-1400nm, C A = 5. For long-term exposure τ p >10s, MPE level is 2*C A W / cm 2 , which is given in terms of optical intensity rather than flux, because a thermal equilibrium between laser-induced heating and conductive cooling is reached during long exposures. For long optical exposures of the eye to collimated near-infrared light at 750-1050 nm, the MPE level for retinal safety is C A mW / cm 2 , where the intensity is measured at the cornea.

[0250] Integrating laser sources into biological tissues or living cells requires independent and compact devices with micrometer dimensions (typical mammalian cells such as HeLa have a diameter of about 20 μm), pump energy sources, and good biocompatibility. Currently, there are only a few examples of successful integration of lasing structures into cells, and the size of the lasing structures is usually larger than 8 micrometers in their largest dimension.

[0251] Over the past few decades, the miniaturization of laser sources has been actively pursued with the goal of realizing integrated optoelectronic devices with fast switching times and / or low power consumption. One of the most widespread options for making small, compact lasers is to exploit whispering gallery modes (WGMs) generated in small structures that have a sufficiently high refractive index relative to the external environment. WGM resonators have been realized in a variety of different shapes and materials; the gain medium can be used to constitute the cavity itself, or its emission can be coupled to the WGM in a resonator made of an inert material. Polystyrene microspheres doped with dyes have been shown to generate lasing when optically pumped. The smallest microsphere lasers reported to date are about 8 μm, which is limited by the decreasing Q factor as the size decreases. Spontaneous emission from dye-doped microspheres as small as about 3.5 μm has been reported, but operation only below the laser threshold has been demonstrated due to limited cavity Q factors, gain, and pump energies.

[0252] Microdisks are a common example of compact lasers fabricated using semiconductors as gain media, since these structures can be obtained from epitaxially grown wafers via photolithographic processes. Their particular characteristics are: lateral dimensions of a few micrometers to tens of micrometers, but their thickness can be reduced to about 100 nm (corresponding to a limit of the order of λ / 4n). Given their high refractive index (about 3-3.5 for the most commonly used III-V compounds), the smallest cavities have been demonstrated with inorganic semiconductors. For example, structures with diameters of 2-4 μm, thicknesses of 100-200 nm and Q factors up to 12,000-17,000 have been achieved for lasing around 900-1000 nm. Although much higher quality factors (over 10) can actually be achieved with appropriate design of the device shape and surface roughness, 8 ), but these structures usually have considerably larger diameters (tens of micrometers). On the other hand, devices as small as ≈600 nm have been demonstrated, albeit with lower Q factors (around 10 3 However, it should be noted that these disk lasers are fabricated on substrates having dimensions much larger than the disk. Of course, no attempts have been reported to coat such disks or to insert disk lasers into cells.

[0253] Several pairs of bioluminescent enzymes and substrates are known that have high transfection efficiency and high brightness in the visible light range. The spectral bandwidth of bioluminescence is typically 50-100 nm, similar to the spectral bandwidth of fluorescence. The lifetime of bioluminescence is also similar to that of typical organic fluorophores, in the order of nanoseconds. Typical bioluminescent reaction rates in biological environments are 0.1-10 photons / second per enzyme molecule. Although this rate is not sufficient to generate stimulated emission, bioluminescence can be coupled to an optical cavity.

[0254] For example, as a preferred embodiment, we have fabricated undoped polystyrene beads coated with luciferase that can generate bioluminescence with an emission spectrum modulated by cavity modes. Biotinylated Gaussia luciferase (GLuc, 19.9 kDa) was coated on the streptavidin-coated surface of polystyrene microbeads. Gluc oxidized coelenterazine (CTZ, 423.46 Da) on the bead surface to generate bioluminescent light that was evanescently coupled to the WGM ( Fig.15 , left panel). When CTZ (25 μM) was added to the medium containing dispersed GLuc-coated beads, bright bioluminescent emission was generated from the surface of the beads ( Fig.15 , middle panel). Representative output spectra taken from 23 to 28 seconds after CTZ addition show well-distinguishable WGM structures ( Fig.15, right). The mode structure in the output emission is different from the standard broad emission of bioluminescence. Compared to optically pumped fluorescent particles, bioluminescent particles can be excited in tissues at certain depths and almost anywhere in the organism by administering substrate molecules, for example via intravenous injection, without being restricted by the penetration depth of optical pumping.

[0255] Once injected into a biological system such as soft tissue, individual laser particles can be detected and localized by optical imaging instruments such as fluorescence microscopy. However, novel microscope configurations can be designed to detect stimulated emission from laser particles. Consider focusing the excitation beam into a medium containing uniformly distributed probes (Figure 16, upper left). For single-photon excitation, the total amount of signal integrated over the cross-sectional area at z is constant (Figure 16, upper right), and therefore, brightfield imaging cannot provide optical sectioning for thick samples. In two-photon microscopy, the signal intensity is proportional to the square of the intensity of the excitation beam; this provides optical sectioning (Figure 16, lower left). In the case of laser particles, lasing can only be achieved at the focus where the intensity exceeds the lasing threshold (Figure 16, lower right); this provides optical sectioning with a spatial resolution below the diffraction limit. This technique may be referred to as laser particle stimulated emission (LASE) microscopy.

