Confocal microscopy system with free-space optical system linkage

Abstract

A rotating disc confocal microscopy system with improved illumination and its components. The system may include (1) an optical engine, (2) a confocal optical system such as a Yokogawa rotating disc confocal optical system, (3) a detector, and (4) a free-space optical system linkage. The optical engine may include at least one light source configured to generate fluorescence excitation light. The confocal optical system can direct the fluorescence excitation light from the optical engine onto a fluorescent sample and collect the fluorescence emission light emitted by the sample. The detector can capture the fluorescence emission light from the sample to form an image of the sample. The free-space optical system linkage can direct the fluorescence excitation light from the optical engine to the confocal optical system at least partially through free space.

Description

Technical Field

[0001] 〔Cross-reference〕 This application is based on U.S. Provisional Patent Application No. 63 / 424,904, filed on November 12, 2022, which is incorporated herein by reference in its entirety for all purposes, and claims the benefit under “35 U.S.C.§119(e)”.

Background Art

[0002] Optical microscopy includes various techniques for generating an image of an object using visible (or near-visible) electromagnetic radiation. Typically, the sample is microscopic (e.g., a cell), and the goal is to generate a high-magnification (500 - 1000×) image with excellent resolution and contrast. Different techniques are distinguished by their methods of generating contrast and / or their resolution.

[0003] Fluorescence microscopy is a dominant form of optical microscopy in the biological sciences. It is sensitive, selective, and equivalent to multicolor imaging of biological samples. In this technique, a fluorescent sample emits radiation mainly in the visible region ( “fluoresces”), and this emitted radiation is captured to generate an image. The fluorescent sample emits light ( “luminesces”) in response to an energy input ( “excitation”) supplied by high-energy short-wavelength radiation (except in the case of multiphoton excitation). Since the input light is spectrally distinct, it can be blocked using a filter. The result is a high-contrast image showing the fluorescent signal against a dark background. Most biological samples are not inherently fluorescent; that is, the sample must be labeled with a fluorescent tag (fluorescent dye). The tag is typically designed to interact with a specific target composition (e.g., within a cell), and thus, the signal originates only from well-defined species or structures.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] Two standard forms of fluorescence microscopy exist. The most basic form, called "wide-field" microscopy, generally illuminates all positions and depths of the sample simultaneously. Unfortunately, this can lead to blurred edges, especially with thick samples, as the image will include contributions from above and below the image plane. An alternative form, called "confocal" microscopy, scans the sample using a focused illumination spot and then eliminates out-of-focus fluorescence using a pinhole filter positioned in the conjugate image plane (placed between the sample and the detector), resulting in an image with significantly reduced edge blur. Laser scanning confocal microscopy (LSCM) uses a single illumination spot and a single pinhole. It is very effective in reducing edge blur. However, it is also very slow, making it less suitable for imaging biological samples. Rotary disk confocal microscopy (SDCM) overcomes the speed limitations and enables imaging of biological samples by using many spots and many pinholes simultaneously. However, dispersing the excitation light across many spots means that the intensity of the excitation light is weakened, fluorescence decreases, and imaging can be slow. Furthermore, suitable light sources are expensive, and their alignment with downstream components can be cumbersome. In addition, imaging functionality may be lost if the illumination fails to excite enough fluorescence from the sample to fill the detector without necessarily overfilling it. Thus, there is a need for confocal microscopy systems with enhanced illumination capabilities, particularly rotating disc confocal microscopy systems. [Means for solving the problem]

[0006] The present invention provides a rotating disc confocal microscopy system with improved illumination and its components. The system may include (1) an optical engine, (2) a confocal optical system such as a Yokogawa rotating disc confocal optical system, (3) a detector, and (4) a free-space optical system linkage. The optical engine may include at least one light source configured to generate fluorescence excitation light. The confocal optical system can direct the fluorescence excitation light from the optical engine onto a fluorescent sample and collect the fluorescence emission light emitted by the sample. The detector can capture the fluorescence emission light from the sample to form an image of the sample. The free-space optical system linkage can direct the fluorescence excitation light from the optical engine to the confocal optical system at least partially through free space. [Brief explanation of the drawing]

[0007] [Figure 1] This is a very high-level schematic diagram of a confocal microscopy inspection system according to an embodiment of the disclosure of the present invention, which may include an optical engine, a confocal optical system, a detector, and a free-space optical system linkage. [Figure 2] This is a more detailed schematic diagram of a confocal microscopy system, such as the system shown in Figure 1, with an emphasis on the optical engine. [Figure 3] This is a more detailed schematic diagram of a confocal microscopy system, such as the system shown in Figure 1, which is linked with a free-space optical system linkage in relation to the upstream optical engine and downstream confocal optical system. [Figure 4] This is a cross-sectional view of a first exemplary free-space optical system linkage having a folded optical path, illustrating how the linkage directs light from a suitable optical engine (left) to a suitable confocal optical system (right). [Figure 5] This is a partially resolved isometric projection of the free-space optical system linkage in Figure 4, rotated approximately 180 degrees around the vertical axis in relation to Figure 4, so that the excitation light enters the linkage in the upper right and exits the linkage in the lower left. [Figure 6] Figure 4 is an exploded isometric projection of the optical engine side components of the free-space optical system linkage. [Figure 7]Figure 4 is a resolved isometric projection of the confocal optical system components of the free-space optical system linkage. [Figure 8A] Figure 4 is an elevation isometric projection of the free-space optical system linkage, showing the location where the linkage, through which the excitation light generated by the optical engine and traveling from the optical engine to the confocal optical system passes, is installed between the optical engine and the confocal optical system, with the optical engine on the left and the confocal optical system on the right. [Figure 8B] Figure 4 is an elevation isometric projection of the free-space optical system linkage, showing the location where the linkage, through which the excitation light generated by the optical engine and traveling from the optical engine to the confocal optical system passes, is installed between the optical engine and the confocal optical system, and the system is rotated approximately 180 degrees around the vertical so that the optical engine is on the right and the confocal optical system is on the left. [Figure 9] This is an isometric projection of a second exemplary free-space optical system linkage with folded light paths, again illustrating how the linkage directs light from a suitable optical engine (left) to a suitable confocal optical system (right). [Figure 10] This is an isometric projection of a second exemplary free-space optical system linkage with folded light paths, again illustrating how the linkage directs light from a suitable optical engine (left) to a suitable confocal optical system (right). [Figure 11] This is a cross-sectional view of the free-space optical system linkage in Figure 9, taken approximately along line 11-11 in Figure 9. [Figure 12] This is a cross-sectional view of the free-space optical system linkage in Figure 10, taken approximately along line 12-12 in Figure 10. [Figure 13] Figures 9 to 12 are isometric projections of the translational mechanism of the free-space optical system linkage. [Figure 14] Figures 9–13 are isometric projections of the free-space optical system linkage, showing the linkage in relation to the mount for the optical engine and a pair of exemplary parallelizing lenses (i.e., downstream lenses for the beam expander). [Figure 15]This is a more detailed schematic diagram of a confocal microscopy system, such as the system in Figure 1, which highlights the confocal optical system and detector that the objective lens and sample are each part of the microscope and can support. [Modes for carrying out the invention]