[0256] Above the threshold, the full width half maximum (FWHM) Δ of the PSF can be derived from Equation (6) for the axial (z) and transverse (x, y) directions: LASE The approximate form of the resolution enhancement factor is shown as:

[0257]

[0258] where Δ0 is the diffraction limit of a Gaussian beam: Δ 0,z =2z R ; Δ 0,x,y =1.67z R NA, where z R and NA are the Rayleigh length and numerical aperture of the excitation beam, respectively. The resolution is always less than the diffraction limit, and at the threshold P th The minimum value is near the threshold (Δ LASE / Δ0) 最小 ≈β 1 / 4 .

[0259] LASE microscopy uses single-photon absorption (although two-photon pumping is possible), generates stimulated emission only in the focal volume, and does not require a tight pinhole. In this sense, LASE microscopy has the advantageous features of confocal and two-photon microscopy to achieve efficient generation and collection of signals, with sub-diffraction-limited resolution.

[0260] Another advantageous feature is the low out-of-focus background. The narrow spectral signature of the laser output is easily distinguished from autofluorescence from other fluorescent molecules that may be present around the laser particle. The decay time of stimulated emission is determined by the cavity lifetime, which is much shorter than the fluorescence decay time. Therefore, lifetime measurements can distinguish the laser mode from the spontaneous background. The low background of LASE microscopy can provide enhanced imaging depths greater than 500 μm or even 1-2 mm in scattering tissues.

[0261] We constructed an inverted imaging setup ( Fig.15 ). The configuration of this setup is similar to that of a line confocal hyperspectral fluorescence microscope, but unlike typical hyperspectral microscopes, this setup uses a high-resolution (0.05-2 nm) spectrometer that includes a tunable entrance slit, a diffraction grating, and a charge-coupled device (CCD) camera with 1024-4096 pixels.

[0262] As laser particles we used lead iodide perovskite (CH3NH3PbI3) nanowires grown to a typical width of 300-500 nm and a length of 3-7 μm. The samples were transferred to a glass slide and air sealed with a cover glass optical epoxy to prevent degradation by moisture and oxygen.

[0263] Given the long shape of the nanowire, the pump beam was shaped to have a matching elongated shape and a measured scan profile across the short axis of the nanowire. The pump light source is a microchip laser emitting light at a wavelength of 532 nm, with a repetition rate of 5 kHz and a pulse duration of approximately 2.5 ns, slightly longer than the fluorescence lifetime of the perovskite (τ≈2 ns). The objective lens is a 0.8-NA, 40X water immersion lens (Nikon). A cylindrical lens (CL; f=500 mm) is used to form an elliptical pump beam profile at the focus, with a FWHM of 2.4 μm along the x-axis and 9.5 μm in the y-axis. The elliptical pump profile is used to illuminate the entire length of the nanowire, and thus achieve effective pumping. The nanowire on the sample stage is oriented parallel to the main (y) axis of the pump beam. The polarization axis of the pump beam is aligned orthogonally to the long (y) axis of the nanowire, where the threshold intensity is lowest. The pump beam is scanned along the x-axis by translating the collimating lens (L1), resulting in a 35-nm step resolution in the imaging plane. The output emission is directed to a camera and a polarization-independent diffraction grating-based spectrometer. The entrance slit of the spectrometer is oriented along the long axis (y) of the nanowire so that the emission of the entire nanowire is collected by the spectrometer. The spectral resolution is about 1 nm.

[0264] Fig.15The inset shows a typical pump beam profile imaged on the CCD, and images of fluorescence and laser emission from a 5 μm long nanowire, collected through a long pass filter (LF) on the CCD, at pump levels below and above threshold, respectively. The laser emission profile exhibits bright spots at both ends (indicating longitudinal laser oscillations), as well as the characteristic interference morphology of coherent laser emission. The polarization state of the laser output at the detector plane is nearly parallel to the short (x) axis.

[0265] Experiments were performed to support the proof of concept of LASE imaging. Fig.16A A typical output spectrum from a nanowire above threshold is shown. The spectrum is decomposed into a broadband fluorescence background ( Fig.16A The broadband fluorescence background has the same profile as the fluorescence spectrum obtained at low pump powers (grey curve in Figure 5) and a narrowband stimulated emission component (magenta curve). The laser power is measured by integrating the stimulated emission spectrum. Fig. 16B The measured output as a function of pump energy is shown. The data reveal a well-defined 2 The laser threshold at β = 1.3 × 10 -3 The best curve fit based on equation (6) was obtained.