[0008] Figure 1 shows an exemplary confocal microscopy system 20 having a free-space optical system linkage according to an aspect of the disclosure of the present invention. The system may include an optical engine 22, a free-space optical system linkage 24, a confocal optical system 26, and a detector 28. The optical engine may include at least one light source, such as a single-mode or multi-mode laser, configured to generate fluorescence excitation light 30. The free-space optical system linkage can direct the fluorescence excitation light from the optical engine through free space to the confocal optical system without requiring an optical fiber or liquid light guide. In particular, a confocal optical system, which may include a pinhole disc and an optional lens disc, may be configured to direct the fluorescence excitation light received from the free-space optical system linkage onto a sample 32 and to collect the fluorescence emission light 34 emitted by the sample. In a rotating disc embodiment, the confocal optical system may simultaneously illuminate at least two discrete locations ("spots") separated by unilluminated areas in the sample and simultaneously collect the light from them, thereby increasing the data retrieval rate. Finally, the detector may be configured to capture the fluorescence emission light from the sample to form an image of the sample. Significantly, free-space optical linkages can offer certain advantages over other optical transmission mechanisms. For example, free-space optical linkages can provide greater light output and / or higher uniformity, thereby improving image quality. Similarly, free-space optical linkages can avoid, among other factors, the optical loss and mode noise associated with optical fibers or light guides that arise due to bending within these optical systems. In addition, free-space optical linkages can simplify and therefore expedite initial alignment. Furthermore, free-space optical linkages can increase stability and thus help maintain existing alignment. Further aspects of the system and its components are described below.

[0009] I. Light engine The optical engine is used to generate fluorescence excitation light that has the function of exciting fluorescence from a sample. The optical engine can include one or two or more individual light sources (e.g., one, two, three, four, five, six, seven, eight, nine, or ten or more light sources). The light sources can include, among other things, lasers, light guides, and / or light-emitting diodes (LEDs). The lasers can include single-mode and / or multi-mode lasers. Each light source can have the function of emitting light mainly at one or two or more single wavelengths (e.g., 488 nm or 514 nm) or over one or two or more wavelength ranges (e.g., from 450 nm to 550 nm). In some cases, two or more light sources can emit light having the same spectral quality, and in this case, the light from two or more light sources is combined to increase the light intensity. In other cases, two or more light sources emit light having different spectral qualities, and the range of available excitation wavelengths can be expanded so that the optical engine can be used in combination with a wider range and a greater number of fluorescent dyes. The intensity of the light from each light source can be independently adjustable, for example, to a relative intensity from 0% to 100%. The light emitted by the optical engine can be light emitted from a single light source or mixed light from two or more light sources. The spectral characteristics of the light emitted by the optical engine can be adapted to its intended use, e.g., exciting fluorescence from a preselected fluorescent tag. The optical engine can optionally include a diffuser and / or a speckle remover for reducing laser speckle and / or other non-uniformities. The optical engine can include reflective elements such as mirrors and / or refractive elements such as lenses for combining light from various light sources and placing it on a single optical path. Exemplary optical engines can include, among other things, the Lumencor ZIVA optical engine. See Appendices A1 and A2 of U.S. Provisional Patent Application No. 63 / 424,904, filed November 12, 2022.

[0010] Figure 2 shows a more detailed schematic diagram of an exemplary optical engine 40. This optical engine includes seven light sources 42a-42g and seven associated mirrors 44a-44g. Other optical engines may include fewer or more light sources as described above. The light from these light sources is combined and guided along a single optical path 46 toward a free-space optical system linkage. The first (most upstream) light source 42a can be positioned directly in the optical path. Alternatively, as shown in this figure, the first light source can be positioned outside the optical path. In this case, the light from the first light source is reflected along the optical path by a properly oriented first mirror 44a. This mirror can be fully silvered (i.e., have 100% reflectivity) to reduce light loss. Alternatively, this mirror can be a dichroic mirror that reflects only the wavelength of interest. Subsequent light sources 42b-42g can be positioned outside the optical path (to avoid blocking the light). Light from these downstream light sources can be directed into the optical path using downstream mirrors 44b-44g. These mirrors can be partially silvered dichroic mirrors or multiple dichroic mirrors, such that light from upstream light sources passes through them, and light added by downstream light sources is reflected into the optical path. Partially silvered mirrors reflect and transmit light at least substantially uniformly across the spectrum (for example, a 50:50 partially silvered mirror reflects half of the incoming light and transmits the other half). In contrast, the degree to which dichroic mirrors and multiple dichroic mirrors reflect and transmit light depends on the wavelength. Dichroic mirrors can be configured to transmit almost or substantially all of the upstream light so that it continues to travel along the optical path, and to reflect almost or substantially all of the incoming light so that it is redirected and travels along the optical path. In some cases, light from two or more light sources can be combined before being directed along the optical path. In the same or other cases, light can follow a more intricate path before being directed into the optical path.For example, the light from the downstream light source 42f can hit one or two or more auxiliary mirrors 44f' and be reflected, and then be reflected from the main mirror 44f and enter the optical path. The light engine can include, among other things, a reflective optical system and / or a refractive optical system, a spectral (e.g., low-pass, band-pass, and high-pass) filter for modifying the spectrum ((relative) wavelength components) of the light, a dimming filter for modifying the overall intensity of the light, and / or an auxiliary optical system 46 such as a polarizer for modifying the polarization of the light. These auxiliary optical systems can be positioned and used in relation to individual light sources and / or can be used in a common optical path. The light engine can include elements such as a speckle remover 48 and / or a homogenizer for removing speckle and / or otherwise homogenizing the light generated thereby. The optical path of FIG. 2 is operationally "linear" in that the light from each additional light source is added to the light from all the preceding light sources. However, in other embodiments, the optical path can be "branched" in that the light from two or three or more light sources can be added before the light from other light sources is added. In that regard, the optical path of FIG. 3 is operationally two-dimensional in that the light source and the optical path are shown as coplanar. However, in other embodiments, one or two or more light sources can be in different planes from each other and / or from the optical path and / or can be shifted out of plane before the optical path exits the light engine.

[0011] The light source in this exemplary embodiment can include one or two or more lasers (including the case where only lasers are present). The lasers can be single-mode lasers, multi-mode lasers, or a combination thereof. The multi-mode lasers can have advantages over single-mode lasers in terms of, for example, lower cost and / or higher output power (brightness or intensity). Generally, only one light source is used at a time to supply excitation light matched to a particular fluorescent dye. However, in some applications, two or three or more light sources can be "turned on" at a given time.

[0012] II. Free-space optical system linkage Free-space optical system linkages are used to direct excitation light from an optical engine to a confocal optical system, and in that process, to adjust the light for use by this optical system. Most fundamentally, a free-space optical system linkage includes a mechanism other than an optical fiber or light guide to couple light from an optical engine to a confocal optical system. More specifically, a free-space optical system linkage includes a mechanism that includes transmitting light through free space. Appropriate lenses, such as achromatic, plan achromatic, fluorite, apochromatic, and / or plan apochromatic lenses, can be used to adjust light toward input into the free-space optical system linkage, to adjust light leaving the linkage toward input into the confocal optical system, and / or to manipulate light within the linkage (e.g., to magnify light). Similarly, appropriate mirrors, such as plane mirrors, convex mirrors, and concave mirrors, can be used to direct light into and out of the free-space optical system linkage. These lenses and mirrors, which have fixed or variable positions, can be part of a linkage and / or shared with or incorporated into an optical engine and / or confocal optical system.

[0013] Figure 3 shows a more detailed schematic diagram of a confocal microscopy system, such as the system in Figure 1, highlighting the relationship between the free-space optical system linkage 60 and the upstream optical engine 62 and the downstream confocal optical system 64. The linkage may include, among other components, a homogenizer 66, a beam expander 68, and / or a baffle or diaphragm 70. The optical path through the linkage can be linear or folded, as shown. The advantage of a folded path is that it facilitates the alignment of the optical engine and the confocal optical system (for example, by allowing light to exit the optical engine at one height and enter the confocal optical system at another height, with the heights measured relative to each other, the benchtop, or other shared support structures). Another advantage is that it can have a smaller shape, reducing the footprint of the linkage. In this case, the upstream components of the free-space optical system linkage, particularly the homogenizer, are shared by the optical engine, and the downstream components of the free-space optical system linkage, particularly the beam expander components, are shared by the confocal optical system.

[0014] The homogenizer 66 may include any component for disrupting or otherwise making the light more uniform (particularly transversely to the propagation direction). Examples include a rigid rod, such as a stretched glass (silica) or plastic rod embedded within a support. The rod may have any suitable length and cross-section. Exemplary lengths may be, in particular, about 10 to 100 mm, about 25 to 75 mm, about 40 to 60 mm, or about 50 mm. Exemplary cross-sectional shapes may be, in particular, at least substantially circular, rectangular, or square. Exemplary cross-sectional dimensions may be, in particular, between about 50 and 5000 microns, between about 100 and 3000 microns, between about 200 and 1000 microns, and between about 300 and 500 microns. For example, a homogenizer with a square profile may be, in particular, about 400 microns × 400 microns. In some embodiments, the homogenizer may be incorporated into the optical engine. In other embodiments, the homogenizer can be housed together with other components of the free-space optical system linkage. In this case, the homogenizer and any associated support or housing can be inserted into the ejection port for the optical engine, and any associated interlocks can be activated during this insertion process.

[0015] The beam expander 68 may include any mechanism for expanding the cross-sectional dimensions of the excitation light transversely with respect to the propagation direction. In addition to expansion, the beam expander may parallelize the excitation light (so that the envelope of the excitation light does not converge or disperse too much as the excitation light exits the free-space optical system linkage). Examples of beam expanders include, in particular, Galilean and Keplerian beam expanders. The beam expander may be telecentric. The excitation light beam can be expanded using a suitable combination of refractive elements (and, in some cases, reflective elements). For example, two lenses 72a and 72b can be used. In particular, a first (upstream) lens 72a, such as a 4×6mm lens, can be positioned close enough to the homogenizer exit port 74 (e.g., within the focal length of this lens, such as about 3.909mm) so that the light passing through it is dispersed. In particular, a second (downstream) lens 72b, such as a 30×300mm lens, can be positioned so that the light 76 exiting it is parallelized and parallel. This can be achieved by positioning the second lens at a distance at least approximately equal to the focal length of the second lens (e.g., about 300 mm) from the first lens. More generally, these two lenses include an upstream lens, which is generally small and generally has high refractive power, located operationally close to the optical engine, and a downstream lens, which is generally large and generally has low refractive power, located operationally close to the confocal optical system. In some embodiments, the magnification or relative magnification of the beam size obtained by the beam expander can be at least substantially equal to the ratio of the focal length of the downstream lens to the focal length of the upstream lens (e.g., in the above example, a magnification of about 300 / 6 = 50x). This magnified beam can be used to simultaneously illuminate multiple small lenses, if present, and also multiple pinholes in the rotating confocal optical system. The magnified beam can be sized to properly illuminate the system's camera sensor and reduce wasted illumination that is not expected to be captured by other sensors.To do this, the system may include a selection of second (parallelizing) lenses, such as second lenses with focal lengths of 200mm, 250mm, and 300mm, in which case the user can select the second lens that best fits the camera sensor. Generally, lenses with shorter focal lengths are used for smaller camera sensors, and lenses with longer focal lengths are used for larger camera sensors. The magnified beam may have a profile that fits that of the homogenizer. For example, a square or circular homogenizer can produce a square or circular magnified beam.

[0016] The baffle or diaphragm 70 may include a mechanism for restricting or shaping the transverse profile of the excitation light beam. The baffle or diaphragm 70 may be positioned at any suitable location in the optical path, for example, downstream of the second lens in the beam expander. The baffle can limit the amount of foreign or unwanted excitation light entering the confocal optical system, and among its advantages, it can reduce scattering and background signals. The baffle, like the homogenizer, may have any suitable cross-sectional shape and size. For example, the shape of the baffle can be adapted to the shape of the homogenizer and therefore the expanded beam. For example, among the possibilities, a square baffle can be adapted to a square homogenizer. Furthermore, the dimensions of the baffle can be fixed (e.g., a static aperture) or variable (e.g., an adjustable iris diaphragm).