[0266] Fig. 16C The results show that the pumping speed of the hysteresis loop is 0.0447 W at two different pumping levels (p(=P / P th ) = 1 and 1.8) of the nanowire. The measured lateral (x-axis) resolution Δ LASE 520 nm at p = 1, which is about 5 times lower than the resolution of the fluorescence detection measured from the scan profile of the fluorescence background emission at p = 1 (FWHM, about 2.5 μm). Fig.16D As shown in , the FWHM of the LASE microscope has a minimum at p ≈ 1 and increases with increasing pump pulse energy. The fitted curve based on numerical simulation using a realistic pump profile and an assumed nanowire size (5 μm × 0.3 μm) shows good agreement with the experimental data. The slight increase in fluorescence FWHM with increasing pumping is presumably due to pump-induced fluorescence saturation or gain depletion (i.e., N0 decreases with q).

[0267] Similar resolution enhancements of a factor of about 5 were consistently measured from many different nanowires using similar lengths and β values. The opening width of the confocal slit in front of the spectrometer only modestly affects the resolution through its effect on the laser output resulting in slightly different β values.

[0268] By using a beam scanner, LASE microscopy can Fig.15 The settings in extend to Fig.17AThe beam scanning imaging setup shown in . Galvano-mirror scanners and acousto-optic beam deflectors are well known.

[0269] LASE microscopy can be further combined with other imaging modalities such as confocal fluorescence imaging, multiphoton imaging, reflectance imaging, and optical coherence tomography (OCT). For example, the system can employ a femtosecond laser to generate and collect fluorescence or harmonic generation signals from two-photon or three-photon excitation of structures surrounding the laser particle, such as cells and tissues. Fig. 17B An exemplary embodiment is shown in . Suitable dichroic filters and beam splitters known in the art can be used to manage the femtosecond excitation beam and pump beam, as well as the fluorescence and harmonic signals and stimulated emission from the laser particles.

[0270] Instead of optical detection, laser particles can be detected and imaged by using photoacoustic microscopy. Laser particles can release a small portion of the absorbed pump energy as acoustic wave energy. This mechanism can allow the particles to be detected by an acoustic transducer as in photoacoustic detection. The amplitude of the acoustic wave is proportional to the pump rate P(t). N g ≈N 总 In the unsaturated regime, the photoacoustic signal is simply proportional to the pump intensity.

[0271] However, the number of molecules in the excited state N1 becomes equal to N 总 Rather, the depletion of the ground state limits the pump absorption and, as a result, the photoacoustic signal is attenuated compared to the unsaturated case of the same intensity. It should be noted that above the threshold, the laser oscillation clamps the number of excited molecules to a constant level, as stimulated emission brings the excited molecules down to the ground state. As the gain molecules recycle faster in the laser, in the saturated regime, this process can generate a higher photoacoustic signal in the laser particle compared to the same gain medium without the cavity.

[0272] Guiding is an astronomical technique that estimates the wavefront distortion due to atmospheric turbulence. Since the density of the air changes when turbulence is present, the inhomogeneity of the refractive index deforms the wavefront of the light, so telescopes in ground-based observatories detect severely degraded star images. To address this problem, a laser beam is directed into the sky, and the wavefront distortion is measured by comparing the difference between the incident and returned light. The return light can be generated by Rayleigh scattering of laser light from air molecules at an altitude of 10 to 15 km or laser-induced fluorescence of atomic sodium at an altitude of 95 km. After guiding-assisted wavefront measurement, the blurred star image can be restored via adaptive optical devices (such as deformable mirror arrays) used to compensate for the wavefront distortion.

[0273] Using the same principles in astronomy, guiding stars also play an important role in biological imaging. Biological tissues, due to their inhomogeneous optical properties, cause scattering and distortion of the wavefront, limiting the imaging of samples placed especially at depth. Since the transmission matrix provides an estimate of the output field behind the scattering medium at a given arbitrary input field, images of the focus point or correction of the degradation of samples in deep tissues can be made by shaping the wavefront of the input light based on the information of the transmission matrix. In this process, guiding stars are useful to measure the transmission matrix of a given tissue. The principle of guiding stars involves feedback and conjugation processes. First, the feedback method utilizes fluorescent particles placed in deep tissues and monitors the fluorescence intensity and finds the maximum intensity. Second, the conjugation method collects the light emitted from the target and synthesizes a phase conjugated beam that can be tracked back to the focus when irradiating the tissue. This process is possible because the scattering and propagation of light in tissues are time reversible.

[0274] Laser particles can be potential candidates that can show better performance as guide stars than ordinary fluorescent labels. For example, the laser emission threshold of laser particles can be a sensitive indicator in feedback guide stars. Laser particles can be an ideal source for conjugate guide stars because they can generate spatially and temporally coherent light. Combined with injection locking that gives a temporal phase to the emitted light from the laser particles, laser particles can be implemented for various applications, such as conjugation-based correction using coherence gating.