[0017] Figure 3 further shows an exemplary transverse beam profile of the widened beam. Generally, a widened beam can have any suitable or desirable illumination profile (e.g., one with a contour that fits the homogenizer and / or baffle, as described above). However, in most contexts, the beam profile needs to be at least substantially uniform (i.e., have at least substantially constant intensity transversely with respect to the beam propagation direction). This uniformity helps ensure that image variations reflect variations in the sample rather than variations in illumination (which are not of interest).

[0018] Figures 4 to 8 show details of a first exemplary free-space optical system linkage 80 having a folded optical path. Figure 4 is a cross-sectional view of the linkage. The linkage includes an optical engine (LE) side component (darker shaded in Figure 4) and a confocal optical system (CO) side component (lighter shaded in Figure 4). These components can be joined together to form a variable-height folded optical path between the optical engine (left) and the confocal optical system (right). Excitation light 82 from the optical engine is focused by lens 84 onto and into the upstream end 85 of a rod homogenizer 86. Light emitted from the downstream end 87 of the homogenizer can then be expanded by a beam expander, such as a two-lens telecentric beam expander. In this case, as in the previous embodiment, the beam expander includes an upstream lens 88 with a small diameter and short focal length, and a downstream lens 89 with a large diameter and long focal length. Even while the beam is expanding, the height Y relative to the common support 92 remains constant. LE Light emitted from the optical engine at (or another suitable reference height) at height Y relative to this shared support COA pair of opposing mirrors 90a, 90b in the optical path adjust the beam height (raise or lower it) so that it is directed into the confocal optical system at (or other suitable reference height). Exemplary supports may include, in particular, a breadboard or table surface associated with a vibration-insulating table or vibration-insulating benchtop. Lowering and / or raising the beam and simultaneously expanding the beam (i.e., performing these functions in parallel) can provide significant advantages over performing the functions sequentially. In particular, this parallel performance can shorten the path length of the beam through the linkage and reduce the overall footprint of the linkage. The upstream (LE side) mirror assembly 90a and the downstream (CO side) mirror assembly 90b used to lower and raise the beam may be the same (as shown) or different. The height and orientation of the homogenizer rod and mirrors may be adjustable using appropriate adjustment mechanisms for fine-tuning both the beam height and direction, e.g., homogenizer setting screws 94 and 96a, 96b, respectively. In some embodiments, one or more of these components may be fixed. For example, one mirror can be adjustable, and another can be fixed.

[0019] Figure 5 shows an embodiment of the free-space optical system linkage 80 of Figure 4, rotated approximately 180 degrees around the vertical axis relative to Figure 4, such that the excitation light 82 enters the linkage from the upper right and exits the linkage from the lower left. The linkage may include complementary boss and channel portions to facilitate assembly and maintain it. More specifically, a protruding boss portion 98 can be received within a recessed channel portion 100 and thereby at least partially supported. In this case, the boss portion is associated with the input of light from the optical engine into the linkage, and the channel portion is associated with the output of light from the linkage to the confocal optical system. The linkage may include one or more interlocks 102.

[0020] Figures 6 and 7 show the optical engine (LE) side (Figure 6) and the confocal optical system (CO) side (Figure 7) of the free-space optical system linkage 80 of Figure 4. Light travels through Figure 6 from roughly left to right and from top to bottom. Components specific to the optical engine side include, in particular, a boss portion 98, a homogenizer rod 86, an extension tip 106 for holding it, a homogenizer setting screw 94 used to set the orientation and position of the homogenizer rod, an upstream beam expander lens 88, and a lens holder 108 for it. Light travels through Figure 7 from roughly right to left and from top to bottom. Components specific to the confocal optical system side include, in particular, a channel portion 100. The channel portion can be used to set the position and angle orientation of the optical engine. The optical engine can be suspended in Z (i.e., perpendicular) relative to the confocal optical system. The length of the channel can further set the distance between the optical engine and the confocal optical system. Both the optical engine side and the confocal optics side of the linkage include mirrors 90a, 90b, such as right-angle mirrors, for steering the light beam upward, downward, and / or otherwise. Similarly, both the optical engine side and the confocal optics side of the linkage may include one or more covers 108. The covers can be formed using any suitable method and material (e.g., 3D printing, machining, and / or metal sheet) and can function to shut out ambient light (and / or confine excitation light) and / or protect both the linkage and the operator. The linkage and covers may further include one or more cooperative switches (interlocks) 102 for turning off the light source if the associated cover is removed, for example. In some embodiments, the covers may include holes and / or other openings 110 for allowing access to set screws, among other options.

[0021] Figure 8 shows the relationship between the assembled free-space optical system linkage 80 from Figures 4-7 and the optical engine (LE) and confocal optical system (CO). Panel diagram (A) shows the connection between the linkage and the optical engine. Various switches, ports, and status lights are visible on the optical engine. Panel diagram (B) shows the connection between the linkage and the confocal optical system.

[0022] Figures 9–13 show embodiments of a second exemplary free-space optical system linkage 120 having folded optical paths. This linkage is very similar to linkage 80 in Figures 4–8. In particular, this linkage includes substantially the same components in substantially the same positioning. Therefore, in order to avoid duplication as much as possible, this description will focus on the representative differences between these linkages and / or features that have not yet been explained (if any) with respect to conventional linkages.

[0023] Figures 9 and 10 show the linkage 120 assembled in two configurations: a descending state (or non-expanded state) (Figure 9) and an ascending state (or expanded state) (Figure 10). The degree of expansion corresponds to the vertical separation width Y (measured at the entrance 124 to the homogenizer 126 (located inside the protective sleeve 128)) between the input light 122 traveling from the optical engine and the output light 130 traveling toward the second lens and confocal optics of the beam expander. The separation widths Y in the fully descending configuration, fully ascending configuration, and intermediate separation state are shown. L , Y R , and sometimes denoted as Y (the variable Y is the difference Y from Figures 4 to 8). LE -Y CO (It can also be used to represent the same thing.) In this embodiment, the homogenizer is more closely associated with the linkage chassis than in the embodiments of Figures 4 to 8.