[0275] By using injection locking ( Fig.18A ), the coherence of the output can be extended to the optical phase level. Injection locking states that when narrowband light enters the laser from the outside, the light can be amplified in the cavity and locked to the output so that the phase of the laser output is phase locked to the injected beam. Injection locking establishes phase coherence relative to the injected beam and should allow the use of coherent interferometry to detect laser particles ( Fig.18B ). Using this principle, we can measure the wavefront of the laser output outside the tissue by exploiting the interference of the injected beam while modulating the pump on and off (blinking the guide star). When a conjugate beam of the same wavelength but with a conjugate wavefront is irradiated onto the tissue, it can trace its path back to where the laser particle was ( Fig.18C ). This technique could be useful for precise deep tissue optical modulation. In addition, in principle, optical coherence tomography could be used to detect injection-locked laser particles, which could potentially be used as contrast agents.

[0276] Laser particles can be incorporated into the cytoplasm of living cells. There are a variety of biological and cellular uptake processes, including endocytosis, phagocytosis, and macrophagocytosis. Cells can be incubated with the medium containing the laser particles for a sufficient time to allow cellular uptake of the particles to occur.

[0277] For cells that do not take up particles efficiently, physical insertion methods such as robotic pipette-based injection can be used.

[0278] A more controlled uptake or laser loading process can be achieved by using a microfluidic droplet device. Several techniques are known in the art to generate droplets in oil that encapsulate micro- and nano-scale objects using syringe pumps. Fig.19A A schematic diagram of an exemplary microfluidic channel device is depicted, in which two droplets each containing a laser particle or a cell are configured to be merged into a single droplet, and the cells in the droplets subsequently internalize the laser particle ( Fig.19A ).

[0279] Fig.19B A schematic diagram of an apparatus using a microfluidic channel device, a pump light source, and a spectrally resolved detection device is shown, which can measure the output spectrum of laser particles in cells. The apparatus is well suited for high-speed reading of the spectral signature of intracellular lasers or high-throughput reading of barcodes.

[0280] Employing a passive or active sorting arrangement such as pillars and high voltage electrodes, the device can be operated to sort laser particles or cells carrying or containing laser particles by using optical information based on the output emission spectrum from the laser particles.

[0281] Due to their narrow emission spectra, lasers and optical cavities embedded in cells have great potential for application in optical labeling of single cells. Cell labeling will be able to distinguish a large number of cell subpopulations or even study cells at the single cell level and be used for highly multiplexed measurements. Current labeling methods include graphic coding, physical coding, lifetime coding, spectral measurement coding, and mass-based labeling using emitters arranged in a specific morphology. Most of these methods are limited to roughly 10 to 100 barcodes. Graphic barcodes can achieve more combinations, but they require imaging, which is slow. Spectrometric methods including brain rainbow and multibow are limited by the wide emission of dyes, quantum dots or fluorescent proteins. The number of unique labels can be increased by multiplexing using intensity, and encoding using up to 500 combinations is commercially feasible. However, intensity coding requires calibration of the instrument and can be changed due to photobleaching, dye degradation, light scattering and absorption, which can be particularly problematic for in vivo studies.

[0282] Conventional fluorescence microscopes and fluorescence activated cell sorters (FACS) cannot resolve fine spectral lines. For high spectral measurement resolution, a flow cytometer device can be configured by using a pump source and a high-resolution spectrometer ( Fig. 20 ).

[0283] Submicron-sized laser particles are coated with binding molecules, such as antibodies, peptides, and click molecules. These laser probes are mixed with cells so that the probes bind to target-specific biomarkers on the cell surface or in the cytoplasm. Fig. 20 ). For each recognition molecule, a laser particle with the same output wavelength is assigned, and different recognition molecules are attached to laser particles with different wavelengths. Subsequently, the number of laser particles of each type can be easily analyzed by measuring the intensity at their corresponding wavelengths. The number is proportional to the abundance of their target molecules.

[0284] To realize microlaser particles, in certain embodiments, photonic semiconductor whispering gallery mode disk lasers can be used. Since the refractive index of semiconductors (n = 3-3.5) is much higher than that of the cytosol (n = 1.33-1.4), semiconductor disk lasers can have a diameter smaller than the vacuum optical wavelength and a thickness that is a fraction of the wavelength. Like quantum dots, semiconductor particles do not photobleach and biodegrade, making them suitable for long-term tracking.

[0285] Millions of identical or incremental particles can be manufactured from a single batch by standard electron beam lithography ( Fig.21A After reactive ion etching and partial chemical wet etching, microdisks are produced on the pillars. This structure is known in the art. When optically pumped with 980 nm, 5 ns pulses, the microdisk laser on the pillar generates a narrow laser emission ( Fig.21A ).

[0286] In example embodiments, we have fabricated semiconductor microdisks with diameters ranging from 0.5 to 3 μm. These microdisks can have an InAlGaAs quantum well structure built on an InP substrate. The disk shape can first be photolithographically engraved using electron beam lithography, reactive ion etching, and then disconnected from the substrate by acidic wet etching to remove the InP sacrificial layer ( Fig.21B The final product is an individual laser disk or "laser particle" ( Fig.21B ). The laser particles can be transferred to an aqueous solution after appropriate filtration.

[0287] When optically pumped at 1064 nm with sufficient pulse energy (1-100 pJ), nearly all harvested laser particles reach the lasing threshold and generate narrowband stimulated emission ( Fig. 21C). Most laser particles emit a single mode corresponding to the WGM mode with l = 10-11. Some particles generate two modes, characterized by two distinct laser lines separated by 50-60 nm, corresponding to the free spectral range of the disk-shaped cavity.

[0288] The laser output from microdisk particles can be measured through relatively thick biological tissues (such as mouse ear skin) with a high signal-to-noise ratio ( Fig. 21C ).

[0289] As described in equation (22), the output wavelength of the laser disk can be tuned by changing the diameter ( Fig.22A ). Using electron beam lithography, it is possible to vary the diameter of the disk with high precision in steps down to 1 nm ( Fig. 22B ).

[0290] In addition to the diameter, several other variables can be used to tune the output wavelength. These include different semiconductor materials for the active and cladding (shell) regions, strain in the semiconductor material, the thickness of the quantum wells, the distance between quantum wells in the case of multiple quantum wells, and the thickness of the passivation layer. Grating structures can be formed on the surface of the laser particle for mode selection and achieving single mode oscillation.

[0291] The material systems for short wavelengths in the UV, blue, and green regions are II-VI compounds and III-V nitride systems such as ZnS, ZnSe, GaN, and AlN. AlGaInP systems (such as sandwiched between (Al x Ga 1-x ) 0.51 In 0.49 P separates Ga in the confining barrier layer y In 1-y P quantum well) can generate visible wavelengths of 600-700nm. For double heterostructures, Al grown on the GaAs lattice x Ga 1-x As is suitable for obtaining lasing wavelengths in the 700-900 nm range. It can be achieved by sandwiching strained In between AlGaAs or GaAs layers. x Ga 1-x As is used to convert the wavelength range of 900-1100nm. x Ga y In 1-x- y In grown on As-InP system 1-x Ga x As y P 1-yCovering the range from 1.1 to 1.65 μm. Other systems include InGaAsN / GaAs quantum well structures.

[0292] For another experimental demonstration of the laser particle and its intracellular operation, we fabricated microdisk lasers from various semiconductor epitaxial wafers. The wafers included InP / InAlGaAs / InP with different alloy compositions for an InAlGaAs gain layer with a thickness of 200 nm (or 350 nm). The nominal gain center wavelengths of the wafers were 1200, 1275, 1350, and 1425 nm, respectively. We also fabricated microdisk lasers from GaAs / AlGaAs / GaAs (840 nm), and InP / InAlAs / InP (840 nm) as well as quantum well wafers.

[0293] Electron beam lithography has high resolution at the 10-nm scale, but requires relatively long scanning times. For rapid mass production, we use optical lithography using custom masks and photoresists. After UV irradiation and reactive ion etching, the size of the disk arrays ranges from 1.5 to 3 μm. The patterned substrate is transferred to an HCl solution for wet etching to remove the InP sacrificial layer. After filtration and washing, we obtain independent laser particles in the colloidal solution. By carefully minimizing the loss of particles, the yield of the wet etching / transfer process can reach 50%. More than 5 million independent laser particles were collected from 10 million microdisks on the substrate.

[0294] Fig.23 The distribution of output wavelengths of 128 laser particles made from three wafers made of slightly different alloy compositions of InAlGaAs is shown. The particle is placed so that one of its flat surfaces is in contact with a flat glass substrate and the other surface is exposed to air. Optical pumping is performed using nanosecond pulses from a Nd-YAG laser (1064 nm), focused to a typical spot size of 1-2 μm. The nominal size of the laser particles is 2.3 μm, but the actual size is different due to limited resolution and random or systematic variations in the optical lithography steps and other possible factors. As a result, the measured histogram shows a wide range of wavelengths over the entire gain spectrum of each wafer. The wide spectral range of 1150 nm to 1500 nm can be completely filled at intervals of 1-2 nm or less by using wafers with appropriate composition and optimized diameter range.

[0295] Semiconductor materials exposed to water and biological environments can generate electrochemical effects that may be toxic. At the surface, the bonding structure is broken. The broken bonding makes the surface highly reactive, resulting in surface reconstruction (surface atomic bonding between them) and surface contamination by absorbing O, C and other atoms and molecules. This can change the energy level of the semiconductor at the surface, which can cause carrier current to leak through the surface to the surrounding environment and generate many undesirable electrochemical processes (such as semiconductor corrosion and water decomposition (split)), as well as the quantum efficiency of the laser particle.