[0024] Figures 11 and 12 are cross-sectional views showing the linkage 120 assembled in the same downward and upward configurations as in Figures 9 and 10, respectively. In these figures, it is easy to trace the path of light 132 through the system from the input light 122 in the upper left to the output light 130 in the lower right. In particular, as in the conventional embodiment, the light passes through the homogenizer rod 126 enclosed in the protective sleeve 128 and the lens 134, which is the first lens of the beam expander, and is then reflected by a pair of right-angle steering mirrors 136a, 136b to adjust its height. Similarly, in these cross-sectional views, it is easy to see how the height between the input and output light corresponds to the height difference between the two mirrors. These mirrors can be adjustable or fixed. In this case, the upper mirror 136a is adjustable and the lower mirror 136b is fixed. Fixing one of the mirrors simplifies the alignment (by reducing the number of variables that need to be adjusted). Fixing the lower mirror means that the adjustable mirror is located above it and is therefore easier to access. The system includes internal light baffles 138 and external light baffles 140 that keep the light localized within the system, while simultaneously preventing leakage of internal light that could potentially cause safety problems and preventing external light that could potentially contaminate the illumination from entering. The light travels a shorter distance in the descending configuration than in the ascending configuration when traveling through the exemplary portion of the linkage. Conversely, this affects the positioning of the second lens of the beam expander such that the path length between the first and second lenses results in parallel light being generated behind the second lens.

[0025] Figure 13 shows a portion of the free-space optical system linkage related to adjusting the separation width between the mirrors (and thus adjusting the height difference between the incoming light arriving from the optical engine and the outgoing light traveling toward the confocal optical system). This translation mechanism 142 includes a movable upper half 144 that fits into the optical engine and a fixed lower half 146 that fits into the confocal optical system. Translation itself utilizes a guide pin 148 associated with the movable upper half, which is received by a corresponding guide groove 150 associated with the fixed lower half and travels within it. The pin-and-groove mechanism can maintain at least substantially the relative orientation of the mirrors when the height is adjusted (up or down).

[0026] Figure 14 shows the relationship between the free-space optical system linkage 120 of Figures 9-13, the mount 152 for the optical engine, and a pair of exemplary parallelizing lenses (i.e., downstream lenses for the beam expander) 154a, 154b having different focal lengths. This figure shows a 30 × 250 mm lens 154a and a 30 × 300 mm lens 154b. These lenses need to be positioned in the beam expander at a distance from the first lens that is at least approximately equal to the focal length of these lenses in order to generate parallel magnified light, as described above. The associated lens holders 156a, 156b are configured in accordance with these positioning. As discussed above, lenses with longer focal lengths are associated with larger magnification or beam diameter. In some embodiments, additional parallelizing lenses, such as a 30 × 350 mm lens, may be present. In this case, as with other parts of the disclosure of the present invention, the first number (30 mm) represents the lens diameter, and the second number (250 mm, 300 mm, 350 mm) represents the lens focal length. Generally, a lens can have any suitable diameter and focal length.

[0027] For alternative expressions of free-space optical system linkages and their relationship to other components of a confocal microscopy system, see Appendix B of U.S. Provisional Patent Application No. 63 / 424,904 filed November 12, 2022.

[0028] III. confocal optics Confocal optical systems are used to achieve confocal illumination and detection from a sample. Figure 15 shows a more detailed diagram of an exemplary confocal optical system 170. These optical systems can be positioned in any suitable configuration, including a folded optical system configuration. In particular, the optical system can be integrated into a microscope or housed in a separate unit that is coupled to it. A rotating disc confocal optical system includes a pinhole disc 172, such as a Nipkow disc, containing tens, hundreds, thousands, or tens of thousands of (generally equal-sized) pinholes 174. The disc and pinholes are positioned in the conjugate image plane. In other words, the pinhole disc, the sample 176, and the image 178 finally formed on the detector 180 are simultaneously in focus. Typically, a subset of the pinholes (e.g., ~1000) is illuminated, and the objective lens projects reduced images 182 of these pinholes onto the sample to achieve multi-beam illumination. As shown by arrow 184, the pinhole disc is rotated at high speed, moving the illumination spot across the sample, and thus eventually illuminating the entire sample. In some embodiments, the entire sample can be illuminated by only a partial rotation of the disc, and this illumination can be completed in as little as 1 millisecond. In other embodiments, the disc may rotate one or two or more times to acquire an image, and / or this process may consume a longer time, including a time considerably longer than 1 millisecond.

[0029] The illustrated embodiment, sometimes referred to as the Yokogawa system, further includes an optional lens disc 186 that fits into the pinhole disc, which confines a set of Fresnel lenses or microlenses 188 that focus excitation light onto pinholes aligned within the pinhole disc. The lens disc rotates rapidly in synchronization with the pinhole disc, as indicated by arrow 190, to maintain the relative alignment of these discs. The use of microlenses in the excitation light path enhances the light compared to systems lacking a microlens array. As a result, image brightness can be greatly improved, especially when using low-intensity light sources. High-speed image acquisition can be achieved using pinholes positioned in a nested spiral set that uniformly illuminates the sample and produces a complete image even after rotation of parts of the disc (e.g., 30° each).

[0030] During use, excitation light 192, generated by the optical engine and transmitted to the confocal optical system via a free-space optical system linkage, is projected onto a portion of the lens disc. The excitation light is focused onto the corresponding pinhole in the pinhole disc by a lens illuminated within the lens disc, passing through a dichromatic (or multiple dichromatic) beam splitter 194 in the process. The excitation light from the pinholes is focused by an intervening objective lens 196 onto individual spots that rotate rapidly across the sample, as indicated by arrow 195. The spots generally rotate rapidly in phase with the rapid rotation of the pinhole disc and, if present, the lens disc. The fluorescent dye within the illuminated spots produces fluorescence emission light 198. A portion of the emission light generated by each spot returns through the same pinhole as the excitation light that induced the emission light, causing the selective exclusion of out-of-focus fluorescence signals. In particular, out-of-focus emission light is out of focus and is therefore blocked by (mostly) missing the pinhole (and adjacent pinholes). Unlike the excitation light, the emitted light is guided towards and projected onto a detector (typically an imaging detector) by a dichromatic (or multiple dichromatic) beam splitter positioned between the pinhole and the lens disc, rather than going to a microlens array (if one exists). The beam splitter allows the excitation light to pass through as described above and reflects spectrally distinct emitted light to it. A tube lens 200 and / or other optical system positioned between the beam splitter and the detector can help focus the emitted light onto the detector. In some embodiments, an excitation light filter and an emitted light filter can be operably positioned between the light source and the beam splitter in the excitation light path, and further between the beam splitter and the detector in the emitted light path, respectively. The excitation light filter can be positioned in particular within the optical engine, within the free-space optical system, and / or within the confocal optical system (e.g., upstream and adjacent to the beam splitter). The emitted light filter can be positioned in particular within the confocal optical system (e.g., downstream and adjacent to the beam splitter) and / or within the detector. An excitation light filter "evens out" the excitation light by allowing only the wavelength or wavelength range suitable for exciting the fluorescent dye of interest to pass through.The emitted light filter, most importantly, also evens out the emitted light overall by blocking excitation light outside the range that could potentially be mistaken for other emitted light.

[0031] In principle, a rotating disk system can acquire up to several thousand frames per second, a frame rate that is significantly superior to that of a LSCM. In practice, other constraints generally reduce the acquisition speed. One example is the need to recover signals of acceptable intensity from low-luminance samples, which often imposes an upper limit of 10 frames per second on the acquisition speed. Therefore, the use of high-quality free-space optical linkages, such as those described herein, is considered crucial for accelerating image acquisition.

[0032] Exemplary confocal optical systems may include, in particular, Yokogawa's confocal scanner units known as CSU-W1® and CSU-X1®. These units can be used with any suitable microscope or microscope platform. Exemplary microscopes may include, in particular, Nikon's Eclipse Ti2® inverted research microscope. See Appendix C1 and C2 relating to confocal optical systems and Appendix D relating to microscopes and microscope platforms of U.S. Provisional Patent Application No. 63 / 424,904 filed November 12, 2022.

[0033] IV. detector Detectors are used to capture fluorescence emitted by a confocal optical system to generate an image. In laser scanning confocal microscopy, point detectors are generally employed because the image is composed of individual points. Examples include photomultiplier tubes (PMTs) and photodiodes. Rotary disk confocal microscopy generally employs imaging detectors. Examples include charge-coupled devices (CCDs), electron-multiplier charge-coupled devices (EMCCDs), complementary metal-oxide-semiconductor (CMOS) devices, and high quantum efficiency back-illuminated scientific complementary metal-oxide-semiconductor (sCMOS) devices. Exemplary detectors may include the pco.edge3.1® scientific CMOS camera. For further details, see Appendix E of U.S. Provisional Patent Application No. 63 / 424,904 filed November 12, 2022.

[0034] V. Selection method This section describes additional selectable embodiments of the disclosures of the present invention, provided without limiting them to a series of items that can be indexed numerically in part or in whole for the purposes of clarity and efficiency. Each of these paragraphs can be combined in any suitable manner with one or more other paragraphs and / or disclosures from other parts of this specification. Some of the paragraphs below explicitly refer to other paragraphs, further limiting them, and provide without limiting examples of some of the suitable combinations. 1. A rotating disk confocal microscopy (SDCM) system comprising: (a) an optical engine comprising at least one light source and configured to generate fluorescence excitation light; (b) a confocal optical system configured to direct the fluorescence excitation light onto a sample and collect the fluorescence emission light emitted by the sample, simultaneously illuminating at least two discrete locations in the sample separated by unilluminated regions and simultaneously collecting light from there; (c) a detector configured to capture the fluorescence emission light from the sample and form an image of the sample; and (d) a free-space optical system linkage that transmits the fluorescence excitation light emitted from the optical engine to the confocal optical system. 2. The system of paragraph 1 in which the free-space optical system linkage does not include optical fibers or light guides. 3. The free-space optical system is the system of paragraph 2, including an optical homogenizer. 4. The optical homogenizer system of paragraph 3 has a square cross-section. 5. The system of paragraph 4, with a cross-section of approximately 400 microns x 400 microns. 6. A free-space optical system, including a beam expander, as described in any of the preceding paragraphs. 7. The system of paragraph 6, wherein the beam expander has a first lens and a second lens, the first lens having a smaller diameter and a shorter focal length than the second lens, and the first lens is positioned upstream of the second lens. 8. The system of paragraph 7, wherein the first and second lenses are converging lenses. 9. A free-space optical system linkage having a homogenizer, wherein the optical path length between the exit port of the homogenizer and the first lens is equal to or shorter than the focal length of the first lens, according to paragraph 7 or 8. 10. A system according to any of paragraphs 7 to 9, wherein the optical path length between the first lens and the second lens is at least approximately equal to the focal length of the second lens. 11. One of the systems from paragraphs 6 to 10 in which the excitation light is parallelized after passing through a beam expander. 12. Any system from paragraphs 7 to 11, wherein the second lens is a first parallelizing lens having a first focal length, and the system further includes a second parallelizing lens having a second focal length, wherein the first and second focal lengths are unequal, and at a given time, only one of the first and second parallelizing lenses is used in the beam expander. 13. The system of paragraph 12, wherein the first focal length is longer than the second focal length, and the first parallelizing lens produces an expanded beam having a larger transverse beam profile than that of the second parallelizing lens. 14. The system of paragraph 13, wherein the detector is an imaging detector, the detector has an imaging area, and the use of either a first or second parallelizing lens in the beam expander depends on which lens maximizes the imaging area without overfilling it. 15. A system of any of paragraphs 12 to 14, further comprising a third parallelizing lens having a third focal length, wherein none of the first, second, and third focal lengths are equal. 16. A system according to any of paragraphs 6 to 15, wherein at least a portion of the light emitted by the beam expander has an intensity profile that is at least substantially uniform in the transverse direction with respect to the beam propagation direction. 17. A system of any of the preceding paragraphs in which a free-space optical system linkage includes a mechanism for adjusting the height of the output excitation light relative to the input excitation light. 18. The mechanism comprises a system of paragraph 17, including a pair of opposing mirrors. 19. A system of paragraph 18 in which the position and / or orientation of at least one of the mirrors is adjustable. 20. A system of paragraph 19 in which both mirrors are adjustable. 21. A system of any of paragraphs 17 to 20, wherein the mechanism for adjusting the height includes at least one guide pin that moves within a corresponding guide groove in a fixed part of the linkage within a movable part of the linkage. 22. A system of any of the preceding paragraphs comprising at least two lasers as the optical engine. 23. The system of paragraph 22, wherein each laser emits light of a different wavelength or wavelength range. 24. A system of light engines comprising at least three separate light sources, two of which produce light having the same spectral quality, and such light being combined to increase its intensity. 25. A system of either of the preceding paragraphs in which the optical engine emits light within at least two different wavelength regimes. 26. A system of paragraph 25 in which the light intensity within each of at least two different wavelength regimes is independently adjustable. 27. A system of paragraph 25 in which the light intensity in one wavelength regime can be kept constant, while the light intensity in the other wavelength regime can be changed. 28. A system of any of the preceding paragraphs in which light from each light source is reflected by a mirror before being combined with light from another light source. 29. The system of paragraph 28, in which the orientation of a mirror can be adjusted to align light generated by one light source with light generated by another light source. 30. The system of paragraph 28, in which the orientation of the mirror can be adjusted to align the light generated by the light source with the entrance to the free-space optical system linkage. 31. A system of any of the preceding paragraphs in which light from each light source is directed along the same optical path. 32. A system of paragraph 31 in which light is directed onto a free-space optical system linkage. 33. A system of any preceding paragraph in which the light source is mounted on a common platform. 34. A system of paragraph 33 in which the light source is positioned within a recess in the platform. 35. A confocal optical system, including a Nipkow pinhole disc, in any of the preceding paragraphs. 36. The system of paragraph 35, wherein the intensity of the excitation light entering the pinhole disk is substantially uniform over at least a portion of the pinhole disk illuminated by the excitation light. 37. A confocal optical system further comprising a lens disc, according to paragraph 35 or 36. 38. A confocal optical system is a Yokogawa optical system, as described in any of the preceding paragraphs. 39. A detector system including an imaging detector in any of the preceding paragraphs. 40. The imaging detector is a charge-coupled device (CCD) in the system of paragraph 39. 41. The imaging detector is a complementary metal-oxide-semiconductor (CMOS) device in the system of paragraph 39. 42. A system of any of the preceding paragraphs, further comprising a controller for controlling the wavelength and / or duration of light emitted by the light engine. 43. A rotating disc confocal microscopy (SDCM) system comprising (a) an optical engine comprising at least one light source and configured to generate fluorescence excitation light; (b) a confocal optical system configured to direct the fluorescence excitation light onto a sample and collect fluorescence emission light emitted by the sample, simultaneously illuminating at least two discrete locations separated by unilluminated regions within the sample and simultaneously collecting light from there; (c) a detector configured to capture fluorescence emission light from the sample and form an image of the sample; and (d) a pair of lenses and a pair of mirrors that magnify and parallelize the excitation light and lower or raise the excitation light, respectively, while simultaneously magnifying the excitation light as it is lowered or raised. 44. A system of paragraph 43 that further includes one or more constraints applicable to any of paragraphs 1 through 42. 45. A method for performing a confocal microscopy, comprising (a) providing or selecting a system from paragraphs 1 to 44, (b) providing or selecting a sample, and (c) forming an image of the sample using the system. 46. ​​The method of paragraph 45, further comprising (a) providing or selecting any system from paragraphs 17 to 21, and (b) adjusting the height of the exit excitation light relative to the height of the input excitation light so that light can travel from the optical engine through the linkage to the confocal optical system. 47. The method of paragraph 45, further comprising (a) providing or selecting any system of paragraphs 12 to 15, and (b) selecting from a plurality of second lenses a second lens that fills the imaging area of ​​the detector without excessively filling it. 48. The method of paragraph 47, further comprising (a) replacing the detector with a new detector, and (b) replacing the second lens with a new second lens having a different focal length that fills the imaging area of ​​the new detector without excessively filling it. 49. The method of claim 45, wherein the free-space optical system linkage has a mechanism for adjusting the height of the exiting excitation light relative to the height of the input excitation light, and the method further comprises adjusting the height of the exiting excitation light relative to the height of the input excitation light so that light can travel from the optical engine through the linkage to the confocal optical system. 50. The method of paragraph 45, wherein the free-space optical system linkage has a beam expander comprising an upstream lens and a pair of candidate downstream parallelizing lenses, wherein the upstream lens has a diameter and a shorter focal length than either of the candidate downstream parallelizing lenses, the focal lengths of the two candidate downstream parallelizing lenses are unequal, and the method further comprises selecting one of the two candidate downstream parallelizing lenses that most substantially fills the imaging area of ​​the detector without excessively filling it for use in the beam expander. 51. The method of paragraph 50, further comprising (a) replacing the detector with a new detector, and (b) replacing the downstream parallelizing lens with a new downstream parallelizing lens having a different focal length that better fills the imaging area of ​​the new detector without excessively filling it.