[0296] A practical solution to this problem is to coat the semiconductor surface with a passivation layer so that there is primarily a bulk junction at the semiconductor surface. Widely used coating materials include SiO2 ( Fig.24A ) or a similar semiconductor with a matching lattice constant but a wider bandgap. The wide bandgap prevents carriers from escaping from the semiconductor. To further reduce the undesirable effects due to surface electrochemistry, the encapsulation material and pump wavelength can be chosen so that the pump light does not optically excite the material.

[0297] Surface coating or surface functionalization with biocompatible polymers such as those used in biocompatible quantum dots can further increase the biocompatibility of the laser particles and make them useful in cells and animals for long-term experiments. Laser particles can be coated with such polymers and biocompatible or bioinert materials ( Fig. 24B In addition, biorecognition molecules (such as antibodies, proteins, various small molecules and drugs) and inorganic nanoparticles (such as iron oxide) can be attached to the laser particles ( Fig. 24B ).

[0298] In addition, laser particles can be encapsulated in liposomes to improve biocompatibility and circulation time ( Fig.24C Liposomes can be further functionalized with biorecognition molecules (such as antibodies) for targeting ( Fig.24C ).

[0299] Genetically labeled probes are useful, for which we can use a hybrid approach. The target cell population in the animal can be made to overexpress certain surface membrane proteins, and the laser particles can be coated with antibodies to them so that the laser particles bind to the cell surface. Next, the cells are transfected to produce a specific intracellular protein, and the particles are designed to produce a certain emission characteristic only when bound to the protein in the target cell.

[0300] For biocompatible coatings, we mixed the disks (1 million / ml in EtOH) with 0.1 μM MPTMS (3-mercaptopropyltrimethoxysilane) to deposit a monolayer of silane on the semiconductor surface after transferring the disks off the wafer by immersion in HCl and filtering to remove the HCl. Subsequently, NH3, H2O, and TEOS were added to the solution to grow the silica shell. The reaction time and TEOS concentration were adjusted to obtain the desired silica shell thickness. Fig.25 Shown are SEM images of two InAlGaAs microdisk laser particles coated with silicon dioxide layers of different thicknesses.

[0301] Cells were incubated with isolated laser disk particles to allow their internalization into the cytoplasm. Fig.26 Various images of HeLa cells with one or two intracellular laser particles in the cytosol outside the nucleus are shown. The number of particles per cell can be varied by controlling the cell density and the concentration ratio of the laser particles in the cell medium, as well as other factors. We found that uncoated or silica-coated laser particles apparently moved freely within the cytosol, changing their localization and orientation. We also observed that the radiation force of the optical pump pulses can move the particles.

[0302] Fig. 27 Typical output-pump-energy curves, spectral stability (<1-2nm), and spectral linewidth (0.3-0.6nm) are shown. The narrow-band single-peak laser emission from the intracellular laser clearly demonstrates the feasibility of using laser particles for multiplexing and cell labeling ( Fig. 27 ).

[0303] refer to Fig.28 , considering laser particles, whose spectral width is less than 1nm (Q factor>10 3 ), and their central wavelength can be anywhere in a wide range of over 1000 nm (e.g., 0.6 to 1.6 μm) with a step size of 1 nm. There can be a thousand different laser particles filled in a thousand spectral bins (N=1000). When a set of five randomly selected laser particles is delivered to a cell, the cell is labeled with a unique combination of the five laser lines. The number of possible combinations is given by: C(N,m)=N(N-1)...(N-m+1) / m! , where m is the number of laser particles per cell. For N=1000 and m=5, we find that there are 8 trillion combinations, enough to label cells in an entire mouse!

[0304] Consider two laser particles attached to form a doublet. There are 499,500 (=C(1000,2)=D(1)=S(2)) combinations of doublets. There are 166 million types of triplet lasers (formed by three lasers). These strategies provide a virtually unlimited number of labels. The key factor for this large-scale wavelength division multiplexing is that N is large. For fluorescence, N is limited to 4, or at most 10.

[0305] Laser-based labeling allows cell lineage tracking. Initially, each cell is loaded with many particles or infected in large quantities, e.g., in doublet particles. <m>= 128 without overlap. This allows labeling of about 3,500 (D(1) / 128) cells. Now, these cells divide over time. Since the set of particles is split roughly in half and half at each division, after the 5th generation, an average of 4 doublets will remain in the cell. Since the same doublets are only present in their direct ancestors and not in others, it is possible to identify their family tree. By counting the number of particles, the approximate number of pathways can also be estimated. Proliferation maps can also be measured.

[0306] The disc-like shape can be used to form doublets and triplets (FIG. 29). There are many different ways to make doublets and triplets. As an example, in Fig.29A An illustration for directly bonding two semiconductor disks is shown in FIG. An insulating layer such as a SiO2 layer may be inserted between the disks to avoid optical crosstalk ( Fig.29B The harvested particles can be further coated with functional molecules or nanoparticles and polymers ( Fig.29B ).