[0035] VI. conclusion The term "and / or" as used in the disclosure of this invention means all combinations of the elements being enumerated. For example, an enumeration of two elements, "A and / or B," means A, B, or both. Similarly, an enumeration of three elements, "A, B, and / or C," means A, B, C, A and B, A and C, B and C, or all three. Extensions to four or five or more elements follow the same pattern.

[0036] As used in the disclosure of this invention, the term “exemplary” means “exemplary” or “contributes as an example,” and is not intended to imply desirability or superiority.

[0037] As used in relation to the disclosure of this invention, the term "fluorescence" means optical radiation emitted in response to the absorption of light. Accordingly, as used herein, fluorescence encompasses any form of photoluminescence, including standard fluorescence and phosphorescence, which elevates an electron to an excited state, regardless of whether the absorption of one or more photons originates from a singlet, triplet, or other state, resulting in the subsequent emission of new photons.

[0038] The headings used within the disclosure of this invention are for organizational purposes only.

[0039] The optical paths and beam profiles shown in the drawings are for illustrative purposes only and may not be to exact scale. However, more detailed paths and profiles can be determined by simple ray tracing or by other techniques using the information provided in the disclosure of this invention, such as focal length and optical path length.

[0040] The disclosure of the present invention as made herein may encompass several distinct inventions having independent utility. While each of these inventions is disclosed in its preferred form, many modifications are possible, so the specific embodiments of these inventions disclosed and illustrated herein should not be limited in meaning. The subject matter of the disclosure of the present invention includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein. The following claims specifically describe certain combinations and subcombinations that are considered novel and non-obvious. Other combinations and subcombinations of features, functions, elements, and / or properties may be asserted in applications claiming priority from this application or related applications. Similarly, such assertions, whether broader, narrower, equal to, or different from the original assertions, are considered to be included within the subject matter of the disclosure of the present invention. [Explanation of Symbols]

[0041] 120 Free-space optical system linkage 122 Input light 126 Homogenizer 130 Emission light 138 Internal light baffle

Claims

1. A photoengine comprising at least one light source and configured to generate fluorescence excitation light, A confocal optical system configured to direct the fluorescence excitation light onto the sample and to collect the fluorescence emission light emitted by the sample, simultaneously illuminating at least two discrete locations within the sample separated by a non-illuminated region, and simultaneously collecting the light from those locations, A detector configured to capture fluorescence emitted light from the sample and form an image of the sample, A free-space optical system linkage that transmits fluorescence excitation light emitted from the optical engine to the confocal optical system, A rotating disc confocal microscopy (SDCM) system including [specific feature / feature].