[0307] Alternatively, the two laser disks can be coated with two different binding chemicals (such as Tz and TCO click chemistry molecules) and glued to each other. Another method is to use a spacer material such as polystyrene beads with a diameter of 0.3-1 μm. With the appropriate chemical glue, a doublet can be produced ( Fig.29B ) and triplet laser particles ( Fig.29C ).

[0308] In addition to cell labeling, the ability to track individual cells to observe their proliferation, migration, and cell-cell and cell-tissue interactions over time in living animals will be very useful. This is a major advantage over viral labeling techniques (known as DNA barcodes), which cannot be visualized by optical imaging and can only be read in vitro using PCR amplification and sequencing after the animal is sacrificed. Laser-labeled cells can be repeatedly imaged in vivo, analyzed by flow cytometry, and sorted for gene profiling and single-cell RNA sequencing. The ability to obtain such comprehensive information from hundreds to billions of cells at the molecular, cellular, tissue, and system levels in a single animal experiment is unprecedented. It should be noted that laser probes can be used with conventional fluorescent labeling techniques and transgenic reporter mice as well as DNA barcodes.

[0309] Another application area for laser particles is biosensing. Different laser cavities are sensitive to changes in the refractive index variation around the cavity, most notably WGM cavities and photonic crystal cavities. Any of these cavities can be operated in a laser emission scheme, or simply as a notch filter to measure the shift in the resonant frequency. Instead of just measuring the refractive index, the surface of the cavity can be functionalized to bind only specific molecules to the surface, in this way specific detection can be performed. WGMs can interact with the environment outside the microsphere through the evanescent field. Particles on or near the surface of the microsphere change the optical path of the light and / or the cavity losses. This results in a shift in the resonant frequency. The resonance condition in a whispering gallery mode resonator is approximately 2πan1=lλ. Here, a is the radius of the resonator, n1 is the refractive index, l is the mode number, and λ is the resonant wavelength of the mode. The attachment of molecules to the surface increases the effective refractive index.

[0310] This refractive index change shifts the resonant wavelength via:

[0311]

[0312] where α ex is the excess polarization of the bound molecule, σ is the surface density of molecules, ε0 is the vacuum dielectric constant, and n1 and n2 are the refractive indices of the resonator and the surrounding medium, respectively. Since the detection of mode shifts will be limited by the fundamental linewidth of the mode, the minimum surface density can be estimated by using Q ≈ Δλ / λ. For example, bovine serum albumin (BSA) has α ex / 4πε0=3.85x10 -21 cm 3 , and using 15-μm polystyrene beads (n1=1.59) in water (n2=1.33) with Q=2,000, it was possible to measure as low as 5.9x10 per square centimeter. 12 The surface density of molecules is similar to that of 10 7 The corresponding molecules.

[0313] For example, the Stokes radius of BSA is 3.48 nm, so the effective binding area on the surface of the resonator is approximately 3.8x10 -13 cm 2 If BSA molecules were bound without any gaps on the surface of 15-μm polystyrene beads, the total number of BSA molecules would be 1.9x10 7 This can be easily detected with Q = 1,000, and this level of sensitivity is comparable to other label-free methods using surface plasmon resonance.

[0314] Spherical microresonators with very high Q factors are among the most sensitive optical systems. 8 In the case of a 100-μm-diameter microresonator, the light travels tens of meters around the sphere, and the particles on the surface are sampled more than 100,000 times.

[0315] The lasing mode is highly dependent on the geometry of the laser cavity. This can be exploited by making a soft laser cavity and using it as a force sensor. The measurement of small forces inside the cell is demonstrated using oil droplet WGM intracellular lasing. When the droplet is subjected to uniaxial stress, its shape deforms, which manifests as a break-up of the laser lines in the emission spectrum. For small forces, the shape can be roughly spherical. The lasing is confined to the equatorial plane, which has the lowest optical losses due to the minimum curvature. By fitting a model for a non-spherical WGM, the equatorial and polar semi-axes are calculated, giving the mean diameter and eccentricity. The flattening stress Δσ is related to the local mean curvature of the droplet surface by Δσ=2γΔH, where γ is the surface tension and ΔH is the difference in curvature. For small eccentricity (ε 2 <<1), the stress is approximately

[0316]

[0317] For example, the lateral stress exerted on an oil droplet with a diameter of 8 μm in a HeLa cell was measured to be Δσ=500 pN / μm 2 (500Pa). Due to cell dynamics, the stress varies with time. The average fluctuation of the internal stress was measured to be about 150pN / μm 2 (Pa). The sensitivity limit of this method, determined by fluctuations in dying cells, is about 20 pN / μm 2 (20Pa), which is roughly an order of magnitude better than direct image-based analysis.

[0318] Solid polystyrene beads can also be used to measure force fields inside cells and possibly tissues, however, due to the high Young's modulus of solid polystyrene beads, the minimum force that can be measured is much larger than in the case of liquid droplets. Forces were measured during cellular uptake of beads, with the minimum measured stress being in the order of 100 kPa. During cellular uptake of beads, mode decomposition due to deformation of the cavity is difficult to separate from decomposition due to local changes in the refractive index.

[0319] The sensitivity of the resonance peak to temperature can be estimated from the material properties.

[0320]

[0321] where α T is the coefficient of thermal linear expansion, and β T is the relative refractive index variation coefficient of the cavity material. For polystyrene, α T ≈7.5×10 -5 ℃ -1 And β T =-8.2×10 -5 ℃ -1 . Therefore, the two effects almost cancel each other. In fact, our measurements show a mode shift of only 3 pm / °C for beads immersed in pure water. This corresponds to a diameter error of 60 pm / °C for 10 μm beads. This temperature-dependent effect is well within the diameter interval of 2 nm. We note that in mammalian cells in culture or in vivo, the ambient temperature is constant to within a few degrees at 37°C.

[0322] For semiconductors, the contributions from thermal expansion and exponential change have the same sign. For example, GaAs and InP have exponential changes at 37°C of 0.59 × 10 -5 ℃ -1 and 0.46×10 -5 ℃ -1 Alpha T , and β T 7.8×10 at 1.15 μm -5 ℃ -1 and 7.2×10 -5 ℃ -1 For GaAs and InP lasers, this produces a wavelength shift of 60-90 pm / °C.

[0323] It should be noted that the terms "substantially" (such as substantially different or substantially the same), "about", "approximately", etc., with respect to a specified value, may indicate: significantly (or not significantly) different, within acceptable manufacturing tolerances, and / or without deviations that would significantly affect the expected operating parameters. In certain embodiments, an acceptable value (substantially the same as a specified value, substantially different from a specified value, about or approximately a specified value) may have a deviation from the specified value of, for example, + / -1%, + / -5%, or + / -10%, depending on the specific application. Other acceptable deviations include, for example, at most 1% or at most 5% (in the case of substantially the same, about, or approximately), or at least 5% or at least 10% (in the case of substantially greater than / less than)

[0324] The present invention has been described according to example embodiments, and it should be understood that, in addition to those explicitly stated, in addition to combining the different features of the aforementioned versions in different ways, many equivalents, substitutions, changes, additions and modifications can also be made within the scope of the present invention. Although the above detailed description has shown, described and pointed out the novel features applied to each embodiment, it will be understood that various omissions, substitutions and changes can be made to the form and details of the device or algorithm shown without departing from the spirit of the present disclosure. As will be appreciated, certain embodiments of the present disclosure described herein can be implemented in a form that does not provide all the features and benefits set forth herein, because some features can be used or practiced separately from other features. The scope of certain disclosures disclosed in this article is indicated by the attached claims rather than the aforementioned description. All changes within the meaning and scope of the equivalent schemes falling into the claims should be covered by the scope of the claims.< / m>

Claims

1. A microscope system comprising: Pump light source; beam scanner; A spectrometer, comprising: Resolution of less than 1 nanometer; and Spectral acquisition rate greater than 1 kHz; a spectrum analyzer configured to distinguish a spectral peak of the laser output from a broadband background due to fluorescence; and a container for containing a sample comprising a plurality of laser particles, wherein each laser particle is configured to emit a narrowband spectrum when excited by the pump light source, the narrowband spectrum comprising at least one narrowband spectral feature having a width less than 1 nm, The spectrometer and the optical spectrum analyzer are configured to distinguish one or more laser peaks emitted by each of the plurality of laser particles contained in the sample contained in the container.

2. The microscope system of claim 1, further comprising a computer configured to generate one or more images of the sample based on an emission spectrum of the laser particles contained in the sample.

3. The microscope system of claim 1, further comprising an arrangement for fluorescence microscopy.

4. The microscope system according to claim 3, characterized in that The arrangement comprises a light source and a detector configured to detect fluorescence.

5. A flow cytometry system comprising: Pump light source; Flow channel; Spectrometer; a spectrum analyzer configured to distinguish a spectral peak of the laser output from a broadband background due to fluorescence; as well as a container for containing a sample comprising a plurality of laser particles, wherein each laser particle is configured to emit a narrowband spectrum when excited by the pump light source, the narrowband spectrum comprising a spectral barcode feature having one or more line widths less than 1 nm, Wherein, the spectrometer and the spectrum analyzer are configured to identify the spectral barcode feature.

6. The flow cytometry system according to claim 5, wherein: The spectrometer has a resolution of less than 1 nanometer.

7. The flow cytometry system of claim 5, further comprising a computer arrangement configured to generate data based on emissions of the laser particles contained in the sample.

8. The flow cytometry system of claim 5, further comprising one or more light sources and one or more detectors for fluorescence cell counting.

Images