2. The system according to claim 1, wherein the free-space optical system linkage does not include an optical fiber or a light guide.

3. The system according to claim 2, wherein the free-space optical system includes an optical homogenizer.

4. The optical homogenizer has a square cross-section, according to claim 3.

5. The system according to claim 4, wherein the cross-section is approximately 400 microns x 400 microns.

6. The system according to claim 1, wherein the free-space optical system includes a beam expander.

7. The beam expander has first and second lenses, The first lens has a diameter smaller and a shorter focal length than the second lens, The first lens is positioned upstream of the second lens. The system according to claim 6.

8. The system according to claim 7, wherein the first and second lenses are converging lenses.

9. The free-space optical system linkage has a homogenizer, The optical path length between the exit port of the homogenizer and the first lens is equal to or shorter than the focal length of the first lens. The system according to claim 7.

10. The system according to claim 7, wherein the optical path length between the first and second lenses is at least approximately equal to the focal length of the second lens.

11. The system according to claim 6, wherein the excitation light is parallelized after passing through the beam expander.

12. The second lens is a first parallelizing lens having a first focal length, The system further includes a second parallelizing lens having a second focal length, wherein the first and second focal lengths are unequal. Only one of the first and second parallelizing lenses is used in the beam expander at a given time. The system according to claim 7.

13. The first focal length is longer than the second focal length. The first parallelizing lens generates an expanded beam having a larger transverse beam profile than the second parallelizing lens. The system according to claim 12.

14. The SE detector is an imaging detector, The detector has an imaging area, Which of the first and second parallelizing lenses is used in the beam expander depends on which lens can fill the imaging area to the maximum extent without excessively filling it. The system according to claim 13.

15. The system further includes a third parallelizing lens having a third focal length, If none of the first, second, and third focal lengths are equal, The system according to claim 12.

16. The system according to claim 6, wherein at least a portion of the light emitted by the beam expander has an intensity profile that is at least substantially uniform in the transverse direction with respect to the beam propagation direction.

17. The system according to claim 1, wherein the free-space optical system linkage includes a mechanism for adjusting the height of the output excitation light relative to the input excitation light.

18. The system according to claim 17, wherein the mechanism includes a pair of opposing mirrors.

19. The system according to claim 18, wherein the position and / or orientation of at least one of the mirrors is adjustable.

20. The system according to claim 19, wherein both mirrors are adjustable.

21. The system according to claim 17, wherein the mechanism for adjusting the height includes at least one guide pin in a movable part of the linkage that moves within a corresponding guide groove in a fixed part of the linkage.

22. The optical engine comprises at least two lasers, according to claim 1.

23. The system according to claim 22, wherein each laser emits light at a different wavelength or range of wavelengths.

24. The aforementioned light engine includes at least three separate light sources, Two of the aforementioned light sources produce light having the same spectral quality, Such light is combined to increase its intensity. The system according to claim 1.

25. The system according to claim 1, wherein the optical engine emits light within at least two different wavelength regimes.

26. The system according to claim 25, wherein the intensity of light in each of the at least two different wavelength regimes is independently adjustable.

27. The system according to claim 25, wherein the intensity of light in one wavelength regime can be kept constant, but the intensity of light in the other wavelength regime can be changed.

28. The system according to claim 1, wherein the light from each light source is reflected by a mirror before being combined with light from another light source.

29. The system according to claim 28, wherein the orientation of the mirror can be adjusted to align the light generated by the light source with the light generated by another light source.

30. The system according to claim 28, wherein the orientation of the mirror can be adjusted to align the light generated by the light source with the entrance to the free-space optical system linkage.

31. The system according to claim 1, wherein light from each light source is directed along the same optical path.

32. The system according to claim 31, wherein the light is directed onto the free-space optical system linkage.

33. The system according to claim 1, wherein the light source is mounted on a common platform.

34. The system according to claim 33, wherein the light source is positioned within a recess on the platform.

35. The confocal optical system according to claim 1, comprising a Nipkow pinhole disc.

36. The system according to claim 35, wherein the intensity of the excitation light incident on the pinhole disc is substantially uniform over at least a portion of the pinhole disc illuminated by the excitation light.

37. The system according to claim 35, wherein the confocal optical system further includes a lens disc.

38. The system according to claim 1, wherein the confocal optical system is a Yokogawa optical system.

39. The system according to claim 1, wherein the detector includes an imaging detector.

40. The system according to claim 39, wherein the imaging detector is a charge-coupled device (CCD).

41. The system according to claim 39, wherein the imaging detector is a complementary metal oxide semiconductor (CMOS) device.

42. The system according to claim 1, further comprising a controller for controlling the wavelength and / or duration of light emitted by the optical engine.

43. A photoengine including at least one light source, wherein the photoengine is configured to generate fluorescence excitation light, A confocal optical system configured to direct the fluorescence excitation light onto a sample and to collect the fluorescence emission light emitted by the sample, wherein the confocal optical system simultaneously illuminates and collects light from at least two discrete locations within the sample separated by a non-illuminated region, A detector configured to capture fluorescence emitted light from the sample and form an image of the sample, A pair of lenses and a pair of mirrors that direct the excitation light from the optical engine to the confocal optical system, while magnifying and aligning the excitation light, and lowering or raising the excitation light, wherein the excitation light is lowered or raised while simultaneously magnified by the pair of lenses and the pair of mirrors, A rotating disc confocal microscopy (SDCM) system including [specific feature / feature].

44. A method for performing confocal microscopy, To provide the system described in claim 1, To provide a sample, and To form an image of the sample using the system, A method that includes this.

45. The free-space optical system linkage has a mechanism for adjusting the height of the output excitation light relative to the input excitation light. The method further includes adjusting the height of the output excitation light relative to the height of the input excitation light so that light can travel from the optical engine through the linkage to the confocal optical system. The method according to claim 44.

46. The free-space optical system linkage has a beam expander including an upstream lens and a pair of candidate downstream parallelizing lenses. The upstream lens has a diameter and focal length smaller than any of the candidate downstream parallelizing lenses, The focal lengths of the two candidate downstream parallelizing lenses are unequal. The method further includes selecting one of the two candidate downstream parallelizing lenses that most substantially fills the imaging area of ​​the detector without excessively filling it for use in the beam expander. The method according to claim 44.

47. Replacing the aforementioned detector with a new detector, Replacing the aforementioned downstream parallelizing lens with a new downstream parallelizing lens having a different focal length that better fills the imaging area of ​​the new detector without excessively filling it, The method according to claim 46, further comprising:

Images